FORENSIC PRACTICE dr Daria Bajerlein (corresponding author) Assistant professor at the Department of Animal Taxonomy and Ecology, Faculty of Biology, Adam Mickiewicz University in Poznań [email protected] prof. dr hab. Maria Wojterska Head of the Department of Plant Ecology and Environmental Protection, Faculty of Biology, Adam Mickiewicz University in Poznań dr Łukasz Grewling Assistant professor at the Laboratory of Aeropalynology, Faculty of Biology, Adam Mickiewicz University in Poznań dr hab. Mikołaj Kokociński Assistant professor at the Department of Hydrobiology, Faculty of Biology, Adam Mickiewicz University in Poznań

Forensic botany: current state of knowledge and possible applications in investigative practice

Summary

Forensic botany is a science that studies biological traces of plant origin with regard to their practical usefulness as evidence used in judicial proceedings. Among the disciplines of forensic botany, the following have the widest application: palynology, plant anatomy, diatomology, plant ecology and plant molecular biology. It has been shown that the knowledge of plants can be used to determine the connections between the alleged perpetrator, victim and . In practice, the methods of forensic botany have been used to identify locations where the hostages were held or the sites of concealment of a corpse, distinguish between the place of the incident and that where the victim was abandoned, identify the perpetrator, the cause and time of death, unravel drug distribution networks, clarify the circumstances of plant and animal smuggling as well as war crimes. Despite the fact that the suitability of forensic botany for determining the circumstances of criminal events has been repeatedly confirmed, this science remains largely underestimated and scarcely used. This article presents the current state of knowledge in the field of forensic botany, characterizes its specific disciplines, possibilities and limitations relating to the application of the methods of forensic botany in investigative practice as well as outlines the perspectives of its further development. Keywords forensic botany, palynology, diatomology, plant anatomy, plant ecology, plant molecular biology, biological traces

Introduction literature, the methods offered by forensic botany remain undervalued and only rarely used, despite As regards investigations, biological traces of human the fact that their suitability for forensic investigations origin, including various body secretions, excretions has been confirmed on numerous occasions [1–6]. and tissues are among the most frequently collected The main reason for insufficient usage of forensic potential evidence at the scene. Notwithstanding the botany methods for law enforcement purposes, foregoing, biological evidence also includes the traces lies in the lack of awareness of the existence of of animal and plant origin. Recent years have shown biological traces as well as lack of knowledge about the usefulness of insects in determining the time, place the methods of identification and preservation of such and cause of death. While the necessity of collecting traces. Nowadays, only in a few countries, such as biological material of human origin is undisputed and New Zealand, Great Britain or the United States, it is awareness of the existence of entomological evidence common practice to consult botanists. The aim of this has been growing among the investigators, biological article is to present the current state of knowledge on traces coming from plants are frequently ignored. forensic botany, possible applications in investigative As has been repeatedly emphasized in specialist proceedings and perspectives of further development.

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Forensic botany is a science dealing with evaluating of literature on the subject of forensic botany is plants for their potential use for law enforcement available in English language. There are relatively few purposes. It comprises several disciplines, such as: works in Polish [18–21]. While the first documented palynology (the study of grains), diatomology application of forensic botany methods in forensics (qualitative and quantitative studies on diatoms), in contemporary times appeared already in the first plant anatomy (the study of the internal structure of half of the 20th century, the intensive development of plants), plant ecology (the study of plant-environment this science started at the turn of the millennium [2]. interactions, plant distribution and abundance) and Generally, particular disciplines of forensic botany plant molecular biology (the study of plant DNA). deal with research on the methods of revealing and Studies have so far shown that single-celled algae recovering botanical traces as well as linking them with and plants belonging to the majority of systematic the circumstances of the event. Notwithstanding the groups, beginning with the simplest forms and ending above, much attention is paid to presenting limitations with angiosperms, can be successfully used to clarify in the use of botanical traces for investigation the circumstances of the offense [7–9]. There is an purposes. extensive range of potential applications of forensic botany in forensics, mostly due to the fact that it helps to find answers to some key questions in the investigation, concerning the time, place and cause of death as well as the perpetrator. In practice, the The earliest and at the same time most intensively methods of forensic botany can be used to identify developed discipline of forensic botany is forensic locations where the hostages are being held or the palynology. The first references to its usability sites of concealment of a corpse, distinguish between in explaining the circumstances of the offenses the place where the murder was committed and that appeared in the 1930s [22], but it was introduced into where the victim’s body was abandoned, identify the forensic practice not until 20 years later [23]. Forensic perpetrator, the cause and time of death, unravel drug palynology undertakes research on pollen grains and distribution networks, clarify the circumstances of plant spores for law enforcement purposes. Pollen grains and animal smuggling as well as war crimes [10–13]. are microscopic structures (invisible to the naked In general, the use of botanical knowledge in eye), ranging in size from 5 to 200 µm, containing investigative proceedings is based on two assumptions. male germ cells and thereby playing a key role in the The first assumption is Locard’s exchange principle, process of fertilization. The spores, produced by fungi according to which, any contact between two objects and cryptogams, including mosses and ferns, are will result in a mutual exchange of matter. This means, responsible for sexual reproduction thereof. Pollen and that botanical can be used to infer links spores are collectively referred to as palynomorphs. between the scene, the victim and the perpetrator. For Forensic analysis is mostly based on the examination example, pollen grains revealed on the suspect’s shoes of pollen, whose certain features predispose it to serve or clothing may help to establish his/her identity as the as evidence. perpetrator [14]. The second assumption conditioning the use of forensic botany methods relates to the distribution of plant species around the world, i.e., the Traits of pollen grains useful for forensic analysis issues covered by phytogeography. Particular plant species are characterized by different environmental Due to the limited size, pollen grains are easily requirements, especially as regards soil conditions, transferable between objects. It has been shown that temperature, humidity and sunlight. Many species are pollen can adhere to practically all kinds of surfaces, tolerant of a broad range of environmental conditions, including organic (leather, cotton) and artificial (latex) hence their wide occurrence. Conversely, less tolerant material [24]. In forensic practice, pollen has been species are present at specific locations, easy to define revealed on clothing [10], shoes and soil in shoeprints and find. Knowledge of geographic and environmental [25] and hair [11]. Especially, the hair of the head and specificities can be used to determine relationships eyebrows make an excellent trap for pollen grains. The between the scene, the victim and the perpetrator. For persistence of pollen in the hair depends on how often instance, the presence of plant species on the victim’s it is washed and also on the use of various cosmetic body that are uncommon in the environment where the products, such as hairspray, gels or waxes (increasing corpse was found, may suggest that the corpse has adherence of pollen) [11, 26]. Pollen has been also been moved after death. revealed on the body surface [26] as well as inside There is an extensive literature on forensic botany, the corpse, during the autopsy [26–27]. Pollen grains including original and methodology papers, review inhaled through the nasal passage are retained by articles and case reports. The most numerous are mucus secreted within the nasal cavity. After death, methodology-case papers and review articles. Also pollen can remain preserved around nasal turbinates, available are several manuals [15–17]. The majority even in skeletonized corpse. There are several

72 ISSUES OF FORENSIC SCIENCE 289(3) 2015 FORENSIC PRACTICE articles on the methods of retrieving pollen from human relationship between qualitative composition of pollen corpse [11, 26–27]. discovered on a corpse and the flowering period, the Pollen is characterized by an exceptional resilience so called “crime pollen calendars” are developed to adverse conditions. Its outer layer (exine) is built of [26]. Moreover, it is worth pointing out that pollen sporopollenin – one of the most durable substances grains originating from different plant species can produced by living organisms. Sporopollenin confers have different durabilities, which is a species-specific extraordinary resilience on pollen grains, i.e., against trait [5]. Some plant species produce more fragile pollen strong acids or high temperature. Owing to this, (e.g. orchids), whereas in other species it is long- pollen grains remain well preserved on the surfaces lived (e.g. alder, lime tree). Because of the above, of various objects and can be revealed and retrieved the probability of finding the former type of pollen in even under extremely unfavorable conditions [28-31]. a sampled material will be lower, whereas the latter Pollen grains of different species display characteristic will be overrepresented. The occurrence of short- size, shape and ornamentation, which features allow lived pollen can lead to a two-pronged approach to their identification and classification into particular investigative reasoning. On one hand, it can be missing genera or species (Fig. 1). from the sample even if its parental plants are present in the area where the sample was collected. On the other hand, the presence of short-lived pollen in a sample can be an indicator of a particular plant species, thus facilitating the identification of an environment from which it originated and testify the short period elapsed between the event and preservation of the botanical trace evidence.

Ways of spreading pollen and its usability for forensic analysis

From a biological point of view, the main role of pollen grains is to participate in plant reproduction. For successful fertilization, pollen has to be transferred onto the female reproductive organs, which occurs by means of wind, water, animals or self-pollination. Plants usually produce large quantities of pollen (often a few to several thousand grains per flower), Fig. 1. Pollen grains: A – Beech (Fagus sylvatica L.), which results from its biological function. However, B – Alder (Alnus glutinosa Gaertn.), C – Common Nettle the number of grains produced, depends largely (Urtica dioica L.), D – Norway Spruce (Picea abies (L.) on the mechanism of pollen dispersal. The largest Karst.). quantities of pollen are produced by wind-pollinated Successful identification of pollen may give plants and the lowest by insect-pollinated and self- a hint to the identification of an environment from pollinating plants. It has been calculated that a single which it originated. Different geographical areas are Hemp (Cannabis sativa L.) flower (wind-pollinated characterized by a specific pollen profile that reflects plant) produces over 300 thousand pollen grains, plant species composition of a given area. One of the while a Marrow (Cucurbita pepo L.) flower (insect- tasks of forensic botany is to develop pollen profiles pollinated plant) approx. 15 thousand [34]. characteristic of different types of environments, the Wind-driven pollen dispersal is not effective, due to its so called pollen , which are very helpful in coincidental nature. Therefore, the low probability of identifying the place of the incident [13, 32]. According a successful fertilization is increased by producing large to literature data, to date, there have been no two quantities of pollen. Insect-pollinated plants produce different areas discovered with an identical pollen less pollen because it can be dispersed more effectively profile [33]. Correct identification of pollen enables by animals who come in contact with flowers. In judicial the determination of an environment from which it practice, pollen grains of insect-pollinated and scarce originated as well as the time when it was released plant species with a narrow range, are most helpful from its parent plant. The majority of plants flower in determining the circumstances of the offenses. during specific seasons, e.g. snowdrops in early spring, Pollen grains of insect pollinated plants are relatively poppies in summer and chrysanthemums in autumn. large (usually above 40 µm), richly ornamented, By knowing the corresponding release dates of pollen viscous (covered with pollenkit) and heavy, hence they grains that were discovered e.g. on a corpse, one can cannot be transported over long distances. Due to estimate the approximate time of death. Based on the its size, pollen of insect-pollinated plants falls much

ISSUES OF FORENSIC SCIENCE 289(3) 2015 73 FORENSIC PRACTICE more rapidly than that adapted for wind dispersal. It that the examination of pollen distribution in enclosed has been shown that pollen of some plant species areas can be very instrumental in investigating the can fall at a rate of 2–3 cm/s. (e.g. light pollen from circumstances of the offence, especially theft, by grass species), or more rapidly for other species facilitating the comparison of pollen found on the [1, 34]. Therefore, finding the second type of pollen suspect’s clothing with that present in the area where grains on a corpse indicates the neighborhood of the offence was committed [8, 30]. Interesting results the parent plant. Less relevant to forensics are pollen are also being brought by studies on pollen preservation grains of wind-pollinated plants, especially those upon contact with various materials. The analysis of commonly found, e.g. grasses. It has been observed pollen on paper surfaces can be useful in determining that 95% of wind-dispersed pollen grains fall within authenticity of documents, e.g. by determining the time 25 m – 2 km of the parent plant, hence their presence of generation, based on pollen composition or on the at the incident scene does not necessarily mean that sequence of events leading up to the generation [29]. the parent plant grows nearby [15]. The results of studies in this field have shown that pollen is best preserved on rough paper surfaces and on ink. Also the envelopes and pen nibs can host large Pollen grains as trace evidence amounts of pollen [29]. If handwriting activity preceded the deposition of pollen, most of the grains will amass There is an extensive literature on forensic palynology, along paper fold lines. Conversely, if pollen appeared including original and methodology papers, review before handwriting, its distribution on paper will be articles and case reports. Predominant among them irregular, driven by hand pressure. are the review articles, which summarize the current The precondition for performing palynological state of knowledge, describe the methods of collecting analysis for investigative purposes is the awareness of pollen as well as problems and limitations related to the evidentiary value of pollen. In addition, it is not only pollen analysis for evidentiary purposes, including the knowledge of pollen grains morphology and how references to particular countries [5, 6, 32, 35, 36]. they spread that is required, but also the understanding There are also numerous case reports describing the of their biological properties and preservation under use of palynology for explaining the circumstances of various conditions. One of the major problems the the offenses [8, 10–12, 33, 35]. In investigative practice, forensic botany expert may come across, is finding an pollen proved its effectiveness in the identification of answer to the question whether the preserved material the scene of crime [11] or the alleged perpetrator [8, is suitable for analysis for investigation purposes and 14, 37], distinguishing between the place of committing how it may be used. For this purpose, experimental crime (rape, murder) and abandoning the victim’s studies are carried out on pollen preservation body [10, 11, 33], determining time of death [26, 27], under extremely adverse conditions, such as high establishing the sources of counterfeit medicines [38] temperature, in order to determine whether this type of and the circumstances of animal smuggling [12]. material could be used as evidence [31]. Original papers of an experimental nature are Although palynology is one of the oldest disciplines relatively few in number. From the perspective of of forensic botany and its usefulness in forensics has investigative practice, such works are most in demand been repeatedly confirmed, it has many limitations as they provide information about pollen deposition that prevent its application. The most often indicated on various surfaces, its durability under different limitations involve: difficulty in finding an expert, environmental conditions and resilience to different, improperly preserved material, insufficient amount of mostly unfavorable, factors. As a consequence, such material for the purpose of analysis, lack of reference experiments enables the development of methods collections and financial issues related to carrying out of pollen recovery from different surfaces and the palynological analysis [1]. assessment of the use of pollen as evidence. To date, studies in this field have addressed pollen preservation in enclosed areas [30], on documents [29], inside motor Forensic diatomology vehicle engines [28], inside burned motor vehicles [31], on clothing and footwear [14, 24, 39], in shoeprints [25], Forensic diatomology is a relatively young discipline of directly on human corpses [11, 26-27] and on the forensic botany that involves the study of microscopic soil surface [40]. The study on spatial and temporal algae as evidence for law enforcement purposes. An distribution of pollen in enclosed areas showed a strong object of interest of forensic diatomology are single- tendency towards decreasing amounts of pollen with celled algae, diatoms. The most distinctive feature increasing distance from its source (in this particular of diatoms is the encrustation of cell wall with silica case, flowers in a vase). Spatial pollen distribution was (hydrated silicon dioxide) forming a shell called influenced by such factors as the air flow, morphology a frustule. A frustule consists of two halves called of pollen grains and the number and arrangement of valves (upper and lower) that overlap forming a box- vases with flowers. The experiments have indicated like structure (Fig. 2). Diatoms are the largest group

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passively, and due to failure of the cardiovascular system, diatoms may reach only to the lungs, without penetrating the internal organs. However, there are known cases of finding diatoms in the internal organs, although drowning was not the cause of death. Such a situation may occur when water carrying diatoms penetrates inside the body through wounds or during corpse decomposition. Some diatom species may be spread by wind and get into the lungs along with the air inhaled. The source of diatoms in the body may also be water and food, as well as cigarette paper [46]. In order to determine the place and time of death and to identify the perpetrators, the knowledge of diatom ecology is used, particularly in terms of their environmental preferences and seasonality [41–42]. Based on a broad knowledge of the ecology of many well known bioindicator species of diatoms, also used in monitoring of surface waters, diatom assemblages may serve as environmental and Fig. 2. Diatoms: A – Epithemia sorex Kütz., B – Sellaphora seasonal indicators. Knowing the structure of such pupula Mereschkovsky, C – Cavinula scutelloides Lange- assemblages, it is possible to compare qualitative and Bertalot, D – Gomphonema acuminatum Ehrenb. quantitative composition of diatoms from the evidence material with a control sample (material collected from of phytoplankton. They are found in various areas of the alleged environment) and to obtain information water reservoirs, both in freshwater environments and confirming or excluding the existence of a link between in the seas. The widespread, all-season occurrence both materials. It appears that diatoms may also serve and high abundance of diatoms, knowledge of the to determine a postmortem submersion interval (PMSI), environmental requirements of individual species i.e. the time that elapsed between body immersion and as well as high resistance of frustules to adverse discovery of the corpse [47–48]. This method is based factors, make diatoms a useful tool in clarifying the on the fact that diatoms are the first algal colonizers circumstances of the offences. In forensic practice, of a corpse in the aquatic environment and constitute diatoms may be used to determine the cause, time, a dominant group at the initial stages of succession. and place of death [7, 41] and to identify the alleged Literature on forensic diatomology mainly includes perpetrator [42–43]. Most of the documented uses of the case reports documenting the usability of diatoms diatom analysis in forensic practice concerned the for forensics as well as original, experimental papers victims of drowning, especially in the cases where showing how diatoms may be associated with the autopsy results were inconclusive as to the cause of circumstances of the event. The intensive studies are death [7, 44–45]. The so-called diatom test allows also carried out on diatom detection and extraction for determination whether the cause of death was techniques [43, 49–51]. Recently it has been proposed drowning, or the body was placed in water after to improve the classical diatom test by applying death. In the case of drowning, death results from more efficient extraction methods, based on the asphyxiation caused by fluid ingress, mainly water, into capacity of DNA molecules to bind silicon present in the respiratory tract. The fundamental principle behind frustules [52–53]. The use of diatoms for evidentiary the use of diatoms in confirming drowning as the cause purposes of law enforcement requires parallel research of death is based on the assumption that diatoms are on the ecology of this group of algae. Qualitative and present at the place of the alleged drowning. Ingestion quantitative composition of diatoms varies in time and of water into the respiratory tract causes diatoms space and additionally, it is a result of human activity. to pass through pulmonary alveoli to the vascular With this in view, the studies on the structure of diatom system, and eventually to the internal organs. It has assemblages should take into account the changes been shown that diatoms accumulate mostly in the in quality of water reservoirs, due to pollution and bone marrow and in the brain, less so in kidneys, eutrophication. stomach and lungs. The characteristic feature is low abundance of diatoms in the myocardium [46]. Finding diatoms within the respiratory tract and their lack in Use of plant anatomy in forensics the internal organs indicates that the death occurred for reasons other than drowning, and the corpse was Knowledge of plant anatomy and the manner of growth placed in the aquatic environment only after death. can be also used for evidentiary purposes. A classic In such cases, water penetrates the respiratory tract example of such an application in investigative practice

ISSUES OF FORENSIC SCIENCE 289(3) 2015 75 FORENSIC PRACTICE are the methods of dendrochronology, a discipline that the circumstances, the disturbance of vegetation deals with determining the age of trees, based on the cover can be manifested in a variety of ways [56]. number of growth rings that correspond to the annual The burial site can host more lush (visibly taller and cycles of stem girth increments. The first example of greener) vegetation than elsewhere. This is due to an using plant material-based evidence for investigative increased concentration of mineral compounds, which purposes in modern times, concerned determining the are released from decomposing body and stimulate origin of a ladder used in kidnapping of a child. The plant growth. The stimulation of vegetation growth analysis involved determining the species and age of can also result from better aeration and loosening of a tree used for producing the ladder [2]. soil, caused by digging, which in turn positively affects In recent years, attention has been drawn to the development of the root system. On the other various species of bryophytes as potentially helpful in hand, digging activity can have an adverse influence determining the time of death [9, 54]. Knowledge of on vegetation, as result of complete destruction of the manner and rate of growth of these plants enables the root system. In such a case, the burial site will to determine the Postmortem Interval (PMI), i.e., the stand out by less abundant vegetation cover or the time that has elapsed between death and finding the lack thereof. It has been noticed that the depth of the corpse. The characteristic feature of the analyzed grave is a key determinant of vegetation response [56]. species of mosses was their monopodial growth For example, a domestic pig carcass – used as pattern (one main growing stem), which allowed the a human body substitute in this type of studies – determination of the number of annual growth units. when buried below the root zone, did not cause any Subsequently, by taking into account the age of changes in species composition of early successional mosses and their absence from the soft tissues, it was plants such as grasses and herbaceous plants [56]. possible to determine the time of skeletonization of Digging activity may mechanically destroy the plants the corpse. In the case described by Cardoso et al. in the vicinity of the grave. Changes in species [9], the knowledge of algal colonization of corpses and composition of vegetation cover, frequently observed accretion thereof by the shrub roots was exploited to at the burial site [56, 57], include the disappearance estimate the time of death. of pre-existing species and the appearance of new Bryophytes have been also the subject of species, uncommon in a particular habitat, i.e. the so methodological studies. The usability of bryophyte called grave indicators or post-burial indicators. In the DNA for genetic analyses was tested on material study conducted in an area covered with herbaceous stored for 18 months in paper bags, at the temperature vegetation in southern Ontario, Canada, Watson and range of + 25˚C to -28˚C. In addition, it was examined Forbes [56] observed different species compositions whether bryophytes adhere to footwear and how they for the burial and undisturbed sites. Two species: preserve on footwear after several hours of walking. Hairy Finger-grass (Digitaria sanguinalis (L.) Scop.) The conducted experiments showed that bryophyte and Witchgrass (Panicum capillare L.) appeared at DNA is amenable to analysis, even when isolated from the burial site as post-burial indicators. Subsequently, specimens stored for several months under adverse the study by Caccianiga et al. [57], carried out in an temperature conditions, and that bryophytes adhere to open area with herbaceous vegetation in southern footwear and remain there, even after several hours of Italy, reported that the burial sites were characterized walking on dry and hard surface [55]. by a higher number of ruderal species, i.e. those typical of highly anthropogenically transformed areas. Conversely, the number of stress-tolerant species, Application of knowledge of plant ecology in such as Spring sedge (Carex caryophyllea Latourr.), forensics Wall Germander (Teucrium chamaedrys L.) or Crested Hair-grass (Koeleria pyramidata (Lam.) P. Beauv.), Ecological studies carried out within the forensic has decreased. These changes were observed in the botany focus mainly on studying the reaction of plant case of both, full and empty (control) graves. This communities to the factors that disturb the ecosystem, allowed for a conclusion that the essential cause of the such as a buried corpse. In investigative practice, such disturbance of plant communities was of mechanical a knowledge can be employed to locate concealed nature, i.e. digging process, and not the presence of corpses or clandestine graves. The studies conducted a decomposing corpse. The above results explicitly so far have shown that the burial of a corpse causes indicated the potential of forensic botany for the local structural disturbances in vegetation cover that search for clandestine graves. The studies on plant can last for a long time [56–57]. From the investigator’s ecology also include the observations on the impact of perspective, it is relevant that the said disturbances carcasses decomposing on the ground surface on the are easily distinguishable visually from vegetation surrounding environment. The researchers focus on patterns of the surrounding sites, undisturbed by soil chemical properties underneath a decomposing digging. Hence, the altered site can provide guidance corpse and the changes in vegetation species in the search for a concealed body. Depending on composition. The observations address spatial and

76 ISSUES OF FORENSIC SCIENCE 289(3) 2015 FORENSIC PRACTICE temporal aspect of the studied phenomenon. The size low mutation rate (deletions, insertions). The inter- of the area affected by the disturbance as well as the species molecular analyses of plant DNA are most time needed for recovery of vegetation cover to the frequently based on plastid DNA, due to its high original state are examined. Results have so far shown level of conservation and low intra-species variability. that changes in vegetation cover at the site of carcass Nonetheless, the identification of a plant DNA decomposition on the ground surface are prominently sequence that would meet all the conditions set out demonstrated in simple structured ecosystems, i.e. low, for a barcode appeared more problematic than in dense vegetation, such as prairie [58] or tundra [59], the case of animal DNA. A range of nuclear (ITS1, whereas they are relatively difficult to observe in the ITS2) and plastid (trnH-psbA, trnL-trnF, rbcL, atpB, environments with more complex structure, such as ndhF, matK) DNA sequences have been considered temperate forests [60]. The factors affecting the extent [67, 68]. In 2009, Ferri et al. [64] proposed the use of disturbance have proved to be the carcass size of two regions of chloroplast DNA: trnH-psbA and and the time of year when decomposition took place. trnL-trnF. In the subsequent years, the same authors The time required for regeneration of vegetation has focused additionally on matK+rbcL region [67]. proved to be different for various types of ecosystems. Eventually, the following combination of plastid DNA On prairie meadows, the disturbance in vegetation sequences: rbcL+trnH-psbA, has been recommended structure was observed up to five years after placing for the purposes of forensic analyses [67]. Research the carcass, while in the case of tundra it persisted for on barcode DNA is actively carried out by the Plant longer periods of time, due to slower decomposition Working Group, acting within the framework of the rate under low temperature conditions. Consortium for the Barcode of Life (CBOL). Forensic botany involves an extensive research on DNA polymorphism. The genome fragments displaying Application of molecular biology in forensic high intra-species variability can be used as molecular botany markers, performing the function of the so called “genetic fingerprints”. In practice, this allows for linking Molecular biology tools can be successfully applied the plant fragments with their corresponding parental to forensic botany, allowing for reliable identification plants. The studies on plant DNA polymorphism involved of the plant material, based on DNA analysis. The the use of various molecular analysis techniques. first report on the use of plant DNA to explain the Among the earliest, were the methods based on DNA circumstances of the offence regarded establishing hybridization, such as the restriction fragment length a link between the alleged perpetrator and the scene. polymorphism [69]. Currently, the most widely used in The analysis has shown that DNA from the seeds of research on plant DNA polymorphism are the PCR- the tree under which the female victim’s corpse has based methods, geared towards the amplification been discovered, matched DNA of tree seeds found in of one or more genome fragments. According to the truck belonging to the suspect [61]. The molecular Nybom et al. [69], in the years 2006–2009, plant DNA methods of plant identification are particularly variability was predominantly identified by an analysis useful wherever the classical methods, based on of microsatellite (or simple sequence repeats – SSR) morphological traits, yield ambiguous results or when polymorphism. The SSR markers found their use in dealing with residual quantities, or damaged plant 36% of the studies (by publication records) of the time. material. Owing to molecular biology methods, plants Choosing this type of markers results from the fact that can be quickly assigned to a particular taxonomic they are useful when working with damaged DNA and group (species, genus, family), various plant fragments the analysis itself is not time-consuming. For example (e.g. leaves, seeds) can be linked to parental plants, the analysis carried out for evidentiary purposes and the geographic origin of plants can be determined. revealed a high polymorphism of microsatellite nuclear In investigative practice, molecular analysis of botanical DNA sequences in the species belonging to the genus material can contribute to establishing links between Quercus [62] as well as in the species Aquillaria crassna the scene, the perpetrator and the victim [62] as well Pierre ex Lecomte [70]. Other techniques regarded by as to solving cases involving smuggling and illicit trade Nybom et al. [69] as frequently used, include random of narcotic drugs and psychotropic substances [63]. amplification of polymorphic DNA (27% of publication In order to facilitate fast and reliable species records), analysis of inter-simple sequence repeat identification, the so called DNA barcodes are (ISSR) polymorphism (13% of publication records) and developed [64-68]. The barcodes correspond to DNA analysis of amplified fragment length polymorphism fragments displaying relatively low intra-species and (AFLP) (11% of publication records). much higher inter-species variability. Other features Since 2005, the so called „new generation of the barcode fragments include the presence of the sequencing (NGS)” has been also used in research on flanking conservative domains, which facilitate PCR polymorphism. The acronym NGS refers to different primer design, moderate length (approx. 700 bp), techniques of high throughput DNA sequencing, which large number of copies within the genome, relatively allow even for whole-genome analysis. An example

ISSUES OF FORENSIC SCIENCE 289(3) 2015 77 FORENSIC PRACTICE of NGS can be a “genotyping-by-sequencing” (GBS) of their potential existence at the scene and have technique or a large-scale RAD tag (restriction knowledge of the methods of their recovery. Practice site associated DNA tag) sequencing. Another has shown that botanical experts have been only popular method is the analysis of single nucleotide rarely and only in a few countries appointed to inspect polymorphism (SNP). SNP markers have been identified the scene. Botanical traces can be also revealed for the majority of agricultural plant species [69]. and recovered by forensic technicians. However, Also gaining popularity is DNA metabarcoding, such persons should undergo appropriate botanical allowing for simultaneous identification of various training covering basic topics related to plant anatomy, organisms (bacteria, protozoa, plants, fungi, animals) development and ecology, types of relationships present in the samples (e.g. of soil) [71]. between botanical material and the scene, persons or Of particular interest to forensic botanists, as objects, and finally, the methods of recovery. A skillful potential molecular biology identification tools are recovery of botanical material is a precondition for its inter alia the common, and therefore often available further processing and additionally, for a long-term as evidence plants such as Knot-grass (Polygonum storage and potential use after many years. Rapid aviculare L.) [72] or grasses [73] which, owing to their development of science entails the development of specific narcotic or functional properties, may be novel tools and methods for analysis of biological subject to illicit trade. In such cases, it is necessary to traces of various origins for evidentiary purposes. This distinguish between the individual plants coming from means that biological traces that are not amenable a legal source and those that have been smuggled, to analysis, and therefore cannot be linked to the which entails determining their geographical area of incident at the current state of knowledge, may prove origin. Individual plants of the same species growing to be more useful as evidence in the future. One of in different geographical areas may have different the basic errors committed when collecting botanical genomes. The differences can be identified by applying traces, is allowing their contamination. In the case of polymorphism analysis. botanical evidence that could be otherwise used to So far, special consideration in respect to molecular discern connections between persons and places, identification has been given to true hemp (Cannabis the contamination practically prevents any further sativa L.) [74–77]. Other studies focused on the varieties of inference. cultivated plants which have significant economic value The next stage of forensic botanist’s work is or those registered as Protected Designation of Origin to classify the identified material into the lowest (PDO), e.g. strawberries [78], grapes [79], olives [80] possible taxonomic level, in most cases the species. or rice [81]. The scope of interest of forensic botany Alternatively, the analysis may terminate at other also includes the plants that are limited to the specific ranks, such as family or genus. This shortcoming areas of the globe and can be attractive to smugglers, can stem from different reasons. First, the secured due to their use in traditional medicine, e.g. Aquilaria plant material can be difficult to identify, based crassna Pierre ex Lecomte [70]. solely on morphological traits. This is the case with the fragments of plants, e.g. leaves or stems, the identification of which may prove difficult or impossible Application of forensic botany methods in forensic due to the lack of relevant structures. Secondly, the practice difficulties with the identification of botanical traces may result from their improper recovery, which leads to Application of forensic botany to investigative their destruction, rendering them unsuitable for further proceedings comprises a few stages: 1) identification of analyses. Wherever the classical, taxonomy-based botanical trace evidence, 2) recovery of trace evidence, methods of identification fail, the molecular biology 3) identification of collected material, 4) establishing techniques can prove helpful. links between forensic trace and the circumstances of the incident. The first two stages are often the most problematic as they rely fully on the experience of Conclusions a person who identifies and collects botanical traces, e.g. a court appointed expert in the field of botany or The knowledge about plants can be useful in a forensic technician. For successful investigation, it establishing links between the scene of the incident, is optimal to appoint an expert in the field of botany, the victim and the alleged perpetrator. The methods who, in addition to possessing academic knowledge, of forensic botany can be used to identify the scene has the ability to recognize botanical traces that are and the perpetrator, determine the cause and time of relevant to the investigation. The latter can appear death, distinguish between the place of death and that burdensome, as botanical material can be microscopic of discovering the corpse, clarify the circumstances in size, and therefore invisible to the naked eye, the of war crimes as well as plant and animal smuggling. best example of which are pollen grains. In order to Nonetheless, as has been repeatedly emphasized be able to identify such traces, one should be aware in specialist literature, botanical evidence is still

78 ISSUES OF FORENSIC SCIENCE 289(3) 2015 FORENSIC PRACTICE only seldom used in the investigations. Although Figures: numerous review and methodological papers as well as case reports of practical applications of Fig. 1: Łukasz Grewling forensic botany have appeared recently, there Fig. 2: Mikołaj Kokociński are still very few original works that could be the basis for admittance of plants to the investigations. In particular, there is a lack of knowledge on the The authors of this article would like to thank the preservation of the plant material under different anonymous reviewer, dr Eliza Głowska (Department conditions and how this material could be linked to of Animal Morphology, UAM) and dr hab. Szymon the circumstances of the case. The above information Matuszewski (Laboratory of Criminalistics, UAM) is essential for using the plant material as evidence. for valuable comments regarding the manuscript. Palynology and plant anatomy, with a particular emphasis on dendrochronology, are the disciplines Bibliography with the longest history of assisting in explaining the circumstances of the offences. Among all disciplines 1. Bryant V.M., Mildenhall D.C.: Forensic of forensic botany, palynology is the most developed palynology: A new way to catch crooks; In: Bryant one, as attested by numerous review papers on this V.M., Wrenn J.W. (eds.), New developments subject. A large potential with respect to confirming in palynomorph sampling, extraction, and drowning as the cause of death is offered by forensic analysis. American Association of Stratigraphic diatomology methods. Both in the case of palynology Palynologists Fundation, Contributions Series and diatomology, future research should focus on Number, 1998, 33: 145. the development of the databases and reference 2. Coyle H.M., Ladd C., Palmbach T., Lee H.C.: collections that will be used for investigation purposes. The green revolution: botanical contributions The creation of this type of resources should be to forensics and drug enforcement, Croatian preceded by studies on the subject of ecology and Medical Journal, 2001, 42: 340. plant and algae distribution. Rapid development 3. Coyle H.M., Lee C.L., Lin W.Y., Lee H.C., of molecular biology methods in recent years Palmbach T.M.: Forensic botany: using plant brought the development of novel tools for species evidence to aid in forensic death investigation, identification that proved useful to forensic science Croatian Medical Journal, 2005, 46: 606. in that they allowed for fast and precise taxonomic 4. Mildenhall D.C., Wiltshire P.E.J., Bryant V.M.: identification or even individual identification of plant Forensic palynology: Why do it and how it material. works, Forensic Science International, 2006, The main obstacle to the use of forensic botany 163: 163. methods for evidentiary purposes on a larger scale, 5. Wiltshire P.E.J.: Consideration of some appears to be the lack of awareness of the existence of taphonomic variables of relevance to forensic botanical traces among the investigators. The proper palynological investigation in the United awareness is indispensable for efficient recovery of Kingdom, Forensic Science International, 2006, botanical traces at the scene of the incident. Also 163: 173. problematic is an insufficient number of forensic 6. Walsh K.A.J., Horrocks M.: Palynology: its botany experts, a resultant of reluctance to perform position in the field of forensic science, Journal expertises, financial issues as well as poor cooperation of Forensic Sciences, 2008, 53: 1053. with investigative authorities. It is important to bear 7. Pollanen M.S.: Diatoms and homicide, Forensic in mind that the results obtained from the analysis Science International, 1998, 91: 29. of botanical materials should be interpreted in the 8. Mildenhall D.C.: Hypericum pollen determines context of the entire investigation process, i.e. with the presence of burglars at the scene of a crime: other available evidence taken into consideration An example of forensic palynology, Forensic as well. With regard to the investigation, it would be Science International, 2006, 163: 231. optimal to simultaneously apply analysis techniques 9. Cardoso H.F.V., Santos A., Dias R., Garcia C., of various evidence. Indeed, as practice shows, the Pinto M., Sérgio C., Magalhães T.: Establishing best results in examining the circumstances of the a minimum postmortem interval of human offenses are achieved by applying interdisciplinary remains in an advanced state of skeletonization approach. using the growth rate of bryophytes and plant roots, International Journal of Legal Medicine, Translation Rafał Wierzchosławski 2010, 124: 451. 10. Mildenhall D.C.: An unusual appearance of a common pollen type indicates the scene of the crime, Forensic Science International, 2006, 163: 236.

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