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Overview

Forensic palynology is an underutilized, but important tool for obtaining trace evidence from suspects, items thought to be associated with a crime scene, or for determining the geolocation of a sample. First used more than 50 years ago in Austria, it is now often used in the United Kingdom, New Zealand, and more recently, in other countries of Europe and Asia, but is still underused in the United States. Forensic palynology has gained importance for its ability to provide information about pollen and spores trapped in clothing or other items of interest needed to help resolve criminal and civil cases, including instances of homicide, terrorism, genocide, bombings, forgery, theft, rape, arson, counterfeiting, manufacturing and distribution of illegal drugs, assault, cases of hit and run, poaching, and identity theft. The foundation for using pollen in forensic applications comes from the discipline of pollen analysis, which began a century ago as a way to search for clues about past environmental changes. A few examples demonstrating the utility of using pollen and spores in forensic applications during the last half century are discussed. Finally, some of the recent analytical techniques that are being used to try to increase the precision of pollen identifications related to a variety of criminal and civil cases are examined.

Introduction

Palynology (the study of pollen and spores) has historically been widely underutilized in forensic science. Traditionally, forensic palynology focuses on legal evidence obtained from the study of pollen and spores that are associated with a crime scene or other aspects related to situations involving the law. As a discipline, the first recorded use was a little more than one-half century ago, but even today the use of this technique is relatively unknown or underutilized in many regions of the world. Even in those areas where forensic palynology is routinely used as a forensic tool, such as the United Kingdom and New Zealand, its utility for forensic science is often questioned.

One of the earliest recorded and successful cases involving the use of forensic palynology occurred in Austria in 1959, where pollen was used to link a suspect with a murder and to the crime scene where a shallow grave contained the murdered victim’s body (Erdtman 1969). A decade later, pollen from the suspected holy relic called the Shroud of Turin was recovered and then analyzed by Max Frei in the 1970s. Using pollen trapped on tapes that had been lightly pressed against the Shroud, Frei claimed the relic could be linked to its purported origin and thus suggested authenticity (Frei 1982). Over the next decade, there were a few additional attempts to use pollen as trace evidence in both civil and criminal cases, but the next use that gained widespread attention occurred in New Zealand (Mildenhall 1990). Dallas Mildenhall, a palynologist trained in geology was asked to help solve a case involving the suspected poaching of deer to obtain the velvet from their newly formed antlers. The recovery of pollen from the seized deer velvet did prove the deer had been killed illegally and thus led to a conviction of the poacher. Finally, forensic palynology in the United Kingdom began when Patricia Wiltshire (1993) first used pollen in helping to solve criminal cases in the United Kingdom. Since the early 1990s, the United Kingdom has led the world in the routine application and use of forensic palynology as part of many criminal investigations.

In the United States, there appeared to be little interest in following the examples set forth in the United Kingdom and New Zealand in using forensic palynology until after the destruction of the Twin Towers of the World Trade Center in New York City on September 11, 2001. Terrorists, using commercial airplanes flown into the two skyscrapers, claimed the lives of over 3,500 innocent victims and created an immediate need to learn more about the terrorists who committed that crime. In a search of information, authorities focused on using every known forensic technique, including the use of pollen as a forensic tool. Although the recovered pollen evidence associated with the terrorists did not produce any immediate useful clues, the addition and use of recovered pollen as a new forensic trace evidence tool was finally accepted as being potentially important for future uses in the United States.

Foundations Of Palynology

To use forensic palynology effectively and to explain its potentials to others, one needs to understand factors that control the type and amounts of pollen and spore production and how those pollen and spores are dispersed. Among the cryptograms, spores are dispersed into either air or water and use those mediums as a transportation vector. Among the gymnosperms, all species produce wind-dispersed pollen. In angiosperms, the vast majority of species now rely on insects, birds, or mammals to vector the pollen from one plant to another. A limited number of angiosperms still rely on wind dispersion for its pollen.

All of the spore and pollen species that rely on wind dispersion generally produce large numbers of spores or pollen because of the inefficiency of wind dispersion. Conifers and some of the angiosperm species are capable of producing hundreds of millions of pollen grains per plant, and some spore-producing species, such as Ganoderma (Kadowaki et al. 2010), reportedly produce and disperse several billion spores during one annual dispersal period of about 6 months. The large number of produced pollen and spores cast into the air, and the inefficiency of that technique provides large numbers of essential clues that are captured as part of the pollen rain at each location. That unique mixture of airborne pollen and a few insect-pollinated pollen and spores at each location is what forensic palynologists primarily rely upon for clues related to a specific crime scene or to assign geolocation to some object of unknown origin (Fig 1). Some pollen grains are tiny, are lightweight, and are aerodynamically designed to travel easily in air currents, while other windpollinated types, which are larger and heavier, do not usually get dispersed far from their source. Some pollen grains have been recovered more than 1,000 km from their nearest known source, but as a general rule, the vast majority of airborne pollen and spores do not travel too far from their dispersal source, which in many cases means that much of the pollen falls to the surface within a radius of about 100 m (Tauber 1965) (Fig. 1).

Although the large number (about 75 %) of angiosperms that rely on some type of insect or animal vector to transport their pollen grains usually produces miniscule amounts of pollen, which often range in number from as few as 10 up to about 1,000 pollen grains per anther, their pollen is often a key component in any forensic sample. By design, the non-wind-borne plant species produce pollen grains that are often highly ornamented, are thick walled, have a heavy mass, and are covered with sticky lipids and waxes. Those are all factors that make for the ideal transport of pollen by some animal or insect vector. The ornate surface ornamentation of these pollen types is designed to hold sticky lipids that will keep the pollen grains firmly stuck to the hairs or bodies of pollinators until those pollinators land on another flower. At that point, the sticky surfaces of the attached pollen grains adhere to the surface of the stigma of a new flower, and if a pollen grain falls in the right location, fertilization will occur. The thick pollen walls and heavy mass of these insect-and animal-pollinated types ensure that the fragile contents of the pollen grains remain protected from abrasion and sudden changes in humidity during the flight time from one flower to another. From the above reasons, only rarely do pollen grains from these types of plants become part of the normal pollen rain of a location. Nevertheless, it is that characteristic that becomes especially useful in many types of forensic situations.

Forensic Palynology, Fig. 1 Pollen grains from (a) Alnus crispa (Aiton) Pursh., (b) Brassica nigra (L.) W.D.J. Koch., (c) Cucurbita pepo L., (d) Eucalyptus calophylla (Lindl.), (e) Helianthus maximiliani Schrad., (f) Taraxacum officinale F.H. Wigg., (g) Spermacoce tenuior L., and (h) Nuphar luteum (L.) Sm

When people or objects come in contact with insect-pollinated plants, especially ones that might be in flower, some of the sticky pollen can often be transferred from the plant’s leaves or flowers to the person or object. Because most of these types of pollen become associated with people or other objects “only through direct contact with the plants,” that type of evidence, when linked to similar plants at a crime scene, can infer evidence of direct association. The highly unlikely argument that such pollen grains were deposited on a person or object by accident from airborne sources as part of the normal pollen rain makes these types of pollen grains an ideal evidence for direct association. Another advantage of many of these insect- and animal-pollinated types is that their sticky surface, durable pollen walls, and ornate surface morphology enable them to adhere firmly to objects they touch. Even after persistent attempts to remove all such pollen traces from objects associated with a crime scene, some of these pollen types often remain and can help to place a suspect at the scene of the crime (Milne 2005).

Utility Of Forensic Palynology

The analysis of pollen and spores (collectively called palynology) is recognized as an effective forensic tool for a number of reasons. First, many types of pollen-producing plants (angiosperms and gymnosperms) and spore-producing cryptograms (algae, fungi, ferns, mosses, liverworts, etc.) disperse vast quantities of pollen or spores as part of their reproductive process. For most of these, they rely upon wind currents to carry these single-celled pollen or spores from the dispersal source to another location where they can carry out part of the reproductive cycle. Because this process is haphazard, the vast majority of these dispersed cells eventually fall to the ground in a thin coating called the “pollen rain.” In some regions, the amount of dispersed pollen and spores is so great that once deposited, they can coat exposed surfaces with a thin yellow layer or form a yellow scum on water surfaces (Faegri and Iversen 1989). Although not a precise measurement of the surrounding vegetation, the pollen rain at each location provides a snapshot of that area’s vegetation. Even though there is not a one-to-one correlation between the percentages of each plant type in an environment with the precise percentages of each pollen type in the pollen rain, the resulting “pollen print” for each area becomes an effective way to identify each particular region of the world. The precision required to recognize a specific region, based on the pollen print from some forensic sample, often depends on the experience and knowledge of the pollen analyst and that person’s awareness of how the presence and various percentages of each pollen type in a sample can often relate to the presence of certain types and quantities of plants.

A second reason why pollen and spores can become effective forensic clues pertains to their tiny, invisible size. All are microscopic in size and the sizes can range from only 1 or 2 m (1,000 mm is equal to 1 mm) for some spores and up to over 200 mm in size for large pollen grains. Because of their tiny size, many are airborne and can then be deposited or become trapped on almost any type of surface. This means that at any geographical location, pollen or spores from local and even some regional vegetation, or in specific cases, the pollen and spores associated with a crime scene can become effective markers for those locations and can often link a suspect or some object with a precise geographical region or a specific crime scene.

Third, there are over 330,000 different pollen-producing plants and thousands more when adding the cryptogram species that produce spores. However, differences in the pollen and spores of closely related species, or in some instances related genera, may appear so similar that precise identification using only the resolution capacity of light microscopy (LM) is tenuous and sometimes unreliable. In those cases, when needed, greater precision of pollen and spore identification can often be achieved through detailed studies using the higher resolution capabilities of a scanning electron microscope (SEM) and/or the resolutions available with a transmission electron microscope (TEM). Nevertheless, obtaining these greater degrees of precision can be costly and time-consuming because they must rely upon comprehensive SEM and TEM pollen and spore reference micrographs for making direct comparisons. Because of the time and cost of using SEM and TEM, other analytical techniques are now being considered to speed the identification of both pollen and spores, which will be discussed later in this research paper.

A fourth reason why most pollen and spores are useful as forensic tools is that the majority of them are highly resistant to destruction or decay. This is why the oldest geological evidence of organic material, related to plants, comes from what is believed to be very ancient spores (Faegri and Iversen 1989). This ability to remain preserved for hundreds or even millions of years means that pollen and spore evidence, if collected and stored properly, can still be used effectively many years after some event has occurred. In one such case, pollen and spore evidence collected more than 30 years ago from the clothing of a murdered victim was examined and then used to help identify where that person may have lived prior to being killed.

It should be noted that even though pollen and spores are resistant to decay, in certain types of oxidizing environments, they can degrade very rapidly and disappear completely in only a few months or years. In addition, studies reveal that in certain types of depositional environments, such as alkaline soils, certain fragile types of both pollen and spores can degrade almost immediately, while other types remain in pristine or recognizable conditions for many decades or hundreds of years (Traverse 2007).

In some regions, and in some countries, law enforcement personnel continue to expand the types of collected evidence they hope can reveal pertinent information based on the trapped pollen and spores. The list of items that can trap pollen and spores, and thus are being collected and examined, has become almost limitless (Mildenhall et al. 2006). During the past decade, a partial list of items that forensic palynologists have been asked to examine for pollen and spore evidence includes the vacuum contents trapped on the pages of books or on various documents, material trapped on the surface of weapons or inside homemade bombs, pollen and spores collected in air conditioning filters or in air filters from different types of vehicles, pollen and spores trapped on or in all sorts of transportation ranging from boats and airplanes to trucks and bicycles, briefcases, illegal and counterfeit drugs, laptops and cell phones, all sorts of clothing and the contents in suitcases, pollen and spores from victims that were buried, and even the pollen trapped on pieces of wire and shoelaces. Pollen and spore evidence examined by other forensic scientists has also been used to resolve situations involving forged documents, fake antiques, authentication of paintings by master artists, removal of artifacts from historic or archaeological sites, illegal poaching of animals or fish, and cases involving the illegal pollution of the environment (Mildenhall et al. 2006).

Although forensic palynology has been accepted as a proven tool by some, it does need to be pointed out that the reliability of using pollen and spore data as forensic trace evidence is often called into question by courts and law enforcement officials, particularly in the United

States. Because forensic palynologists often present their results as relative percentages of various pollen types within the total pollen and spore composition of a sample, some question the uniqueness of such data, especially when a forensic palynologist attributed a sample to a single location or to an actual crime scene. Questions often arise about the precision of the overall pollen data and the precision of identification of individual pollen taxa.

Forensic pollen specialists generally present their conclusions in terms of probability of association and often cite the importance provided by the combination of many pollen and spore taxa in a single sample. Often when challenged to produce “statistical data” that could be used to support or negate the importance or relevance of a pollen sample, or the potential link of a pollen sample to either a suspect or to a crime scene, the palynologist cannot effectively do so. This inability to provide statistical confirmation results from an almost endless list of variables that influence the deposition of pollen types and their ratios within a single sample deposited on some surface or person. Even though some scientists have championed the limited use of statistical techniques, most types of statistical analyses cannot be adequately adapted in ways that will test the multiple unknowns or conclusions based on forensic pollen assemblages (Mildenhall et al. 2006).

One question that is frequently raised during investigations or in court focuses on whether or not two different locations will signal identical pollen spectra. The chance of that occurring is virtually impossible and thus far, an identical match in the pollen spectra at two different locations has yet to be proven. The unlikeliness that this possibility would occur depends to a great extent on how precise the attempt might be. Experience has shown. Minimal pollen counts of only a few 100 pollen grains from comparative samples collected at two separate locations might suggest that both locations appear nearly identical in terms of their pollen signatures. However, as the pollen signature of each sample is refined by counting larger numbers of pollen grains and spores from each sample, the potential of both samples providing nearly identical pollen spectra disappears. The list of variables at any given location is numerous, and each one contributes in some way to the formation of a pollen spectrum for that location, which in turn produces a pollen spectrum unlike those from similar locations.

Instead, the focus should be on the knowledge, skill, and research experience of the pollen analyst and on the discussion of which variables may have been a factor in producing the pollen assemblage in question. The reputation, previous work, and the acceptance of the person’s skills by courts and by other forensic palynologists should serve as a guide to support the conclusions of competent scientists and also prevent evidence and testimony being accepted from less-skilled or under-qualified individuals.

Types Of Forensic Palynologists

There are essentially two types of forensic palynologists, based upon the types of information or investigations that are being usually undertaken. Although some forensic palynologists are called upon to work in both areas, often each group tends to specialize and focus mostly on one type of needed information. The primary type of forensic pollen specialist becomes directly involved in the investigation of crimes as they pertain to situations involving victims, suspects, actual crime scenes, and items suspected of being associated with one of the previous situations. These individuals generally work directly with local, state, and national law enforcement agents, they usually work within a law enforcement agency or established forensic lab, and they are generally included as part of the forensic teams that are the first to reach crime scenes. This type of forensic palynologist relies on his/her botanical knowledge, previous experience, and an ability to evaluate a crime scene and/or other places of forensic interest. He/she should determine what types of comparative pollenreference samples need to be collected to properly reflect the pollen print of the crime scene and can also be used later for comparisons between the crime scene and evidence recovered from potential suspects and other items. These forensic palynologists need to have training in palynology, plant ecology, and plant taxonomy and should also understand the relationships of those botanical areas to other crime scene features such as, soils, past vegetational changes, and the topographic features of the landscape in and around the general region.

The other type of forensic palynologist worked mostly with questions related to geolocation. In other words, this type of individual is often called upon to examine items related to crimes, items of unknown origin, or items associated with threats to national security or terrorist activities. The goal in each of those types of studies is to determine the geographical location where such items originated or were used. For scientists working in this second area of forensic palynology, their background and training should include all of the areas and aspects needed to conduct the first type of forensic work, but in addition, they also need other related types of expertise. These individuals should have a comprehensive understanding of pollen and spore dispersal patterns, pollen productivity as it pertains to different taxa, knowledge about how topography and climatic conditions can affect the potential pollen rain in a region, pollen sinking speeds and potentials of key types of both pollen and spores being recycled, and access to an extensive pollen database covering many potential pollen and spore types found in different geographical regions from the Arctic to the equatorial tropics in all of the major continents.

One of the problems that also affect potential geographical interpretations, based on recovered pollen and spore data, is the never-ending process of expanded and new plant distributional ranges related to the introduction of agricultural or ornamental plant species into new regions of the world, the natural catastrophes such as volcanic eruptions and fires, or the widespread changes in landscapes created by human modifications. Before the Age of Discovery and the worldwide expansion by European nations, if pollen from key types of plants, such as maize (Zea mays) and chili peppers (Capsicum) (native to the New World), Melaleuca (native to Australia and some areas of Southeast Asia), and tamarind (Tamarindus indica) (native to Africa), were found in a forensic sample, it could be linked quickly to some specific geographical location. Today, however, those plants, and hundreds of other ones, have become almost pandemic and their pollen is now found in many regions far beyond where those plants were once restricted. Therefore, a major problem confronting these types of forensic analysts is an understanding of the potentials in each world region for the introduction of foreign ornamentals that are not native to those areas.

Collecting Forensic Samples

For those forensic palynologists, who work closely with law enforcement personnel, an early arrival at a crime scene is often essential. Pollen and spores at a crime scene are easily disturbed, removed, or contaminated with unassociated pollen and spores that arrive on the clothing of law enforcement personnel and forensic teams investigating the crime scene. Ideally, the forensic palynologist should be given early access to a crime scene so that careful studies of the plants and pollen sources can be collected, noted, and photographed. Normally, the forensic palynologist will need to collect undisturbed comparative samples of surface soils of various locations in and around the actual crime scene, which, when analyzed, form the basis for comparisons with future pollen evidence thought to be related with the crime scene. Unfortunately, potential comparative samples collected improperly or gathered by untrained personnel can often endanger the reliability of the pollen evidence or render samples useless as evidence. Such problems can also provide a basis for the dismissal of evidence in court.

One should collect many reference or comparative samples from a crime scene. Sometimes control samples need to be collected from both sides of a path or walkway or from a series of closely spaced areas in and around a crime scene.

Without this type of precise knowledge, it becomes difficult to argue either for or against the confirmed association of a pollen assemblage found on a suspect’s shoes, or car, or clothing, with the pollen types found at the actual crime scene. Therefore, even though a large number of comparative samples might be collected from a crime scene, not all of them may need to be examined. However, because the pollen spectrum in each comparative sample may vary slightly in reference to the pollen types and percentages of each taxon, the combined pollen spectra from a variety of comparative samples provide a potential range of pollen variation that can be expected in and around the actual crime scene. Once a crime scene has been trampled upon by law enforcement personnel, teams of forensic specialists, and eventually by others who might be curious about the events that took place, the value of collected pollen evidence is reduced and those samples become subject to suspect from post crime pollen contamination or removal.

An example that illustrates the importance of collecting adequate numbers of control samples focuses on samples collected from the ground where a sexual attack occurred. A woman was kidnapped at knifepoint while jogging in a New Zealand city park. She was then sexually assaulted and killed in a nearby wooded area. A man who lived near the park had been seen near the jogging trail in the park just before the woman disappeared. Later, when questioned by the police, the man admitted being in the park, but denied he had seen the victim and said he had never been in the nearby wooded area. A search warrant was granted and items of clothing were gathered from the suspect’s apartment. Of particular interest were a dark woolen sweater and a pair of soiled pants, which might have been worn during the kidnapping and assault. Pollen studies of the sweater and pants revealed that each garment contained a rich combination of pollen and spores from pines and ferns. Comparison samples collected from the crime scene revealed a dominance of both pine pollen and fern spores. Likewise, the jogging shorts worn by the murdered woman also contained pine pollen and fern spores.

The pollen spectra from all three groups of samples (suspect’s clothing, comparison samples, jogging shorts from the murdered woman) were consistent and revealed that the only way so much pine pollen and fern spores could have become trapped in the clothing was that both the victim and suspect must have struggled on the ground in the wooded pine forest where there was only one local area an understory covering of fern plants. Because pine trees are not native to New Zealand and are used as ornamentals in parks and grown for its timber, there was only one local area near the park where both pine trees and ferns grow together. Thus, the pollen evidence did not prove the suspect was guilty, but it did show he had been in the same wooded area where the crime scene occurred; an area he denied he had ever visited (Mildenhall 1992). As with all types of forensic trace evidence, it is essential to keep accurate records and photographs of how and where each comparison sample was collected, ensure that complete records of the chain of custody are maintained, and provide detailed information related to the method of analysis and the steps taken to provide for security of the samples when not being directly examined. When doubt can be raised about the possible contamination of a forensic pollen sample at any point between the time of collection and the time of final analysis, then that sample is compromised as evidence, even if it might clearly establish a link between a suspect and a crime.

Once all control samples have been collected, they should be stored in airtight, contamination proof containers that are clearly marked. Because comparative reference samples might contain moisture, which could encourage the growth of microbes, a microbe-killing solution, such as alcohol, needs to be added to each sample. Another alternative, which is effective, is to place samples in cold storage at or below 0 C, which will retard the growth of most microbes and will not damage the pollen or spores in the sample (Holloway 1989).

There are three potential areas where forensic pollen samples could be ruined or compromised. The first area is during the collection phase, the second area is during the extraction of pollen from the matrix material of collected samples, and the third area is in the identification and analysis of the collected sample.

Forensic palynologists often only have very small amounts of evidence material available for analysis; the exception is the collected comparative samples at a crime scene where ample amounts of soil can be collected for each sample. There is almost no limit to what types of pollen and spore samples one can collect as potential evidence because their tiny size enables them to become trapped on or in many different types of surfaces (Mildenhall et al. 2006). For instance, items sampled for pollen evidence include a tissue wipe of a tabletop, leather shoes and sandals, a shoelace from a boot, a piece of crumpled electrical wire from an explosive device, the vacuumed contents from pages in a book, dust vacuumed from the keyboard of a laptop computer, pollen trapped on tape used to wrap a package of illegal drugs, the lint trapped in the pockets of a pair of pants, and pollen and spores vacuumed from all sorts of clothing, backpacks, suitcases, rugs, and vehicles.

Samples used for forensic pollen evidence offer several major challenges during the extraction process. First, often there is very little material sample and thus the extraction procedures selected must be the right ones for the sample. Although some samples do not present much of a challenge, others do. Samples containing microscopic charcoal flecks or soot and samples collected from various types of oils and grease can present problems when trying to remove the pollen from those matrix materials. Other types of difficulties arise when examining samples vacuumed from polyester clothing or other synthetic materials, which contain many fibers and fiber fragments that resist most forms of chemical digestion or turn into partly digested globs that stick to pollen trapped in the sample. Second, because most samples cannot be replaced, great care must be taken not only to prevent contamination but also to prevent the loss of the pollen sample during the extraction process. Lab accidents must be avoided such as the accidental spilling or breaking of beakers or test tubes containing a sample, mixing of samples, using incorrect chemical reagents, or not keeping samples tightly sealed throughout each stage of processing. The primary objective in most cases is to remove as much debris in the samples as possible, thereby freeing the pollen grains for analysis and preventing remaining debris from obscuring part or all of the individual pollen grains during analysis. This means a forensic sample’s matrix material will not be available later for other types of forensic testing. If planned studies of the DNA, isotopes, trace elements, hairs and fibers, or other sample contents are planned, they should be completed before pollen extraction occurs. In those types of situations, however, great care should be taken to avoid any pollen or spore contamination of the sample. There are a number of reliable extraction procedures that can be used to prepare forensic pollen samples for both light microscopy and for scanning electron microscopy analysis (Faegri and Iversen 1989). The choice on which procedures to use will be dictated by the type of sample and by the type of matrix materials in the sample.

Microscopy And Other Analytical Techniques

The primary analytical techniques used in forensic palynology is light microscopy because it is easy to use, and most of the identification keys and atlases (printed and on web sites) rely on LM images as guides to the identification of pollen and spore types. LM is also a fairly fast technique that, in most cases, is adequate for determining the ratio of each pollen or spore type in the total pollen and spore spectrum of a sample. Thus, it is often the combined identification of both the pollen and spore taxa and the abundance of each type that provides the critical data needed for analytical precision. The primary problem with LM is that the resolution capability is limited to about 0.3 mm even using the best optimal microscopic lenses available (Jones and Bryant 2007). That resolution ability typically limits the identification of pollen and spore in some groups to the family level (i.e., Cupressaceae, Poaceae, and Chenopodiaceae) and others to the generic level (i.e., Pinus, Quercus, Artemisia, and Eucalyptus). Unfortunately, when using LM analysis, it is rarely precise enough to identify most pollen types confidently to the species level. Therefore, when added precision is needed, the forensic palynologist must rely upon some other technique, such as the higher resolution ability provided by both SEM and TEM (Milne et al. 2005).

Some analysts have been asked to examine entire forensic pollen samples using only SEM in an effort to gain the added precision of pollen and spore identification to the species level. Analyzing samples using only SEM is time-cosuming, costly, requires access to expensive equipment, and assumes that the forensic palynologist has the expertise and familiarity with SEM usage to ensure accurate results. An added problem of conducting SEM analyses of forensic samples is that one must have access to a large databank of SEM images of all the potential species in a genus in order to ensure the correct identification, to the species level, of pollen grains found in samples. Although this can be done, and has been done (Jones and Bryant 2007), these techniques are rarely feasible based on the funding constraints facing most investigative agencies.

The search for new techniques that could increase the accuracy and identification of pollen and spores in forensic samples has focused on a number of analytical techniques often used with other types of forensic samples. One of these is the use of Raman spectroscopy.

Raman spectroscopy has gained praise for its use in forensics as a successful analytical tool. This praise is based on a number of advantages of Raman spectroscopy, which makes it ideal for applications in the field of forensics. First, the techniques used in Raman spectroscopy are rapidly changing as improvements broaden its potential uses. Second, Raman spectroscopy is a nondestructive technique meaning that samples do not have to be destroyed or changed in order to enable analysis. Third, Raman spectroscopy is quick because it does not require any special type of sample preparation prior to examination. This means that forensic pollen samples can often be examined first using Raman spectroscopy rather than having to wait until other types of analyses have been completed, each of which might alter or contaminate the original sample. Fourth, only minimal amounts of material are required for a complete analysis, which means that tiny amounts of a sample, containing pollen and spore evidence, can be effectively examined. Fifth, Raman spectroscopy is highly sensitive to slight differences in the molecular and chemical composition of materials, which enables it to detect very small differences in samples. That type of high-resolution potential is important where minor differences are essential to distinguish one type of pollen or spore from all others (Ivleva et al. 2005).

A significant drawback of Raman spectroscopy for use in forensic pollen studies, however, is that the identification of pollen and spores down to the species level requires the construction of high-resolution spectral maps for each taxon. To construct such spectral maps for just 1 pollen or spore species requires an estimated 360–720 h of time gathering and storing the needed data. Therefore, the amount of time and cost expenditures required doing this type of analysis on samples containing a variety of pollen and spore species is usually considered unreasonable. An additional problem is that conducting and interpreting the signatures of Raman spectroscopy require a special expertise, which most palynologists may not have.

Although a few studies have shown that successful spectral signatures can be used to identify specific types of pollen using Raman spectroscopy (Manoharan et al. 1991), the use of this technology in forensic palynology is hindered by a number of problems. First, very few pollen species have spectral signatures that could be used for comparative purposes. Even if more pollen spectral signatures were desired, the very large investment of time and effort to obtain these signatures for each new species would be impractical. Second, a major problem with Raman spectroscopy is that for any given forensic sample, it might provide identification for some of the pollen and spores, but it could only offer statistical ubiquity rather than quantitative percentages of each taxon in the entire forensic sample. Finally, in some cases, it appears that Raman spectroscopy cannot identify some pollen types with certainty. After mounting fresh Celtis (hackberry) and fresh Cannabis (hemp, marijuana) pollen, it was noticed that some of the pollen grains in each group were folded, deflated, broken, and overlapped with other grains or were partly covered by pieces of debris. When setting the laser beam on specific targets within each group, the resulting Raman data for each group and between both pollen types was not conclusive enough to provide the certainty of identity that would be needed for accurate identification of those pollen types to the genus level.

Another technology that can aid in the identification of pollen grains in forensic applications is Fourier transform infrared spectroscopy (FTIR). This technique uses infrared rays (IR) to stimulate different chemical bonds in pollen grains that vibrate at certain known frequencies when excited. When the surface of a pollen grain is bombarded with an IR beam, the transmittance and reflectance of the IR beam occurs at different frequencies, which is measured and translated into an IR absorption plot. By matching the IR plot of an unknown pollen grain with IR signatures of known pollen taxa, the identity of the unknown type can be confirmed. The advantage of using FTIR technology is that it does not require complex sample preparations.

When targeting the surface of a pollen grain, an average depth of 2–3 mm is excited by the IR beam. Experiments using FTIR technology for pollen identifications note that individual pollen grains within a single genus and species will produce similar, yet sometimes slightly different, IR absorption plots depending on various factors such as slight chemical variations in the composition of the pollen wall, and the level of maturity of the pollen grain. These variations within a single pollen or spore taxon can be recognized by developing a FTIR database from multigrain spectra for each known pollen and spore species. Similar to Raman spectroscopy, there are several drawbacks associated with using FTIR technology for pollen identifications in forensics. The number of IR absorption plots for known pollen taxa in the current FTIR databases is limited, thereby decreasing the chance of successfully identifying individual pollen grains. Also, it has yet to be determined if pollen grains in various stages of degradation continue to send the same, or different IF absorption signatures, meaning it is unknown if degraded pollen grains will give the same signature as that of a pristine grain of the same species. Finally, there remains the problem of quantification of pollen and spore taxa in terms of the ratios of each type in a forensic sample to all others types present. Similar to Raman, FTIR can determine the “presence/absence” of individual taxa, but it cannot determine how many individual grains are present in a sample (Gottardina et al. 2007).

DNA analysis, a common technique used by many forensic laboratories, could potentially aid in pollen and spore identification. Recent evidence has demonstrated that a DNA signature can be recovered from a single pollen grain. DNA in pollen grains, however, can only be found in the cytoplasm inside the walls of a pollen grain. Once the cytoplasm degrades or is lost through rapid decay, the DNA signature is lost. Since this technique, as related to individual pollen grains, is still fairly new, the recovery methods, techniques to isolate individual pollen grains from the surrounding matrix materials, and adequate polymerase chain reaction (PCR) replication and identification database constructed from existing pollen DNA are still in the testing phase. Another problem concerns the type of pollen evidence that is used for forensic interpretations. Since DNA can only be recovered from viable pollen, using DNA signatures one could not identify nonviable pollen or degraded pollen in a sample (Zhou et al. 2007). Therefore, this type of data recovery would provide only limited results in terms of the total pollen present in a forensic sample. Another potential problem with using the DNA of individual pollen grains is that even with an adequate databank of information, it still may not be possible to identify individual pollen grains in a sample. That potential exists because certain species of plants can easily hybridize with other species of the same genus producing an F1 generation. Studies of the DNA signatures from other types of plant material, such as leaves and wood, reveal that the DNA of F1 plants is not identical to either of the parent plant sources (Schaal et al. 1998).

Unfortunately, DNA, Raman spectrometry, and FTIR analysis methods will provide only ubiquity types of data related to the pollen types in a sample. Instead, what are essential elements in forensic pollen studies are quantitative counts of the relative frequency of each pollen taxon.

Finally, aside from the other already mentioned limitations created by the data obtained from the DNA of individual pollen grains, the ultimate cost and time needed for this type of study make it unsuitable for forensic pollen analyses.

Similar to DNA analysis, stable isotope analysis is another important analytical technique that is gaining added use in forensic science. The advantage of this principle is that isotope ratios in organic and inorganic materials retain a record, in their molecules, of conditions that existed when that material was created (West et al. 2006). Using that principle, studies reveal that pollen grains contain recoverable isotopic signatures that can be recovered and mapped. During the last decade, scientists have attempted to recover the isotope signatures recovered from individual fossil pollen grains and then use those data to provide information about the environmental and climatic variations that existed where the pollen grain was generated. Further studies have revealed that stable isotopes of individual pollen grains from different plant families and different genera, and in some cases even individual plant species, generate unique isotope signatures. However, the major problem those scientists have encountered when trying to do isotope studies of individual pollen grains is how to isolate and concentrate the pollen grains in a forensic sample and remove them from the various types of surrounding matrix material. Thus far, the various chemical treatments designed to concentrate pollen and remove non-pollen debris in forensic samples alter the molecular composition of the pollen wall (Loader and Hemming 2000, 2001).

Similar to Raman spectroscopy, DNA, and FTIR, this type of study using isotopes would provide only ubiquity data. Finally, isotope studies of individual pollen grains would be very costly, time-consuming, and extremely complicated, thereby limiting its effectiveness as an analytical technique in forensic palynology (Loader and Hemming 2004).

Case Studies

Forensic palynology has been used to assist in the solving of a variety of criminal cases. In one instance, a suspect with a large quantity of cannabis resin was arrested at a port in New Zealand. Authorities believed that the cannabis was grown outside of New Zealand and they wanted to test their hypothesis. Several subsamples of the cannabis were tested for pollen and spores, which were then analyzed to determine whether or not the cannabis came from New Zealand. All subsamples yielded a similar pollen and spore spectra that included plant sources that are not indigenous to New Zealand. Based on the pollen and spore evidence, authorities were able to confirm that the cannabis was imported from Asia and was not from a locally grown crop (Mildenhall 1990).

Forensic palynology can also help to resolve murder cases. In 1999, the body of a man was discovered in a mountainous region north of Wellington, New Zealand. An eyewitness claimed to have seen another person with the victim in the area where the murder took place. Based on evidence, a suspect was arrested and a search revealed that the suspect had the distinctive type of clothing, which has been described by the eyewitness. To verify the eyewitness’ claim, the suspect’s clothing, camping gear, and other accessories were analyzed for their pollen and spore content. The recovered pollen and spore assemblage from the suspect’s clothing and backpack gear closely matched that of a mountainous region where the victim had been found. Although the pollen and spore evidence did not confirm the guilt of the suspect, it did show that the suspect had been in the immediate area where the crime had occurred and where he had been seen by the eyewitness (Mildenhall 2004).

In another case, a murder victim was found in an excavated depression wrapped in a bedcover in West Yorkshire, England. To determine whether or not the victim was murdered where the body was discovered, investigators sampled the bedcover and the victim’s hair for pollen. Upon analysis, the recovered pollen from the bedcover and victim suggested that the murder had taken place in a household garden and not where the body was discovered. Wood ash was also recovered from the victim’s hair, suggesting that the victim had been near a fire at the time of death. About a year later, a suspect was identified, and the recovered pollen spectra that had been recovered from the murdered victim and from the bedcover were compared with comparison soil samples collected from the suspect’s garden. Multiple samples were taken from different locations around the suspect’s garden and property. Not only did the pollen recovered from the bedcover and victim’s clothing match that of the suspect’s garden, but it also contained wood ash that matched an area next to a bonfire that had been constructed by the suspect. The pollen evidence did not confirm the suspect was the murderer, but it was important because it linked the victim and the bedcover to the suspect’s garden and property (Wiltshire 2006b).

Summary

Forensic palynology is a predominantly underutilized field. Only recently have some governments and law enforcement agencies around the world begun to explore and appreciate the utility of pollen and spores in forensic cases. Among these, the United Kingdom and New Zealand still lead in terms of the number of cases and applications they have explored using pollen and spore evidence. The potential uses of forensic palynology are many and have already been used effectively in instances involving homicide, terrorism, genocide, bombings, forgery, theft, rape, arson, counterfeiting, manufacturing and distribution of illegal drugs, assault, cases of hit and run, poaching, and identity theft. Pollen and spores are microscopic in size and are often produced in large numbers. They can be found in almost any environment and can become attached to almost any type of surface; these are reasons why they have become useful as trace evidence of places where items originated or were used. Although forensic palynology has played an important role in helping to convict criminals and in some cases support the innocence of some suspect, most countries have been slow in adopting this technique. This hesitation often comes from the discipline’s lack of notoriety and often an unfamiliarity with how the use of pollen and spores can contribute to as forensic evidence.

More law enforcement agencies should consider using pollen and spores in forensic cases and come to understand the importance of how to collect samples without contaminating them and whom to depend upon for help with the analyses. Pollen evidence has a bright future as one of the types of materials that can be useful in determining the geolocation of some person or object. Currently, there are limited numbers of scientists trained in the field of forensic palynology, mainly because there has not been a demand for their services. This, however, could change if the need to conduct these types of analyses was to broaden. Unfortunately, the training of these individuals cannot be accomplished overnight and thus when the time comes for new forensic palynologists, they will need to be able to meet the needs of law enforcement and others requiring their services.

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