Mycology in palaeoecology and forensic science

FUNBIO744_proof ■ 5 August 2016 ■ 1/19 f u n g a l b i o l o g y x x x ( 2 0 1 6 ) 1 e1 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 journal homepage: www.elsevier.com/locate/funbio Q8 Mycology in palaeoecology and forensic science Q7 Patricia E. J. WILTSHIRE* Department of Geography and Environment, University of Southampton, Southampton Road, Southampton, SO17 1BJ, UK article info abstract Article history: Palynology (including mycology) is widely used in palaeoecological and bioarchaeological Received 21 April 2016 studies. Lake and mire sediments, soils, and the deposits accumulating in archaeological Received in revised form features, invariably contain plant and fungal remains, particularly pollen and spores. 14 June 2016 These serve as proxy indicators of ancient environmental conditions and events. Forensic Accepted 8 July 2016 palynology has been successfully employed in criminal investigations for more than two Corresponding Editor: decades. In recent years, it has included fungal palynomorphs in profiling samples from Geoffrey Gadd crime scenes, and from exhibits obtained from suspects and victims. This contribution outlines the main features of palynology, and gives examples of case studies where fungal Keywords: spores, pollen, and plant spores, have enhanced the interpretation of ancient landscapes Archaeology and land-use, and provided pivotal intelligence, and probative evidence, in criminal Palynology investigations. Pollen Crown Copyright ª 2016 Published by Elsevier Ltd on behalf of British Mycological Society. Proxy indicators All rights reserved. Spores Trace evidence Introduction Ecology is the interdisciplinary study of the distribution and abundance of organisms, their interactions with each other, and with their physico-chemical environment. The ecological literature involving fungi is vast, and only a selective and brief view of the growing appreciation of the importance of mycology in palaeoecology and forensic science can be presented here. Rather than whole plant and fungal remains, the main emphasis will be on palynomorphs1 and palynological2 profiles. Palynology provides the basic tool for a wide range of scientific studies, including those of: ancient environments (as in palaeoecology and bioarchaeology: Birks & West 1973; Dimbleby 1985; Huntley & Webb 1988; Edwards 2000; Innes et al. 2013) and contemporary ones (as in forensic investigations: Wiltshire 2016a). Palaeoecology is the study of changes in the environment over time, and particularly those caused by the impact of human activity. Demonstration of these changes is generally achieved by studying the subfossil macro- and microremains of plants in sediments, and palaeosols.3 Palynology has been established for over 100 y (von Post 1918) with internationally accepted conventions and analytical methods (Ertdman 1921, 1943, 1969; Faegri & Iversen 1964, Faegri et al. Q2 1989; Nilsson & Praglowksi 1992; Jansonius & McGregor 1996; Brown 2008; Wiltshire 2016b). Forensic science relates to, or deals with, the application of scientific knowledge to legal problems; to be termed ‘forensic’, any scientific information must be prepared for, and/or brought to, a court of law. Analysis of pollen and spores has been applied to forensic studies in a meaningful way for * Corresponding author. Tel.: þ44 (0) 1372 272087. E-mail address: [email protected] 1 Palynomorph: Any microscopic entity dispersed away from its origin. 2 3 Palynology: The study of palynomorphs. Palaeosol: Ancient, usually buried, soil. http://dx.doi.org/10.1016/j.funbio.2016.07.005 1878-6146/Crown Copyright ª 2016 Published by Elsevier Ltd on behalf of British Mycological Society. All rights reserved. Please cite this article in press as: Wiltshire PEJ, Mycology in palaeoecology and forensic science, Fungal Biology (2016), http:// dx.doi.org/10.1016/j.funbio.2016.07.005 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 FUNBIO744_proof ■ 5 August 2016 ■ 2/19 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 P. E. J. Wiltshire approximately 30 y, and it has reached its most varied and highest level of development and utility in the UK over the last 22 y. GNS Science in New Zealand, and Texas A&M University (Department of Anthropology), are the only other centres that have significant published record in forensic palynology (Mildenhall et al. 2006). Both specialise in provenancing illicit recreational and counterfeit medical drugs, and counterfeit honey. In the UK, palynology has been used in cases of murder, rape, missing persons, aggravated burglary, theft, and insurance fraud. Palynology is also the basis for: aerobiology (allergens; Hyde 1969); melissopalynology (honey; Jones & Bryant 1992); oil prospection (Hopping 1967); palaeobotany (ancient plants and their evolution; Stuessy 2009); plant taxonomy (Harley & Ubara 2012); and climate change (Kutzbach & Guetter 1986; Chambers 1993). Any pollen grain, plant spore, or fungal spore acts as a proxy indicator of the environment from which it was derived. If sufficient proxy indicators are identified and quantified, it is possible to reconstruct the nature of the past environments embedded during accumulation of the sediment over time. In a similar fashion, proxy indicators picked up from modern vegetation, soils, and other deposits, can allow the forensic practitioner to envisage the kind of environment from which the proxy indicator was produced. The value of reconstructing ancient and modern environments from proxy indicators is shown in the examples presented here. The background materials needing removal are humic acids, cellulose, lignin, and silica, and this is achieved by passing the sample through a series of digestions. After an initial boiling in potassium or sodium hydroxide to eliminate humic acids, the sample is sieved through a mesh (apertures typically 120 mm diameter) to retrieve any macro-remains. It is then centrifuged to form a pellet to which treatments are applied sequentially by boiling in: glacial acetic acid, hydrochloric acid, acetolysis mixture (concentrated sulphuric acid and acetic anhydride), and hydrofluoric acid. In between each treatment, the sample is washed and centrifuged and finally neutralised, and stained. It is then embedded in a preferred mountant to make a permanent preparation. In palaeoecological samples is sometimes possible to obtain very ‘clean’ preparations, where nothing impedes a clear view of the various pollen grains and spores (Fig 1), but in archaeological or forensic samples, the background material is often impossible to remove completely. This makes palynomorph identification and quantification particularly difficult, especially where charred fragments, and other recalcitrant materials, are abundant (Fig 2). Details of the most appropriate methods of preparation for palaeoecological, archaeological, and forensic studies are outlined in Moore et al. (1992), Clarke (1994), Wood et al. (1996), and Wiltshire (2016b). Pollen, plant, and many fungal palynomorphs are able to withstand the stringent preparation treatment because of the robust polymers embedded in their cell walls, sporopollenin in the case of plant palynomorphs, and chitin in fungal ones. Relatively few decomposer microorganisms possess Methods Pollen and spores may be embedded in various matrices such as soil, organic and inorganic sediments (e.g. peat, silt, mud, clays), and other palyniferous4 materials (e.g. leaf litter, humus, food, gut contents). They may also be embedded in natural and synthetic fabrics, and may form films, or be included in debris on various surfaces (e.g. footwear, various parts of vehicles, skin, hair, furniture, weapons, luggage, paper, and vegetation). Palynomorphs are widely distributed and are routinely retrieved from many different kinds of material and object (Wiltshire 2009). In palaeoecology, a restricted number of materials are processed to obtain palynomorphs, and analysis is concentrated on organic and inorganic sediments, and palaeosols. The retrieval of palynomorphs from forensic soils or sediments uses the same procedures as in palaeoecology and archaeology, but when they need to be recovered from surfaces or fabrics, they must first be extracted from those items and then subjected to standardised chemical treatments. The aim is to obtain and present palynomorphs such that their gross and fine structure can be observed by light microscopy (with or without phase contrast), so as much background material as possible in the sample has to be removed. Palynomorphs are washed from the sample or specimen; the washings are then sieved and centrifuged to obtain a pellet of palyniferous material (Wiltshire 2016b). 4 Palyniferous: Having palynomorphs coating the surface, or being embedded in, a sample. Fig 1 e A mixture of palynomorphs in a ‘clean’ preparation which palynologists aim to produce for ease of analysis. Please cite this article in press as: Wiltshire PEJ, Mycology in palaeoecology and forensic science, Fungal Biology (2016), http:// dx.doi.org/10.1016/j.funbio.2016.07.005 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FUNBIO744_proof ■ 5 August 2016 ■ 3/19 Mycology in palaeoecology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3 Q1 a representative profile of the contents of the sample. Thus, scanning electron microscopy (SEM) is not used routinely as it is impractical and unnecessary (Jones & Bryant 2007). The diversity in size, shape, structure, and wall sculpturing, aid the identification of pollen and plant spores (Fig 4). A guide to useful literature for identification is given in Hawksworth & Wiltshire (2015). Palaeoecology Fig 2 e A preparation from a forensic sample, showing background material resistant to removal by standard methods. Charred fragments resist chemical removal and obscure the material needing analysis. This shows the difficulty in analysing archaeological and forensic samples. enzymes capable of hydrolysing these complex polymers and, in hostile environments (those with extreme temperature, low water potential, and/or low oxygen tension) pollen, plant spores, and fungal spores can persist in rocks and certain sediments for millions of years and, in soils, for decades. Palynological preparations are rarely processed for fungal remains alone, and this can cause problems where spores are thin-walled or have delicate appendages. Some pollen may disappear during processing because of the small amount of sporopollenin in the cell wall, and fungal spores with very thin walls, or thin-walled appendages, can be lost. Some pollen taxa may even have some degree of size alteration, although experience has shown that this does not appear to be the case with thick-walled fungal palynomorphs. A range of spores found in various criminal investigations is shown in Fig 3. Analysis involves systematic scanning of prepared microscope slides, and identification of all palynomorphs encountered in equidistantly spaced traverses. Magnifications of 400e1000 are necessary, and phase contrast microscopy is favoured by many practitioners where appropriate. In many cases, a view of the interior structure of the pollen grain wall is necessary for identification, and thousands of palynomorphs may need to be identified and counted to obtain Apart from the oil industry, palaeoecology and bioarchaeology are the disciplines where palynology is used most. Peat, lake muds, ice, or inorganic sediments, receive airborne, or colluviated,5 pollen and spores from organisms (alive and dead) within the catchment of the sampling site. As the sediment accumulates, information about older biological communities becomes buried progressively deeply, while a record of recent vegetation change is found in the shallower strata. Depending on the nature of the sediment, samples are obtained with various kinds of coring equipment. Coring starts at the surface of the sediment (Fig 5), in both terrestrial and aquatic habitats, and continues by bringing up sequences of overlapping sections usually of 50 cm in length (Fig 6). As the depth of sample increases, rods are added to the corer to accommodate the depth (Fig 7). Where the bottom of the sediment or soil sequence is accessible, samples can be obtained by cleaning an excavated face of the deposit and inserting monolith tins (Fig 8). The depth of sample depends on the site of accumulation and, in archaeology, a core may be less than 0.2 m in depth, while in a mire, more than 10 m of sediments may be retrieved. Soil samples, in contrast, are rarely deeper than 0.5 m (Moore et al. 1992; Lowe & Walker 2015), while palaeosols usually become compressed to a few cm beneath overlying deposits. Fig 9 shows an excavated area under London’s Guildhall (Macphail et al. 2008). The ‘dark earth’ deposits developed over the Roman amphitheatre and continued until the 11th century before becoming buried by 3 m of later Mediaeval, Tudor, and modern strata. The 40 cm of black deposit thus represents nearly 1000 y of accumulation, and contains a great deal of environmental information. This urban buried soil is much thicker than most found in rural sites, many of which may be only 2e3 cm in depth. Because palynomorphs represent ecological communities, efforts are made by some palynologists (often working in teams of specialists) to identify as many indicators in a preparation as possible. As well as plant and fungal material, palynomorphs include: pre-Holocene fossil spores; eggs; cysts; plant hairs; diatoms; foraminifera; testate amoebae; acritarchs; dinoflagellates; fragments of chitinous animals, and starch grains (Jansonius & McGregor 1996; Stoermer & Smol 1999; Lowe & Walker 2015; Wiltshire 2016a). The greater the number of proxy indicators included in an analysis, the more accurate will be the reconstruction of any ecological community and, frequently, a palynologist will seek help to achieve accurate identification from experts in specific groups of organisms. The reconstruction of past ecological 5 Colluviation: Process of soil and sediments being washed down a slope. Please cite this article in press as: Wiltshire PEJ, Mycology in palaeoecology and forensic science, Fungal Biology (2016), http:// dx.doi.org/10.1016/j.funbio.2016.07.005 Q3 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FUNBIO744_proof ■ 5 August 2016 ■ 4/19 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 P. E. J. Wiltshire Fig 3 e (AeJ). Examples of fungal palynomorphs encountered in forensic samples (not to scale). (A). Caryospora callicarpa ascospore. (B). Chaetomium cf. mollicellum terminal hairs. (C). Choiromyces meandriformis ascospore. (D). Clasterosporium flexum conidium. (E). Dictyosporium toruloides conidium. (F). Endophragmiella sp. conidium. (G). Melanospora zamiae acospores. (H). Ustilago echinata ustospores. (I). Thermomyces lanuginosus. (J). Thecaphora frezzii ustospores. Not to scale. communities has allowed climate, hydrology, and the nature of soils to be envisaged at specific locations. This has made research on temporal changes feasible, and palynology has been one of the most important means by which changing ecosystems, climate change, and the effect of natural and human impacts on the environment, has been studied. In most cases, palyniferous deposits are obtained by coring or monoliths and then sampled systematically and sequentially in the laboratory; the palynomorphs at any one level are then identified and counted. The array of different taxa found in any sample is termed the ‘assemblage’ and, collectively, the relative quantities of these taxa are termed the ‘profile’. The relative values for each taxon are plotted on a diagram, with palynomorph abundance on the ‘x’ axis, and sediment depth on the ‘y’ axis. Sedimentation rates can be variable, and it is essential to obtain chronological estimates (via e.g. radiocarbon, tephra,6 or even artefacts) of the deposits throughout the cores so that sediment depth may be equated with time. Changes in the pollen and spore profile may then be related to prehistoric or historical events. Conventionally, radiocarbon results are calibrated in ‘years BC/AD’ (before Christ and Anno Domini),7 and standard Bayesian methods are used to 6 Tephra: Volcanic ash. Some palynologists were relatively late in adopting calibration, and there are publications where radiocarbon estimates are expressed as ‘before present’ (with ‘present’ being the fixed point of 1950). Some use the system BCE/CE (before common era/common era) with ‘common’ representing ‘current’. The two systems are numerically identical. 7 obtain corrected time/depth curves; the ranges of probability for radiocarbon estimates are then plotted diagrammatically (Bayliss et al. 1997; Reimer et al. 2013). Although pollen and plant spores are essentially botanical markers, palynology has been used extensively by geologists, geographers, and bioarchaeologists, for reconstruction of past environments. Traditionally, bacteriology and mycology were studied in botany departments, and botany students were the most likely to be introduced to micro-organisms and their biology. Nevertheless, irrespective of their background, it is only relatively recently that palynologists have considered fungal spores, and other non-pollen microscopic entities, to be valuable proxy indicators. Graham (1962) recognised fungi, and other taxa, as potentially valuable indicators of past environments but, in the main, only pollen and plant spores were considered for palaeoecological studies. The inclusion of other classes of palynomorph was even discouraged by some senior botanical palynologists in the past (Faegri & Iversen 1975), and the willingness to accept this negative influence may have been attributed to the relatively narrow biological skills base of most other palynologists. Fungal remains in Holocene sediments were first studied in a systematic way at Amsterdam University in the late 1960s, and resulted in a pivotal and much-quoted paper (van Geel 1972). To date, more than 1300 scientific papers have been published on ‘non-pollen palynomorphs’ (NPPs) (Miola 2012) and, increasingly, fungal spores and other microscopic structures are regarded as important indicators of past ambient environments. When correlated with temporal changes in Please cite this article in press as: Wiltshire PEJ, Mycology in palaeoecology and forensic science, Fungal Biology (2016), http:// dx.doi.org/10.1016/j.funbio.2016.07.005 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FUNBIO744_proof ■ 5 August 2016 ■ 5/19 Mycology in palaeoecology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 5 Fig 4 e (AeJ). Examples of pollen and spores encountered in forensic samples (not to scale). (A). Anthoceros sp, (B). Polypodium vulgare. (C). Pteropsida sp. monolete. (D). Pinus sp. (E). Corylus avellana, (F). Alnus glutinosa (G). Eucalyptus-type (H). Prunus spinosa-type, (I). Salix sp. (J). Calluna vulgaris tetrad. (K). Pollen from different genera of Acanthaceae (scanning electron micrographs). A, C, E, F, G, H, I (Courtesy Dr Judy Webb), B, D, J (Courtesy Professor Vaughn Bryant), K. (Courtesy Professor Robert Scotland). vegetation (as recorded by fluctuations in pollen patterning in the sediments being analysed), fungi have given additional evidence of vegetation change and land-use in ancient times (Bakker & van Smeerdijk 1981). For example, spores of Fig 5 e Palynologists at the commencement of coring. Considerable strength is needed for effective penetration of the deposits. coprophilous fungi, and those associated with burnt ground, have been taken as markers of woodland clearance and domestic husbandry (Wicklow 1988); others have been taken as indicators of certain plant taxa (van Geel 1978), the prevalence of dry conditions, and soil erosion (Anderson et al. 1984). A restricted number of literature studies have been carried out to assess the validity of certain fungal taxa being reliable Fig 6 e A core of sediments in the chamber of the corer. The sediments were obtained from considerable depth and show variation in sediment composition. The dark band consists of glass shards from an old volcanic eruption. Please cite this article in press as: Wiltshire PEJ, Mycology in palaeoecology and forensic science, Fungal Biology (2016), http:// dx.doi.org/10.1016/j.funbio.2016.07.005 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FUNBIO744_proof ■ 5 August 2016 ■ 6/19 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 P. E. J. Wiltshire Fig 7 e Additional poles have been added to the corer so that sediments of greater depth can be obtained. Depths in excess of 6 m are often sampled. indicators of large herbivore dung in palynological profiles, and 33 types have been commended as reliable proxies (Baker et al. 2013). However, these authors have not characterised fungi to species level, and this is crucial as precise substrate preference operates at specific level rather than generic (Dix & Webster 1995). Neither have they addressed the phenomenon of the same species being found on dung produced by a wide range of animals. To refer simply to genera is potentially misleading. Over the last 40 y or so, with fewer botanists being engaged in palaeoecological studies, geographers, and archaeologists have taken the discipline forward. However, currently, many botanists and non-botanists lack a broad biological and ecological training, and many have been reluctant to exploit the huge range of fungal, algal, and animal palynomorphs in palynological preparations. When able to work in teams, multiproxy profiles have been produced but, invariably, those teams do not include mycologists. Many palynologists have restricted their studies to pollen grains and plant spores. It is unfortunate that few palynologists ever liaise with professional mycologists, and most rely on relatively few papers which provide pictures and descriptions of identified, and unidentified, non-pollen palynomorphs (e.g. van Geel 1978, 1986, Fig 8 e An excavated face of a soil profile with monolith sampling tins inserted into the sequence. Complete sequences may be obtained by inserting overlapping tins. 2001; Miola 2012; Prager et al. 2012; Revelles et al. 2016). The unidentified palynomorphs have been assigned ‘type numbers’ and these are quoted as identifiers for communication purposes; these numbers are retained even when the spore has been identified. Invariably, most palynologists lack ready access to comprehensive fungal reference material and, for identification, rely on published descriptions and illustrations for identifying unknowns. Inevitably, misidentifications are possible (Hillbrook 2012) and corrections have to be published (Hawksworth et al. 2016a). An example of the role of fungi in palaeoecology Fig 10 is a summary diagram of a data set of over 60 taxa (including unknowns). The results were obtained from sediments taken from a site near a large, ruined Norse farm Please cite this article in press as: Wiltshire PEJ, Mycology in palaeoecology and forensic science, Fungal Biology (2016), http:// dx.doi.org/10.1016/j.funbio.2016.07.005 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FUNBIO744_proof ■ 5 August 2016 ■ 7/19 Mycology in palaeoecology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Fig 9 e An excavated hole beneath the Guildhall in London. The black layer (Dark Earth) is 40 cm in depth and lies directly over the arena of a Roman amphitheatre. The black layer represents urban accumulation from the Roman period to the 11th century. At greater depth, a layer or mortar can be seen. These sediments lay 3 m beneath the modern building (Macphail et al. 2008). complex in Greenland (Schofield & Edwards 2011). Just four plant and four fungal taxa are plotted here, and the diagram demonstrates how the data complement each other e and are useful for interpretation. The method of construction for this kind of diagram is given above. A column showing the depth, and the nature of the deposits throughout the sequence, is drawn on the left of the diagram, along with radiocarbon estimates. The first plot is loss on ignition, and this indicates the ratio of organic peat to inorganic sediment; greater amounts of inorganic material are indicative of local land disturbance and colluviation into accumulating peat deposits. Microscopic charcoal fragments were quantified and plotted to the right of the diagram. These are indicators of local fires at the site. For ease of reference, horizontal lines are drawn to create three major zones (QIN 1, QIN 2a/2b, QIN 3). Zone QIN1 covers the period just before, and around the time of Norse settlement. The high levels of Sordaria-type and Cercophora-type were interpreted as being indicative of wild herbivore dung, and it is known that reindeer were formerly abundant in this part of Greenland (Ingstad 1966). At the end of the zone, the influx of inorganic material to the peat may have been caused by removal of surface turf for roofing by the settlers. These events are accompanied by a fall in Betula pollen, an increase in Poaceae, and increase in microscopic charcoal. The full diagram also records the appearance of native plants favoured and spread by human activity, as well as aliens introduced by settlers (e.g. Rumex acetosella). The authors considered that early Norse settlers were exploiting birch for a wide range of domestic purposes, and burning the wood locally, and this resulted in the relative fluctuation in abundance of grasses, Betula and Salix. In Zone QIN-2a, there is a marked rise in microscopic charcoal, with an increase in the spores of the Sporormiella-type, 7 and a decrease in Cercophora-type, almost to extinction. The changes in relative spore abundances, along with an increase in microscopic charcoal, was interpreted as the settlers’ management of landscape, and the exploitation of wood for fuel and other domestic purposes. The decline of Cercophora may have been due to a reduction of its woody substrates while Sporormiella may have increased in response to greater inputs of dung from stock animals. Zone QIN-2b represents the middle to later phases of Norse settlement, and fungal spores fell to a very low level, an effect seen at other Norse sites (Schofield et al. 2008). Schofield & Edwards (2011) suggest that there was great pressure on Norse subsistence agriculture at a time when deteriorating climate and progressive soil impoverishment inhibited the maintenance of animal husbandry at previous levels. There appears to have been a drastic decrease in stocking densities of animals, reflected in a marked reduction of dung, the appropriate substrate for Sporormiella- and Sordaria-type spores. Stable isotope studies have indicated that there was a greater reliance on marine foods during this period (Lynnerup 1998). There is also evidence of climatic deterioration (Dugmore et al. 2007) soil degradation and erosion, probably from over-grazing (Fredskild 1992; Mainland 2006). The radiocarbon results show that there was an hiatus in the sediment from the late 14th century to about 1950 AD; i.e. approximately 500 y of the palaeoecological record is missing. Zone QIN-3 represents the vegetation from a time when sheep farming was reintroduced to the area in 1924 (Fredskild 1988). There was less Betula wood, but Salix, and Poaceae gradually increased throughout the zone. Sordaria- and Sporormiella-types also increased markedly, but microscopic charcoal was at lower levels than during the Norse settlement phase. Although shown only in the full diagram (Schofield & Edwards 2011), pollen of Hordeum-type was found in this zone and may represent the attempt to increase hay yields by the local Greenlandic farmers. The considerable increase in both Sordaria- and Sporormiella-types was interpreted as showing the increase of dung from large flocks of sheep. The absence of Cercophora-type was taken to indicate that it might be associated more with wild herbivore dung than that of domestic animals, as was the case in Zone QIN-1 before Norse settlement. It is only relatively recently that palynologists have attempted to identify fungal spores routinely and, even so, relatively few attempt to do so. This is primarily because they lack mycological expertise. Few use standard keys and reference material for identification but instead rely primarily on photographs and diagrams in various publications. Inevitably, this means that their mycological data have a relatively low level of resolution. For example, in Fig 11, Sordaria-, Cercophora-, Sporormiella-, and Podospora-types were used rather than identifications to specific level. This precludes detection of heterogeneity in substrate preferences of the actual fungal species represented by the spores. The underlying reasons for fluctuating spore deposition in the sedimentary sequence can only be surmised. The authors’ interpretation was based on what is known of these fungal genera in general terms but, in the case of Cercophora-type, for example, it would have informative to know if wood- or dung-inhabiting species were Please cite this article in press as: Wiltshire PEJ, Mycology in palaeoecology and forensic science, Fungal Biology (2016), http:// dx.doi.org/10.1016/j.funbio.2016.07.005 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FUNBIO744_proof ■ 5 August 2016 ■ 8/19 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 P. E. J. Wiltshire Fig 10 e Summary diagram of relative abundances of four plant and four fungal taxa plotted against sediment depth (time). This was taken from a much more detailed diagram and is intended to demonstrate the value of fungal spores as indicators of herbivore stocking densities at a site in Greenland (Schofield et al. 2008). involved (Lundqvist 1972). It is easy, therefore, to misinterpret the ecological role of any particular fungal ‘type’ in a palynological diagram, and this could lead to an erroneous interpretation of the environmental history of a site. As palaeoecologists become more adept at identifying fungal spores, correlations between the plant and fungal records will be better understood; many are now making great efforts to refine identifications. In spite of the low resolution in identification, there is little doubt that the inclusion fungi in the palynological profile of the Greenlandic site enhanced the interpretation of the changes in vegetation and land-use during Norse times, and provides added depth and interest to the study(Fig 12). Forensic palynology It is easy to accept that the modern ecologist/palynologist is able to reconstruct and envisage modern ecosystems from modern samples, just as the palaeoecologist does for ancient ones. One main difference is that, in addition to sediments, the forensic palynologist must be prepared to retrieve palynomorphs from any organic or inorganic material or object from crime scenes, suspects, animal corpses, and human remains. The range of taxa requiring identification is also much wider. In the developed world, for many centuries plants and fungi have been transported globally. The growth in export and import of agricultural and horticultural products, and the growing popularity of gardening, has meant that it is essential to include native and alien taxa in reference collections. Until approximately eight years ago, forensic mycology had been employed mainly for: (a) providing information on the growth and spread of fungi in buildings in cases of civil litigation; (b) identification of neurotropic or poisonous species in criminal investigations, or in cases of accidental death. In addition, there are reports of fragments of basidiomes or lichens providing trace evidence, as well as fungal colonies on human remains providing intelligence and probative evidence in the estimation of post mortem interval (time since death). There have been claims that certain basidiomes might indicate the location of buried human remains (Carter & Tibbett 2003; Tibbett & Carter 2003) but no such instance is known, though some macromycetes can indicate disturbed ground. Fungi have also been of concern as biological warfare agents (mycotoxins). These aspects are reviewed by Hawksworth & Wiltshire (2011, 2016), who endeavoured to provide comprehensive bibliographies covering the range of applications of mycology to criminal and civil investigations. Please cite this article in press as: Wiltshire PEJ, Mycology in palaeoecology and forensic science, Fungal Biology (2016), http:// dx.doi.org/10.1016/j.funbio.2016.07.005 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FUNBIO744_proof ■ 5 August 2016 ■ 9/19 Mycology in palaeoecology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 9 Fig 11 e (A). Cannabis pollen grain (19 mm diameter). (B). Spores of Psilocybe semilanceata (10.5e15 mm long axis). (C). Seed of Papaver somniferum (longest axis 0.7 mm). (D). Fruiting head of Papaver somniferum showing oozing latex which contains opiates (Wiltshire et al. 2015a). Botany and mycology have now become recognised as being critically important disciplines in forensic ecology and, most significantly, are accepted as admissible by the British judiciary. The merging of mycological and botanical skills, and collaboration between the mycologist and the forensic ecologist/palynologist, has proved to be synergistic and powerful (Hawksworth & Wiltshire 2011, 2015; Wiltshire et al. 2014, 2015a). Forensic mycology can be employed independently, particularly for estimation of post mortem interval, and the provision of other temporal information, but it has proved most powerful when combined with palynology. Both disciplines have been pivotal in the successful application of ecological principles to forensic investigation, and they have provided probative evidence for many kinds of investigation (Table 1). Joint palynological data have frequently provided the only forensic evidence available, and they have been the key to securing convictions, confessions, or notguilty, verdicts. There has been no case where the results obtained from botanical and mycological evidence have been at variance with the facts of a case as established by a court. They have provided a novel and powerful addition to forensic science. General considerations The forensic palynologist must be able to approach any criminal or civil case holistically, and in an unbiased way. In any criminal investigation, some background information about a case is often required in order to construct the most effective working strategy; but every caveat must be applied in interpretation, and a conscious attempt made to avoid cognitive bias (Dror 2011, 2013). The forensic ecologist must have at least some knowledge of most communities of organisms (particularly those associated with scavenging and decomposition), the habitats in which they occur, and their intra- and interspecific interactions. In the forensic context, the practitioner must understand the variable impacts of temperature, humidity, oxygen tension, and light quality/intensity on the growth and development of plants, invertebrate animals, and fungi. A sound knowledge of soil science, and the environments offered by various bodies of saline and fresh water, is also valuable. In police investigations, ecosystems may be artificial (inside and outside buildings), and all the investigative components need to be considered. These include: (a) the crime Please cite this article in press as: Wiltshire PEJ, Mycology in palaeoecology and forensic science, Fungal Biology (2016), http:// dx.doi.org/10.1016/j.funbio.2016.07.005 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FUNBIO744_proof ■ 5 August 2016 ■ 10/19 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 P. E. J. Wiltshire Fig 12 e Summary of palynological data obtained from rape case in Devizes, Wiltshire. These show: comparator samples from the park (alibi site), wooded area (putative crime scene), clothing and footwear of the female (claimant), clothing and footwear of the male (defendant) (Wiltshire et al. 2014). scene, (b) other pertinent locations, (c) possessions (exhibits) seized from suspects, and (d) the bodies of victims. Where appropriate, the scientist must personally evaluate all relevant locations, any in situ corpses and associated soils, animals, and vegetation, and the skin, hair, nails, nasal passages (turbinates), intestinal tract, vomit, faeces, and clothing of the corpse in the mortuary (Wiltshire 2009, 2016a). The practitioner should also be able to recognise anomalies associated with any aspect of the investigation. To recognise what is not right, it is important to know what is right, and this necessarily takes many years of experience. In the diverse areas of palynological endeavour (see above), palynologists generally focus on analysing a restricted range of materials, such as sediments, soil, honey, spore-traps, fossiliferous rocks, and fresh plant material. This contrasts with the large range of items, substances, and deposits tackled by the forensic palynologist. Many different sediments and soils, vegetation, objects, human remains, gut contents, vomit, and even slime extracted from a corpse’s lung, have all been analysed in recent years (Wiltshire 2016a). Trace evidence Locard’s Exchange Principle is a concept attributed to Edmond Locard (1877e1966) who suggested that every time a person has contact with someone else, a place, or a thing, it results in an exchange of physical materials; it is often paraphrased as ‘every contact leaves a trace’. While there may be exceptions, it is indisputable that this is often the case, and contact trace evidence has been the main source of incriminating evidence in numerous cases. Trace material enables an analyst to show likely contact between objects, people, and places. Plant and fungal palynomorphs are readily transferred to textiles (particularly woven, synthetic ones), any article of clothing and footwear, digging implements, vehicles, hair, fur, plastic, paper, and even hard surfaces. Transference may be primary, secondary, or even tertiary and, with each transfer, the original profile becomes reduced in the numbers of taxa and individual pollen grains and spores; but because of Please cite this article in press as: Wiltshire PEJ, Mycology in palaeoecology and forensic science, Fungal Biology (2016), http:// dx.doi.org/10.1016/j.funbio.2016.07.005 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FUNBIO744_proof ■ 5 August 2016 ■ 11/19 Mycology in palaeoecology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Table 1 e The range of cases in which palynology has contributed to successful outcomes by analysing botanical, palynological, and soil samples. (a) Estimation of body deposition time (b) Estimation of post-mortem interval (c) Demonstration of offender pathways and events at, or in, the environs of an offence (d) Linkage of people, objects, and places (trace evidence) (e) Location of clandestine human remains and graves (f) Differentiation of a kill site from the place where a corpse is deposited (g) Providing information for possible cause of death (h) Providing information of peri-mortem events (i) Demonstrating false rapes, and highlighting other erroneous claims (j) Provenancing origins of objects (k) Elimination of locations as being relevant to enquiries (l) Eliminating suspects (m) Biological warfare mycotoxins (n) Poisoning (plant and fungal) (o) Involvement of neurotropic substances (plant and fungal) their ability to adhere strongly to most materials, palynomorphs are particularly valuable where investigations have been ongoing for many years (cold cases). Many classes of evidence, such as fibres and mineral particles, are easily and quickly lost, but there have been examples of pollen and spores being retrieved after more than 20 y of storage. Even machine washing fails to remove all pollen and spores from fabrics, and normal, domestic hand-washing is very inefficient (Wiltshire 2016). If a suspect denies having visited a place, their statement can be tested by comparing trace evidence profiles from personal belongings (clothing, footwear, vehicles, digging implements, etc.) with those from samples of the soil and vegetation (or other pertinent material) from that location, and any other locations the suspect might have visited. Species lists of all observed live and dead plants (and plant debris if possible) need to be made so that the feasibility of their pollen and spores being found on an article may be anticipated. A plant may be overlooked in a vegetation survey, and its pollen fail to be picked up in samples collected from the place. Here, and in the absence of a record of the plant at the location, the palynomorphs from that plant would not be useful trace evidence if found on a suspect. If, however, spores of a fungus, only associated with a limited range of hosts, one of which was the plant in question, the fungus acts as a secondary proxy indicator. The presence of a plant species can be inferred by the presence of the fungus that depends upon it. In forensic palynological profiles, over 100 taxa are often represented, and both primary and secondary proxy indicators may contribute to the overall picture. They have proved important in several cases in the UK. Samples of soil, sediment, vegetation, and other material obtained from these sites, are termed ‘comparator’ samples rather than ‘controls’. Unlike controls, they are deliberately selected for comparison, and not be collected randomly, or 11 in proscribed transects, as would be necessary for an experiment to investigate one or more variables. Sufficient comparator samples must be obtained in order for a ‘picture of place’ to be obtained. Pollen and spores are distributed heterogeneously on the ground and other surfaces, so sufficient samples must be taken to account for inter-sample variation. Unfortunately, forensic investigations are normally constrained by the available resources and time, and the practitioner often has to undertake work with what is available and/or what is affordable. Palynological samples should also be obtained from a victim’s clothing, footwear, hair, and any items carried at the time of the offence; internal bodily samples should be obtained in cases of rape. It is imperative that attempts are made to obtain comparators from any surface or material likely to have been contacted by the offender and, if appropriate, the victim. The profiles from the suspect can then be compared with those from the victim, and from comparator samples. Every attempt must be made to collect sufficient samples to answer essential questions posed by an inquiry. The sampling strategy at any location, however, will ultimately depend on context and the nature of the crime; consequently, it cannot be too prescriptive. It must be stressed that the exact place of contact can often only be estimated, but with a few exceptions, these are: (1) soil around a grave; (2) backfill of a grave; (3) actual place of an attack (i.e. as described by the victim, or where there is other physical evidence), and the approach path to a crime scene, if this can be established. A valuable use of palynology is in differentiating the places contacted by a corpse after death (Wiltshire 2006b). Brown (2006) demonstrated that victims deposited in seven mass graves in N.E. Bosnia had been exhumed and reburied in a large number of secondary sites. Palynological and mineralogical analyses of sediment, that had been in intimate contact with the bodies, provided environmental profiles of the original burial sites which had been located in areas of contrasting geology, soils and vegetation. Although fungal spores were not considered by Brown at that time, it is easy to see how they could have enhanced the resolution of the analytical data in such cases. Palynology has been useful in locating clandestine graves and other features. A palynological profile will often allow a palynologist to determine a geographical location if the palynologist has a good knowledge of ecology and biogeography. Fungal spores can enhance this ability where the practitioner has knowledge of regional and/or global fungal distributions (Wiltshire & Hawksworth, unpublished). DNA as trace evidence DNA provides powerful evidence of contact, but as sensitivity of the techniques increases, it is emerging that there are sometimes serious problems in interpretation (Goray et al. 2012). This is especially so where partial profiles and low copy number DNA are involved (Gill 2001; Butler 2011, 2014). The relative experience of some forensic scientists in the application of Bayesian statistics may also be problematical, and the complexity of secondary and tertiary transfer of DNA may not be considered sufficiently (Balding & Steele Please cite this article in press as: Wiltshire PEJ, Mycology in palaeoecology and forensic science, Fungal Biology (2016), http:// dx.doi.org/10.1016/j.funbio.2016.07.005 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FUNBIO744_proof ■ 5 August 2016 ■ 12/19 12 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2015). Palynological evidence may be included as part of a police investigation at the commencement of a case, but with the progressive decrease in police budgets, it is now largely sought when DNA, fingerprint, and other physical evidence is poor, lacking, or inadmissible as evidence. A novel approach has been tried in the US whereby the geographical origins of dust samples have been identified by molecular analysis of fungal spores within the dust. In a citizen science project, they obtained house dust samples from the 50 States and obtained 40 000 fungal isolates from them by molecular methods. Statistically they determined that many fungal taxa exhibited a degree of geographical endemism, and they predicted that they might locate the origin of a dust sample based on the molecular profiles in their database. So far, the database consists of fewer than 1000 dust samples and, while this approach looks interesting, they would need very many more to make the approach viable (Grantham et al., 2015). Interpretation Multiple variables are involved in palynological investigation, and the comparators are examined for similarity or dissimilarity of palynological profiles between objects and/or places. No attempt is made to eliminate variables as the aim is observational rather than experimental. If there are many points of similarity, it is more likely that there has been contact; if there are low levels of similarity, the case for contact is weaker. These aspects are discussed by Bryant & Mildenhall (1998), Bull et al. (2006), Wiltshire (2006a, 2006b), and Wiltshire & Black (2006), so are not treated further here. The profile must be sufficiently robust to withstand crossexamination. There is, however, unlikely ever to be a perfect match between the samples, but there should be sufficient commonality to infer contact (Wiltshire et al. 2006a, 2006b). The presence of palynologically rare pollen and spores can also add value to the likelihood of contact (Hawksworth et al. 2016b). Palynological taphonomy8 is highly complex, and understanding the taphonomic parameters surrounding any pollen/spore profile is one of the greatest challenges in interpreting forensic palynological data (Wiltshire 2006a). For interpretive purposes, the forensic palynologist needs to know: (1) the identity of the plants and fungi that have produced the pollen and spores; (2) the way the spores and pollen are dispersed from the parent plant or fungus; (3) the relative pollen/spore productivity of any taxon; (4) the geographical and ecological distributions of the organisms producing the spores and pollen; (5) the phaenology of the organisms involved; and (6) the characteristics of the locations pertinent to the enquiry (hydrology, geology, soil type, plant taxa growing in situ, and the presence of barriers to dispersal (e.g. buildings, walls, fences, hedges, and other obstructions). Pollen is produced only by gymnosperms and angiosperms for fertilisation and seed production. All other 8 Taphonomy: The sum total of factors which determine whether an item (such as a pollen grain) will be found at a particular place at a particular time. P. E. J. Wiltshire groups of land plants (pteridophytes and bryophytes and their allies) produce spores, and their dispersal tends to be limited to their immediate locality; the vectors often being insects or rain splash. Pollen, plant spores, and fungal spores are produced in different ways, and are of varying mass, size, and structure, and these characters affect dispersal patterns. Some fungi, considered ‘weed’ species, such as certain species of Aspergillus and Penicillium, produce vast numbers of easily-dispersed dry spores. Others might have poor dispersal, travelling no further than a few mm. Many taxa are exuded in slime and do not travel far unless carried by vectors such as insects (Gregory 1966; Ingold 1971; Lacey 1995, 1996). Even in the case of agaricoid basidiomes, most spores may fall within 1 m of the sporophores (Galante et al. 2011). However, if the fungus is growing high above the ground as, for example, on the leaves of trees (e.g. Alternaria and Cladosporium); their spores can enter the airspora,9 even if they are passively released. Interestingly, only a relatively small proportion of fungal species are represented in the spore traps of aerobiologists (Lain & Bustillo 2003; Lacey & West 2006; Agarwal 2009; Robertson & Brandys 2011). Robust ascospores, vegetative hyphae, various kinds of hyphal structures, some types of ascomata (e.g. thyriothecia), ascomatal setae, and sclerotia are most commonly found, although, occasionally, distinctive and unusual structures, which are obviously fungal in origin but defy identification, are present in samples. If they have not been previously recorded although are present in both comparator samples and on exhibits, a description, and photographic record would suffice. Identification is preferred because the ecology of a species may be known, and this might provide some level of intelligence. Pollen dispersal is mainly achieved by wind, insects, or mammals and birds as vectors. Where wind is the main vector, the plant invariably produces copious amounts of pollen which finds the female by chance. Where animals are the vectors, generally small amounts of pollen are produced and, waste is minimised by targeted pollination. After release, pollen and spores travel various distances away from source, depending on release and dispersal mechanisms of the taxon concerned; but dispersal patterns can be highly variable and caution must be taken with generalised distances quoted in the literature. There has been a large amount of interest and research on relative pollen production and dispersal in recent years (Davis et al. 2013), but many of these studies depend on mathematical modelling, and extrapolation. It would be inappropriate to use these techniques in forensic investigation, as previous work has shown that each site is unique; forensic applications of palynology need precision in the application of analytical results. Moreover, comparator samples in forensic palynology are not obtained from artificial traps, as many experimental ones are. The ground (soil, vegetation, and other materials) actually touched by a suspect’s belongings, are needed for establishing potential similarity between objects and places. 9 Airspora: Pollen and spores carried by, and suspended in, the air. Please cite this article in press as: Wiltshire PEJ, Mycology in palaeoecology and forensic science, Fungal Biology (2016), http:// dx.doi.org/10.1016/j.funbio.2016.07.005 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FUNBIO744_proof ■ 5 August 2016 ■ 13/19 Mycology in palaeoecology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Wind-pollinated plants, such as Pinus, Betula, and Corylus, invest in the production of vast amounts of pollen which can potentially travel long distances (Fig 9). Even so, most of the pollen of any plant falls near the parent, and dispersal may be limited by physical barriers, especially in urban environments. In insect-pollinated plants, such as Digitalis purpurea, Lonicera spp., and Trifolium repens, it is rare for dispersal to be beyond the plant itself as the pollen is transferred directly to the insect’s body, or just its proboscis. Thus, some pollen and spores are palynologically rare (they virtually never reach the airspora) though the plants that produce them may be common. Rarity is a function of the amount produced, and the way it is dispersed. Rare palynomorphs are often the most potent indicators of contact between an object and the specific plant or fungus that produced it. The areal extent of the palynological catchment around any sampling point is variable, and whether a pollen or spore is found at any particular location, at any particular time, will depend on many taphonomic factors including: (a) variable time of anthesis10; (2) level of decomposer activity in palyniferous deposits; (3) level of plant maturity at the time of sampling; (4) ambient and micro-climates, and weather patterns; (5) human activity; (6) various kinds of physical barriers; (7) residuality, with previous years’ pollen and spores being preserved and redistributed, even becoming airborne again; (8) whether the palynomorph is present as a subfossil in the soil; and (9) the input of allochthonous11 palyniferous material into a sampling site. When assemblages and profiles of these proxy indicators are examined, the origin of the material from which the preparation was made can be deduced, but this can only be achieved if the identification of palynomorphs is at the highest possible level of resolution. In the analysis of over 4000 samples over more than 20 y, no two locations have generated the same palynological profile; each one is evidently unique. A large assemblage of palynomorphs allows the palynologist to envisage the kind of place represented by that assemblage. When the component taxa within the assemblage are quantified and a profile is obtained, an even clearer ‘picture of place’ is produced. This is important where geographical origins are being searched. The overall profile should be similar between places and items being compared, but the presence of rare markers is particularly important as they confer specificity, and demonstrate the uniqueness of the palynological status of each location and item. Case studies A large number of case studies have already been presented and summarised elsewhere (Hawksworth & Wiltshire 2011, 2015) which demonstrate the value of combining botanical and fungal palynomorphs in actual criminal investigations; but to illustrate the power of fungi in forensics, some examples are presented here in outline. 10 Anthesis: Opening of anthers and release of mature pollen. Allochthonous: Not belonging to that place e originating from some other place. 11 13 Multiple consumption of psychotropic plants and fungi In some groups of people, it has become fashionable to meet and engage in mysticism and the consumption of exotic, psychotropic substances under the supervision of a ‘shaman’. During such a session, some men and women in the west of England were given an infusion of ‘Ayahuasca’, administered by a British ‘shaman’ (Wiltshire et al. 2015a). This substance is made from soaking the leaves and stems of two South American climbers (usually Psychotria viridens and Banisteropsis caapi). P. viridens produces dimethyltryptamine (DMT) and B. caapi yields a monoamine oxidase inhibitor which prevents the gut from breaking down the DMT. The psychotropic substance passes straight into the blood stream and across the blood/brain barrier. In the session mentioned above, the participants enjoyed the experience except for one young man who became demented and needed restraint. He was taken home by his friends who nursed him through a coma, dealing with his bodily functions, and feeding him orange juice. On the fourth day after the ‘ceremony’, the man died, and a police investigation was held into the suspicious death. One of his friends stated that the deceased was in the habit of using ‘magic mushrooms’ (Psilocybe semilanceata), and his home was searched for evidence. Two flasks, a tin, a plastic box, and drawers from an item of furniture were provided for palynological analysis. In the post mortem, a standard sample of stomach contents was taken but, in this case, samples of the contents of the lower gut were requested in addition. This was necessary as the man had been kept alive for four days, peristalsis had continued, and defaecation had occurred. Whatever he had consumed on the day of the ceremony would have moved from the stomach into the ileum and colon. One sample was taken from the stomach, three from the ileum, one from the transverse colon, and one from the descending colon. All samples were subjected to palynological analysis. The results of the analysis are shown in Tables 2e4. The first flask contained liquid and mint tea-bag, the latter being identified from its odour and appearance of leaf fragments inside the bag. Table 2 shows that the fluid contained some plant debris, but it consisted mainly of a dense pollen suspension of Cannabis (Fig 10A), and some garden weeds. It is likely that an infusion of hemp plants (including males) had been made and poured into the flask after ‘brewing’. The second flask was dry but washings showed it to have contained a very dense spore suspension of P. semilanceata (Fig 10B). The tin and box also contained very abundant spores, and it is likely that fungi had been stored in them. The drawers also contained the fungal spores as well as some plant debris and pollen of Cannabis, some trees, ferns, and various weeds. It is likely that the deceased had used the cabinet for storing fungi and Cannabis plants. The weeds associated with the flask containing Cannabis probably possibly indicated that the plants had been grown in an untended garden. The pollen in the drawers suggested that fungi had been collected from some natural habitat near to woodland. Table 3 shows the full range of plant material, pollen, and spores found in the man’s gut. As expected the stomach and ileum yielded very little residual material, and most Please cite this article in press as: Wiltshire PEJ, Mycology in palaeoecology and forensic science, Fungal Biology (2016), http:// dx.doi.org/10.1016/j.funbio.2016.07.005 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FUNBIO744_proof ■ 5 August 2016 ■ 14/19 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 P. E. J. Wiltshire Table 2 e Pollen, spores, and other items found in various containers taken from the deceased man’s home. The plus signs indicate presence: increasing plus signs indicate increasing abundance (subjective assessment). Contents of sample Flasks Macroscopic remains Plant debris (unidentifiable) Angiosperm protoxylem Angiosperm fibre bundles Angiosperm vessels and tracheids Epidermal cells Fibres Leaf fragments Sphagnum leaf Trichomes Fungal spores Psilocybe semilanceata Tree/shrub pollen Corylus Fagus Picea Pinus Quercus Ferns Pteropsida monolete Pteridium Herb pollen Cannabis-type Aster-type Apiaceae Artemisia Cerastium-type Cerealia-type Papaver Poaceae Amaranthaceae Ranunculus-type Rumex Urtica-type Geranium Other containers 1 þ þ þ 2 þ Tin þ Box þ þ þ þ þþ þþþþþ þþþþþ þþþþþþ Drawers Middle & bottom þ þ þ þ þ þ þ þ þþ 1 1 1 1 1 1 1 1 2 þþþþþþ 4 3 3 3 2 2 2 1 1 1 1 palynomorphs had moved down to the colon. A summary of the findings from the colon of the deceased is given in Table 4. The data from the transverse and descending colon have been combined. A single orange pip and lentil testa were found along with considerable numbers of Papaver somniferum seeds (Fig 10C). The orange can be explained although the poppy seeds were unexpected. They were highly unlikely to have been from bread (bakers coat bread rolls with them) because he would have been unable to eat while in the coma. The pollen of poppy was also found along with large quantities of P. semilanceata spores, and Mentha and Cannabis pollen. The deceased had obviously drunk from the flasks within days before his death. The mixed assemblage of trees and herbs in the ‘Adventive’ category probably reflects the places from which the Cannabis and fungi were harvested. Of great interest is the number of taxa which are known to be favoured by bees. Rosaceae, Brassicaceae, Fabaceae, and Boraginaceae were well represented. It is known that the Ayahuasca mixture is very bitter and that honey is usually added to it before consumption. It is likely, therefore, that this mixture of pollen was derived from honey being added to the infusion at the ceremony. 2 1 1 1 A feasible explanation for the seeds of P. somniferum was that the deceased had been sucking poppy capsules to obtain the opiate-containing latex (Fig 10D). He was obviously willing to ingest a wide range of psychotropic substances at the same time and the use of the poppy capsule cannot be discounted. The police had charged the ‘shaman’ with manslaughter, but when the results were provided from the man’s colon, it was impossible to implicate Ayahuasca as the main cause of death, especially as none of the other participants had exhibited any ill-effects. The defendant was eventually given 15 m custodial sentence for possession of a Class A drug. Without the analysis of several areas of the man’s gut, and the finding of pollen, fungal spores, and seeds, there would have been no evidence of other drug consumption (Wiltshire et al. 2015a). Rape in Devizes, Wiltshire, UK A girl of 16 reported that her boyfriend had raped her after an evening out together. He had walked her home but, instead of leaving her at her parents’ house, he forced her to walk about another about 100 m to a small strip of land between two Please cite this article in press as: Wiltshire PEJ, Mycology in palaeoecology and forensic science, Fungal Biology (2016), http:// dx.doi.org/10.1016/j.funbio.2016.07.005 Q6 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FUNBIO744_proof ■ 5 August 2016 ■ 15/19 Mycology in palaeoecology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 15 Table 3 e Pollen, spores, and other items found in the gut of the deceased. Contents of sample Stomach contents Macroscopic remains Papaver somniferum seeds Citrus sinensis seed Lens culinaris cf testa Fungal spores Psilocybe spores Puccinia urediniospore Tree/shrub pollen Rosaceae indet. Rosaceae (Crataegus-type) Acer Ligustrum-type Fraxinus Quercus Eucalyptus Betula Pinus Corylus Fagus Picea Herb pollen Brassicaceae (Sinapis type) Urtica-type Boraginaceae (Borago cf) Trifolium-type Poaceae Mentha-type Vicia-type Cannabis-type Cereal-type Fabaceae indet. Ranunculus-type Papaver Brassicaceae indet. Geum Stachys sylvatica-type Chenopodiaceae Rumex Geranium Mercurialis Primula cf Melampyrum cf Aster-type Apiaceae Artemisia Cerastium-type Ferns Pteropsida monolete Pteridium Ileum Colon Flasks Upper Mid Terminal Transverse Descending 1 4 1 Others 2 Tin Box Cabinet Drawers 1 1 1 þþþþ þþþ 1 30 1 4 6 4 2 1 1 1 1 þþþþþ þþþþþ þþþþþþ þþ 1 1 1 9 9 8 6 3 4 1 1 1 1 10 1 1 2 1 4 1 3 2 2 1 þþþþþþ 2 1 2 2 2 2 1 1 1 1 1 1 2 1 1 2 2 1 1 1 4 3 3 2 1 1 1 1 þ ¼ present (not counted). þþþþþþ ¼ increasing number of plus signs shows relative abundance (too abundant to count). roads. The small area was dominated by trees and shrubs; the girl claimed that he raped her by forcing her to the ground onto her back under a large oak tree. The putative rape scene was surrounded by shrubs and the ground was littered with twigs, dead wood fragments, and leaves. A path ran through the woodland, only 10 m from the crime scene; no-one witnessed the offence. The male insisted that they had had consensual sexual relations on the lawn in a local park before he left her at her home; he claimed that this accounted for his semen having been found during the girl’s medical examination. Please cite this article in press as: Wiltshire PEJ, Mycology in palaeoecology and forensic science, Fungal Biology (2016), http:// dx.doi.org/10.1016/j.funbio.2016.07.005 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FUNBIO744_proof ■ 5 August 2016 ■ 16/19 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 P. E. J. Wiltshire Table 4 e A summary of categorised botanical and mycological findings in the colon of the deceased. Macroscopic remains Colon Papaver somniferum seeds Citrus sinensis seed Lens culinaris cf. testa Taxa favoured by bees Rosaceae (total) Brassicaceae indet. Fabaceae (total) Boraginaceae (Borago cf.) Ligustrum-type Stachys sylvatica-type Geum Geranium Melampyrum cf. Taxa here associated with drugs Psilocybe semilanceata Cannabis-type Papaver Mentha-type Adventive taxa Cerealia-type Acer Poaceae Ranunculus-type Betula Eucalyptus Fraxinus Quercus Amaranthaceae Convolvulus Mercurialis Total palynomorphs counted þþ þ þ 30.7 15.0 8.6 6.4 2.1 1.4 1.4 þ þ þþþþ 9.3 2.9 2.9 4.3 3.6 2.1 1.4 þ þ þ þ þ þ þ 140 þ ¼ presence. The park was located approximately 250 m to the south west of the girl’s home. Thus, there was only about 350 m between the park and the site of the putative crime scene. A vegetation survey was carried out in both the park area and the putative crime scene, and a record of the vegetation at each place was made. The whole area was examined to identify any stand of vegetation that would resemble the park or the wooded strip of land. None was found. The male had been wearing cotton over-trousers on top of his jeans, and the police found these discarded on a Cotoneaster bush in a nearby housing estate. Comparator samples were taken from the surface of the ground from the site in the park and in the wooded strip. The grass had been cut since the alleged offence and the cuttings had been left on the ground. As the couple would have contacted these rather than the new grass, samples were taken of the loose cuttings. Standard surface sampling was carried out at the wooded site (Wiltshire 2016b). Palynological analysis was carried out on the footwear, over-trousers, jeans, and upper garments of both parties as appropriate (Table 5), and the profiles of the two sets of comparator samples were examined against those obtained from the exhibits. A large amount of quantitative data were generated from the analysed samples (Wiltshire et al. 2014) although just a simple diagram is given, with a few key taxa (Fig 11). It is immediately obvious that, in spite of the park having large trees around its perimeter, little airborne tree pollen Table 5 e Exhibits from the claimant and the defendant, after a rape allegation. All were analysed for pollen and spore profiles. A summary of the results are shown in Fig 11. Forensic samples for analysis Female Vulval and vaginal swabs Soles of claimant’s shoes Claimant’s shorts Claimants top Claimaints tights Male Penile swabs Knees of suspect’s jeans Bottoms of suspect’s jeans Footwear Trunk and legs of cotton overtrousers Three swabs combined Two combined Buttock area Back of garment Entire garment Six swabs combined Two combined Two combined Soles and uppter Entire garment had entered the ‘alibi site’ (a body-sized area near a path, even though prolific pollen producers, such as Betula and Pinus, were present. The dominant herbaceous taxa were Fabaceae (actually Trifolium-type), and Poaceae. At the wooded site, the dominant pollen taxon in the profile was Betula, followed by Quercus, and Pinus, while only a single grain of Fabaceae and moderate amounts of Poaceae were found. The fungal profile from the grass cuttings was dominated by Epicoccum nigrum (97 %) and very few spores of Bactrodesmium betulicola, and Pestalotiopsis funerea. At the wooded site, a richer assemblage of species was found, dominated by Clasterosporium flexum, Pseudovalsella-like spores, and Pestolotiopsis funerea. It is of considerable interest that only one spore of E. nigrum was found. Long experience of casework has shown that, to accumulate significant amounts of pollen and spores, fabrics and footwear need to have direct contact with palyniferous surfaces. It can be seen, therefore, that both the female and the male had had direct contact with surfaces coated with tree pollen. There were smaller amounts of Betula and Quercus than found in the comparator samples but they were well represented on both parties’ clothing. The high value for Rosaceae on the male is because of the considerable amounts of Cotoneaster pollen transferred from the shrub over which he discarded his over-trousers. Only single grains of Fabaceae were found on the female and the male and, there is little doubt that they would have accumulated much higher values if they had lain on the grass in the park. The high levels of Poaceae and Urtica on both of them was not surprising as there were abundant nettles and grasses around the edges of the area of the crime scene. They are shown as only moderate levels in the woodland comparator samples and this indicates that, during the attack, both parties contacted the plants as well as the ground. The fungal profiles are of particular interest as only single spores of E. nigrum were retrieved from both the male and female and if they had lain on the grass cuttings, they would have picked up high loads of the spores. The fungal profiles of the claimant and the defendant are similar to that of the wooded area. It is likely that the girl accumulated more C. flexum and B. betulicola than her assailant because her back directly contacted the ground which was littered with dead Please cite this article in press as: Wiltshire PEJ, Mycology in palaeoecology and forensic science, Fungal Biology (2016), http:// dx.doi.org/10.1016/j.funbio.2016.07.005 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FUNBIO744_proof ■ 5 August 2016 ■ 17/19 Mycology in palaeoecology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 twigs. The swabs from the girl proved useful as pollen of Quercus was retrieved from the vagina and this was additional evidence that she had been raped under a tree of this genus. These profiles convinced the legal representatives of the defendant that he had not been telling the truth and they advised him to plead guilty to the charge of rape, and he was given a custodial sentence. This was yet another case where plant and fungal palynomorphs resulted in a confession. This obviated the huge public cost associated with criminal court cases. Discussion Only in the UK have fungal and plant palynomorphs been simultaneously analysed in the same forensic samples. Two distinct and separate bodies of forensic information are generated, and these may be used for providing both intelligence and probative evidence (Wiltshire et al. 2015a, 2015b). They are complementary, and serve as independent checks of the data sets. To date, they have not yielded divergent results in any case, but have corroborated each other. Further, one data set may provide information the other cannot, for example, through rare pollen and spore types (Hawksworth et al. 2016). The judicial systems in most of Europe are inquisitorial, whereas the Anglo-American systems are adversarial, with the onus of proving guilt beyond any reasonable doubt lying with the prosecution. If compelling evidence is brought against a defendant, the defence must rebut that evidence as robustly as possible. It is for the jury to decide on the guilt or innocence of the accused, and for the judge to distil the information presented in court to enable the jury to come to a decision. Whether a palynologist works for the prosecution or defence, there is always greater confidence in the testimony when there are two or more independent classes of evidence presented, especially if they are from the same samples. Invariably, any investigator will seek to obtain as many independent lines of evidence as possible in order to make the strongest possible case for presentation to court. Plant evidence is often stronger when supported by mycological evidence, and vice versa. Both are excellent sources of trace evidence, but fungi are particularly valuable because of their ability to grow in seemingly unpromising, and often unexpected, situations if there is even a miniscule amount of suitable substrate available. They have been retrieved from glass, clothing, wood, plastic, painted surfaces, books, metal, and many other objects and substances. Actively growing fungal colonies have helped to determine: post mortem intervals; time of abandonment of premises; sources of contamination in insurance fraud; legal liability; child neglect; and culpability for negligence in the work place. Apart from providing excellent trace evidence, fungal bodies and spores have provided information on: accidental and intentional poisoning; attempted acts of terrorism; cause of death by candidiasis, aspergillosis, mucormycoses, and other mycoses (where these have been unsuspected, and deaths have been deemed suspicious). Biofilms have been shown to be due to negligence in medical establishments, and in other situations where tubing or piping is involved. Fungi are highly diverse and exploitative. Most fungi of forensic importance are 17 the microfungi which may not even be noticed, and investigators are now coming to realise their potential role in forensic investigation. One great limitation of the application of mycology to palaeoecology, bioarchaeology, and forensic science is the lack of mycologists with appropriate experience and knowledge of a wide range of taxa. Modern research tends to be highly focussed, and reflects current teaching. Even if more mycologists were traditionally trained, the individual would need to have stamina, a strong stomach, and be robust enough to cope with sometimes hostile cross examination in the court system. 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