Crime & Clues

Vaughn M. Bryant, Jr.
Palynology Laboratory
Texas A&M University
College Station, Texas 77843

Dallas C. Mildenhall
Institute of Geological and Nuclear Sciences
P.O. Box 30368
Lower Hutt, New Zealand

Introduction

The term "forensic palynology" is rarely used in the United States. Forensics pertains to evidence used in courts of law. Palynology is the term first used by Hyde and Williams (1944) for the collective study of pollen grains and spores. Years later, the discipline of palynology was expanded to include the study of a number of other acid-resistant microorganisms as well.

Today, the science of forensic palynology traditionally focuses on legal evidence derived from the study of pollen and spores, both fossil and modern. In its broader application, the field of forensic palynology also includes legal information derived from the analyses of other palynomorphs such as dinoflagellates, acritarchs, and chitinozoans. However, in most sampling situations forensic palynologists rarely encounter these other types of acid-resistant palynomorphs because most are marine and many are restricted only to fossil deposits.

It is difficult to establish precisely when the field of forensic palynology began. Attempts made prior to the 1950s, or those that may have failed, probably did not gain public attention and therefore were not reported. Or, it is possible that if earlier attempts were made, the results may have been purposely hidden from the media so as not to alert criminals about the use of this technique.

One of the earliest successful cases where forensic palynology was used, and one of the most dramatic, pertained to a criminal case in Austria in 1959 (Erdtman, 1969). In that case the solving of a murder and the conviction of the criminal was based primarily on the evidence recovered from a pollen sample associated with the crime. The details of the case are worth noting.

A man on a journey down the Danube River disappeared near Vienna, but his body could not be found. Another man, with a motive for killing him, was arrested and charged with murder. Without a confession or a body, however, the prosecutor's case seemed hopeless. As the investigation proceeded, mud found on a pair of the defendant's shoes was given to palynologist Wilhelm Klaus of the University of Vienna for analysis. Klaus determined that the mud contained spruce, willow, and alder pollen as well as a fossil hickory pollen grain 20 million years old which had eroded from an exposed Miocene-age deposit. Only one small area 20 kilometers north of Vienna along the Danube Valley had soils that contained this precise mixture of pollen. When confronted with the identity of this location, the shocked defendant confessed his crime and showed authorities where he had buried the body, which indeed was in the region pinpointed by Klaus (Erdtman, 1969; Newman, 1984).

In a recent book by Moore and Webb (1978), and in the latest textbook published on palynology by Faegri et al. (1989), the authors gloss over or almost totally ignore the subject of forensic palynology. Both books devote only one paragraph to the topic. Faegri's text uses only two sentences to discuss its only example of forensic palynology--a case in which an expensive item had been stolen and its space had been replaced with straw. By conducting a forensic pollen study of the straw, the experts were able to determine where the straw was grown and find the locale where the theft and substitution had taken place.

Pollen Studies

Today the study of fossil and modern pollen has many uses from helping people identify their various allergies to providing us with information about the location of oil, coal, and gas resources needed to run our modern civilization. Yet, of the many uses of pollen data, one of the least known and utilized is the application of pollen studies to the science of forensic analysis.

The study of palynology has, by necessity, been closely associated with the development and later improvements of microscopes. Because pollen grains are microscopic, mankind had to wait until the invention of the compound microscope in the mid 1600s before he could see pollen grains in any detail. During the next two centuries following the invention of the microscope, botanists studied the morphological features of pollen grains, their form and structure, and began to develop taxonomic keys for their identification (Wodehouse, 1935). It was during this period that botanists learned that some plants were wind pollinated while others were insect pollinated. Geologists, like Christian Ehrenberg, were among the first to realize that pollen grains remained preserved in ancient sediments and that many of them looked similar to pollen types still being produced by plants growing today (Traverse, 1988).

The beginning of modern palynology occurred in the early 1900s when researchers like Gustav Lagerheim suggested that fossil pollen might provide a useful clue to past vegetation cycles. A few years later his student, Lennart von Post, added the use of statistical standards and set forth the basic theory of pollen analysis (Faegri and Iversen, 1975).

By the 1920s and 1930s ecologists and geologists were using fossil pollen to date late Quaternary deposits and reconstruct sequences of past environmental cycles from sediments taken in lake bottoms and from peat cores in deposits throughout Europe. A few archaeologists also began using pollen data from their excavated sites as a dating technique for the artifacts they recovered.

Prior to the 1940s, other applications for pollen studies were slow in coming. It wasn't until the early 1940s that archaeologists discovered that fossil pollen could tell them more than just the probable age and environmental setting of a site. Johannes Iversen was the first to show how fossil pollen records could provide archaeologists with information about the introduction of agriculture, ancient diets, the establishment of permanent dwellings and villages, and the beginning of animal domestication in regions of Denmark (Iversen, 1941).

In the late 1940s a geologist, T. F. Grimsdale, pointed out that fossil pollen could be used to correlate sediments of similar age in deep underground deposits in areas being tested for oil exploration (Hopping, 1967). After refining this new application of palynology, petroleum companies were able to drill for oil and gas locked in deep deposits where stratigraphic correlations were a critical part of the exploration and recovery process (Wilson, 1978). Thus, the period of the early 1940s was the beginning point when palynologists began looking for all sorts of new and creative ways of applying pollen data to the solving of scientific problems.

Palynomorphs

Pollen Production and Dispersion

Pollen and spore production is an important consideration in the study of forensic palynology. First, if one knows what the expected production and dispersal patterns of spores and pollen (called the pollen rain) are for the plants in a given area, then one will know what pollen assemblage should be found in samples collected from that given locale. If a palynologist examines a sample of material (i.e., mud, soil, clothing, etc.) thought to come from a specific geographical region, yet the expected assemblage of pollen and spores is not found, or if other pollen and spore types are present in unusual numbers, then those results suggest something is wrong. In such an example it is the "out of the ordinary" pattern which gives the forensic palynologist clues and suggests that he ought to search for the reasons why the sample is different from the expected pattern of pollen. Like any detective, the palynologist must use the knowledge of his discipline to link a specific pollen sample to a precise location or event. Knowledge of pollen dispersal and productivity often plays a major role in helping him solve such problems. There are a number of different methods by which plants disperse their pollen or spores. Many flowering angiosperms that live completely submerged in water release their pollen underwater and rely upon water currents to transport the pollen from the male anther to the female stigma of a neighboring flower. This method of transport, like the wind, is a hit and miss method of pollination. For this reason these plants tend to have high productivity levels of pollen with each anther producing thousands of pollen grains. However, since these plants produce pollen types that consist only of a single-layered cellulose wall, the pollen is almost never preserved in lake sediments and generally oxidizes rapidly if removed from water. Because of these limitations, these types of pollen are of little potential value for forensic work.

A small group of plants, called "autogamous" because they are self-pollinating, is so efficient that little pollen production is needed. Most plants in this category produce less than 100 pollen grains per anther. Pollen from these plants is rarely dispersed into the atmosphere even though their pollen preserves well and has durable outer walls, called an exine, made of a stable chemical compound called "sporopollenin" (Shaw, 1971). Like pollen produced by submerged plants, the pollen of autogamous plants is generally of little value in forensic work because it exists in such minimal numbers in the deposits of most regions.

In a large number of plants, called "zoogamous" plants, pollination is dependent upon the transport of pollen from the anther of one plant to the stigma of another by some type of insect (i.e., bee, wasp, beetle, moth, ant) or animal (i.e., hummingbirds, lizards, nectar-feeding bats, or other small mammals). The pollen from zoogamous plants generally has a thick exine that offers essential protection from humidity changes and possible abrasion during transport (Wodehouse, 1935).

Because of the efficiency of zoogamous plants, pollen productivity is often very low, yet not as low as is found among the autogamous plants. The potential value of zoogamous pollen in forensic work is excellent for two reasons. First, zoogamous pollen grains have some of the most durable exines. This means their grains often will remain preserved in deposits for long periods of time and are generally less susceptible to destruction than pollen grains dispersed by other methods. Second, zoogamous pollen is produced in low amounts and thus is not normally a potential contaminate found in the pollen rain of an area. This last point is both good and bad. It is good because if the pollen of a given species of zoogamous plant is found in a forensic sample, there is a high degree of confidence that the pollen belongs with the sample and is not an atmospheric contaminate. It is bad because so little pollen is produced by each zoogamous plant that the chances of its pollen getting into any sample are reduced.

The last category of pollen is the wind-pollinated types from anemophilous plants. This group includes a wide range of pollen producers such as the gymnosperms and a significant number, but not a majority, of the angiosperms. Also included in this group are the spore- producing plants such as fungi, ferns, and mosses. Because wind pollination is the most inefficient and most archaic method of dispersion, anemophilous plants must produce vast quantities of pollen or spores and must produce light weight grains that will travel easily in air currents. Some species of wind-pollinated plants, such as marijuana (Cannabis), produce as many as 70,000 pollen grains per flower. When large fields of these plants grow together, their flowers can produce millions of pollen grains that are dispersed daily during the flowering season. Thus, there is a high probability, as in the example of marijuana pollen, that these pollen types will settle on or become mixed with everything in close proximity to these blooming plants.

Many of the plants that produce wind-pollinated grains, such as ragweed, grasses, some species of eucalyptus, pine, oaks, pecan, hickory, birch, alder, and elm produce anthers which each contain between 10,000 and 100,000 pollen grains. Even anemophilous types producing low amounts of pollen will still produce more than 10 times the amount of pollen per plant than almost all species of zoogamous plants.

Because of the vast volume of pollen produced by the anemophilous plants, their types are the most common found in the pollen rain of most regions of the world. As such, their types are the most common in the fossil pollen record of a region and are also the most common types analyzed for forensic studies.

Pollen Sinking Speed

Another factor that determines how much pollen actually becomes part of the pollen rain of a region is the "sinking speed" or rate at which a pollen grain falls to earth. Marijuana, alder, juniper, and birch pollen are very small and very light. Their average fall rate is about 2 cm per second (Traverse, 1988). On the other hand, maize and fir produce pollen that are large and heavy and have a rate of fall 15 times faster than the lighter ones. Using just these two examples one can see that the potential distribution area of maize and fir pollen grains will be much smaller and more restricted than the dispersion area covered by the plants in the first category (Tauber, 1967). From the standpoint of forensic studies, this means that when maize and similar types of large and heavy pollen grains are found in samples, small dispersion areas are indicated and greater precision in identifying the source region may be possible.

Pollen Degradation

When examining forensic samples, palynologists must consider how much, and which types, of pollen may be missing due to differential preservation and degradation. Studies by Havinga (1964, 1984), Sangster and Dale (1961, 1964), Holloway (1981), Hall (1981), and Bryant and Schoenwetter (1987) have noted that all pollen types do not preserve equally well in sediments. Some pollen types such as grass, goosefoot, composites, and pine are very durable and often remain preserved long after other pollen grains have oxidized or become so degraded that they are no longer recognizable. Often, however, pollen samples used in forensic studies are from modern deposits and thus degradation or differential preservation is not generally a problem.

Pollen Recycling

Pollen recycling is yet another problem one must consider when examining forensic samples. Sometimes pollen eroding from earlier deposits can become incorporated into contemporary samples, such as the example of the 20-million-year old Miocene-age pollen grain that became a key factor in the forensic study from Vienna, Austria. In some cases, recycled pollen can provide valuable information about specific locales and can be used to pinpoint regions precisely. In other cases, recycled pollen may be of modern varieties and thus obscure the desired data.

Depending upon how badly recycled pollen grains have been degraded, they may, or may not, be able to be separated from the original pollen rain. If recycled pollen grains cannot be distinguished from the normal pollen rain, the addition of recycled pollen may mask the true identity of a target locale. This could occur when the combined pollen assemblage (modern + recycled) suggests a flora quite different from floral reconstructions based on modern pollen control samples collected from the same location.

There are several techniques palynologists use to identify recycled pollen in standard samples. In a recent study of the pollen rain from Arizona, O'Rourke (1990) separated recently released pollen from recycled pollen by staining them with basic fuchsin and then noting which grains contained an intine, and which did not. Those that still had an intine were counted as being part of the recent pollen rain; those that did not were considered recycled pollen.

Other techniques often used to identify recycled or reworked pollen in standard samples is differential staining and fluorescence microscopy (Traverse, 1988). In a study conducted by Stanley (1966), he noticed that recycled pollen grains absorbed different amounts of safranin-O stain. In some cases the recycled pollen absorbed less stain and in other cases they absorbed more stain. This led Stanley to propose this technique as a useful method for isolating recycled from normal pollen in samples. Fluorescence has also been used with some success to recognize which pollen grains are recycled and which are not. Pollen and other types of organic materials in different stages of preservation, or in different stages of carbonization, will emit light of varying intensity and wavelengths (Rolfe, 1965). These differences can be detected under ultraviolet light during fluorescence studies and make the different pollen grains appear as different color hues.

The theory behind using both techniques is the belief that recycled/reworked pollen have different depositional histories than the normal pollen assemblage and thus will make the recycled grains appear as being different. We have found that although each of the above mentioned techniques is useful, and each has helped us separate modern from recycled pollen in some forensic samples, in each case we have always proceeded with great caution and with reservations concerning the analytical results. We have found that not all recycled pollen will behave in the ways listed above.

In laboratory experiments conducted at the Texas A&M Palynology Laboratory "spikes" of modern pollen and spores were added to pollen samples of known age collected from the Boriack peat bog (Bryant, 1977). After processing and counting the pollen in each peat sample, we added modern pollen and spore spikes to other peat samples from the same core samples and then processed them. Prepared pollen slides were then counted by a palynologist who did not know which types of modern pollen and spores had been added to the fossil samples. The results of his counts demonstrated that for the peat samples we examined, that ranged in age from 2,000- 15,000 years old, only a few of the added pollen could be recognized using staining or fluorescence. A similar study by Shellhorn et al. (1964) revealed that when using ultraviolet light both modern and fossil pollen (20,000+ years old) in lake deposits of the Wilcox Playa in Arizona glowed with the same fluorescent color hues.

Collection And Extraction Of Palynomorphs From Forensic Samples

In forensic palynology the collection and extraction of pollen and spore assemblages from samples are critical aspects. Improper collection of samples and/or the accidental contamination of forensic samples will produce inaccurate results. Not only can this lead to misinformation, but improper collection and handling of forensic samples can be used to dismiss resulting data as invalid evidence.

Ideally, forensic pollen samples should be collected by a competent palynologist knowledgeable in the field of forensics. Such individuals will know how to collect contamination-free samples. They will also know what precautions should be taken to ensure that forensic pollen samples remain contamination free throughout the storage, laboratory extraction phase, and during the analysis process. If it is not possible to have samples collected by a forensic palynologist, then there are guidelines others should follow to ensure that such samples are collected properly and remain contamination free. It is also important to keep accurate records of how each sample is collected and what has happened to each sample from the time of collection until examined by a forensic palynologist.

Security should be an essential concern. To ensure the court admissibility of forensic pollen evidence, it will be critical that the palynologist working with the sample be able to state under oath that the materials, and the subsequent pollen samples collected from those materials, were stored in a locked and secure location. If any hint of contamination, either natural or intentional, can be proven or implied as being possible, then doubt can be cast upon the pollen results and the resulting interpretations.

A major problem concerning the collection and extraction of forensic pollen samples is the amount of material that is available for collection. In most cases very little dirt, mud, or other debris is available for collection and analysis. Therefore, most forensic palynologists face several immediate problems. First, he will generally not have enough sample to try a series of different extraction techniques to determine which one might work best. And, second, he will often not have enough sample to conduct a second test if something goes wrong (i.e., a centrifuge tube breaks, a beaker spills, or a microscope slide is broken).

Collection of Samples

When determining what kind of materials one should select for pollen forensic studies, three aspects should be considered: 1) what type of materials should be collected, 2) how should the materials be collected and by whom, and 3) how should samples be treated once they are collected. The most important consideration that should always be foremost in the mind of the person collecting forensic samples is to make sure that all collecting tools and all collection containers are free of pollen contamination. If improper sampling procedures are used, or contaminated tools are used during collection, or samples are not placed in sealed, sterile containers, the value of a collected sample will be compromised.

Listed below are some examples of common materials often sampled for pollen forensic samples:

Soil and Dirt. Dirt, mud, or dust thought to be associated with a crime, are generally good sources of pollen information. Samples of dirt collected from the clothing, skin, shoes, or the car of a victim might prove useful in linking the victim with the location where the crime occurred. The same would be true of any suspects thought to be associated with a crime. Mud found on a stolen vehicle, or a vehicle used in a crime, could link the vehicle with the scene of a crime or link it to the place from which it was stolen. Dirt found associated with other objects or other types of conveyances (i.e., bicycle, motorcycle, boat, etc.) thought to be associated with a crime also might yield pollen evidence useful in linking those items with a specific crime or a specific geographical locale.

In each of these examples great care must be taken when collecting samples for forensic analysis. In instances where dirt and mud have dried on objects, one should use a soft, clean, paint brush to clean the surface before collection. We have found that soft, sable hair brushes designed for applying cosmetic facial rouge are the best suited for this type of work. This procedure will remove possible surface contamination of ambient pollen that may have settled on the dirt or mud sample after it dried. The paint brush one uses must be clean and free of pollen. This can be achieved by thoroughly washing it with a mixture of distilled water and detergent and then rinsing it first with distilled water and then alcohol prior to using. This procedure will ensure that the paint brush is clean and dry and does not add its own pollen contamination to the sample being tested. One should keep a supply of spare brushes each in a separate, sterile, sealed, plastic bag. We have found that sterile, self sealing, zip-lock plastic bags are ideal for this purpose. Only distilled water should be used for cleaning the brushes because many municipal water systems may be free of microbes, but often their water is not free of pollen.

Once the dirt or mud is cleaned, the collector should wear a clean pair of sterile, surgical gloves while collecting each sample to ensure that pollen on one's hand does not contaminate the sample. Actual collection should consist of picking up the dirt or mud fragments or gently scraping them with a clean implement. The size of the sample collected will vary. Small samples are easier to protect from possible contamination, yet the smaller the sample the more difficult it will be for the palynologist to extract sufficient numbers of pollen for analysis. When in doubt, it is wiser to collect a bit more sample than is needed than not enough. If possible, a sample size of from 15-30 grams of dirt should be collected.

Once collected, each sample should be placed in a sterile, plastic bag and tightly sealed. This will ensure that no pollen contamination occurs between the time of collection and the laboratory analysis of the sample. We recommend the use of plastic bags even though there are two potential drawbacks. First, if a sample of dirt, mud, or other material is moist it will not dry properly if sealed in a plastic bag. The solution to this is to seal the bag when the sample is collected and later carefully open the plastic bag only slightly and place it in an oven at low heat until the sample is dried. The other alternative is to add sufficient alcohol to the sample to kill any microbes that might cause damage to the pollen in the sample.

The second drawback to the use of plastic bags is static electricity. When atmospheric conditions are dry, it might be difficult to get dust into a plastic bag full of static. If after several tries there is still too much static in the plastic bag, put the dust into a clean, paper envelope. Next, seal the envelope and put it inside a sealed plastic bag. We prefer not to use paper bags or envelopes because they sometimes contain pollen accidentally incorporated into the paper making process.

In situations where there is only dust present, and there is not enough dust to sweep into some type of container, then one should try collecting pollen samples using transparent cellophane tape. A roll of one-inch wide cellophane tape is ideal. Using sterile gloves, one should tear off long pieces of tape and stick them on the surfaces to be sampled. Next, remove the tape carefully, with the dust attached, and stick it together, one half stuck on the other half. This protects the sampling surface of the tape and prevents further contamination of the once sticky surfaces. When sampling is completed, place each tape in separate, sterile, zip-lock plastic bags to prevent potential contamination of the sample. Once in the laboratory, forensic palynologists use solvents to loosen the material stuck to the tape.

An alternate method can also be used. For convenience, after strips of sticky cellophane tape have been used to collect dust, they can be stuck to the inside of a sterile, disposable, plastic plate. This procedure has an advantage for certain kinds of collection needs. In situations where serial samples are required, each tape strip can be stuck to the same plastic plate in sequential order. A felt-tip pen can then be used to write the serial order of collected samples on the plate. When sampling is completed, the plate or plates should be placed into a sterile plastic bag and sealed to prevent further contamination from atmospheric pollen. Collect as many of these tape samples as needed remembering that it is better to collect more than will be needed than not enough.

Hair. Hair is an excellent pollen trap. When wind blows through hair, pollen in the wind becomes trapped in the open spaces between individual strands. In humans, the addition of various types of hair sprays and tonics makes hair surfaces sticky and provides an even better trap for pollen. Hair can be sampled for its pollen contents by carefully washing it with detergents and warm, distilled water. This process will loosen trapped pollen and free it from sticky hair surfaces. Once collected, the wash water should be stored in a sterile container that is tightly closed and frozen or kept at a temperature near freezing in a refrigerator to retard microbe growth. If refrigeration is not possible, an alternative storage method is to add sufficient alcohol to the sample to prevent the growth of bacteria and fungi. Usually, if the solution contains about 10% alcohol, it will be sufficient to kill microbes that might damage the pollen. Do not use hydrogen peroxide in place of alcohol because hydrogen peroxide is an oxidant that is known to damage pollen.

Sampling hair need not be restricted to humans. Fur rugs found at the scene of a crime might have been used to wipe shoes and thus may be rich in pollen. Sheep, cattle, or other stolen fur-bearing animals might be traced to their original owner through the pollen analysis of hair samples washed or shaved from their bodies. Hair on fur coats, blankets, felt hats, or sheep skins sometimes used as car seat covers each acts as an excellent pollen trap and should be considered for their potential forensic value. Each of these samples should be placed in a sterile, plastic bag that is sealed and remains unopened until it can be analyzed. Woven Cloth, Woven Bags, Baskets, Ropes, and Clothing. Woven materials have many of the same pollen trapping properties as hair. Pollen in the atmosphere is constantly settling on exposed surfaces. Woven materials left exposed to the air become coated with airborne pollen that is trapped in the fibers of the material. Woven burlap or cloth bags used to transport products become exposed to the pollen rain of the region where their contents are being produced and packed. Thus, coffee beans picked and packaged in burlap bags in Costa Rica will contain a pollen assemblage representative of Costa Rica. Likewise, rice sacks packaged in Texas or Louisiana should contain a pollen assemblage from those regions.

If the whole item cannot be placed into a sterile, plastic bag and then sealed, then each item should be repeatedly sampled using pieces of transparent, cellophane tape. The same procedure explained above, for sampling dust, should be used. However, because much of the pollen often becomes trapped in between the weave of woven fabrics, it is much better to let a forensic palynologist examine the whole item (i.e., burlap sack, sweater, shirt, pants, etc.) whenever possible. Sampling the surfaces of fabrics using the cellophane tape procedure should be used only when the item in question cannot be retained as evidence for later examination.

Wicker baskets used to transport products often contain pollen grains trapped in the spaces between their weave. These trapped pollen types are often indicative of the place where the baskets were made. Likewise, products stored in baskets (i.e., tea, marijuana, coca leaves, coffee beans, etc.), often contain pollen that will become trapped in the basket's weave. Like baskets and woven bags, the weaving of clothing also becomes a natural trap for ambient pollen from the atmosphere. This is especially true of coarse weave garments made of burlap, cotton, or wool where the coarse fibers act as natural pollen traps.

When sampling for pollen trapped in woven bags, baskets, or items of clothing, the item should be placed in a large, sterile, plastic bag and sealed. Later, in the lab, a forensic palynologist can remove the pollen from these items by thoroughly rinsing them in a solution of hot, soapy, distilled water. As described earlier, this process will dislodge trapped pollen. Once removed, the wash solution should be processed for its pollen contents or frozen until it can be processed.

Packing Materials. Many products are packaged with various types of packing materials. In instances where products are packaged outdoors, or in open-air warehouses, ambient pollen in the atmosphere can enter the packaging area and settle on the packaging material being used. Later, a pollen analysis of these packing materials might reveal clues about the locale where certain products were produced or packaged.

Small, portable vacuum cleaners are effective tools for collecting pollen from packaging materials. The main advantage of a portable vacuum is that it can clean a large area quickly and the suction it produces will generally dislodge most trapped pollen and suck it into the vacuum cleaner's internal lint trap. Another advantage of a vacuum is that it enables a person to collect samples easily from the inside lining of a container and especially from the corners of wooden or cardboard boxes. When using this technique it is important that special, sterile, lint bags be used and that the vacuum be thoroughly cleaned between each use. Fiberglass or paper filter bags are preferred, but cotton bags can be used if they have been thoroughly cleaned. Fiberglass and paper filter bags are convenient because they can be collected after the vacuum process is completed and the entire bag, contents and all, can be chemically processed in a laboratory to ensure that no trapped pollen escapes analysis. Fiberglass and paper fibers quickly dissolve in certain acids yet this process leaves the collected pollen undamaged.

Illegal Drugs. One of the most useful applications of forensic pollen samples is in the search for, and identification of, illegal drugs. Often it is important to link specific individuals with specific shipments of illegal drugs, determine if drugs found in various locales are part of the same original shipment, or it may be important to trace and identify shipments of drugs coming from a specific processing laboratory or a specific geographical locale.

As mentioned above, marijuana plants are one of nature's most prolific pollen producers. Because marijuana growing, harvesting, and packaging often occurs in the open, ample amounts of marijuana pollen, as well as other pollen from the local pollen rain, will become incorporated as part of the material being packaged. On the other hand, if marijuana plants are grown, harvested, processed, and shipped from locations that are entirely indoors, then few if any examples of the local pollen rain will become incorporated in the harvested product. This makes it difficult to determine the precise geographical location where the plants were being grown or processed. However, because even indoor marijuana plants produce millions of pollen grains, samples of dust, clothing or furniture in a room, dust filters on the return ducts used for air conditioning or heating units, or even the dirt on the floor of greenhouses, underground growing basements, or enclosed processing locations will confirm, beyond doubt, that marijuana plants were being grown and/or processed. Because marijuana plants are prolific in their ability to produce and disperse pollen, everything their pollen grains contact will be contaminated.

In a related example, the process of turning coca leaves into cocaine begins when the leaves of the coca plant are picked, dried in the open, processed in outdoor areas, and then refined into cocaine (Weatherford, 1987). Because most of this occurs in, or near, the place where the coca leaves are grown, pollen from other plants in the area should be reflected in samples of the refined cocaine. Likewise, the first step in the production of heroin is to scar the outer surface of a poppy's immature seed pod. This scarring produces a sticky sap that becomes an excellent pollen trap until it dries. To our knowledge, recovery of pollen from cocaine, crack, or heroin has never been attempted, but in theory, these products should contain pollen.

Bags, baskets, packing materials, vehicles, the clothing of individuals, and even the paper money associated with drug transactions are potential sources for collecting samples for pollen forensic studies. The resulting pollen information from such studies could link individuals or items with illegal drug shipments, can determine whether or not several shipments have a different or common geographical origin, and can often pinpoint the geographical origin where the drugs were made or processed. Finally, one should remember that collection of pollen forensic samples related to drug transactions must be conducted with the same degree of care and skill as other kinds of materials.

Stomach and Intestines. In situations where a victim has been killed, forensic pollen samples should be collected from the stomach, small intestines, and colon areas during an autopsy. The length of time between the death of a person and the time when an autopsy is performed should not affect the preservation or validity of results obtained from forensic pollen samples.

When investigators find a victim's remains have become severely decomposed, or the victim was buried and only skeletal material remains, forensic pollen samples should still be collected. In burials one should collect the thin layer in actual contact with the underside of skeletal bones in the chest and pelvic regions. Such samples might provide clues as to the time of year when the victim was buried or could contain pollen that had been on the victim's clothing at the time of death. Samples of dirt should also be collected from the region inside the pelvic area because these samples may reveal clues about the victim's diet and possibly where the victim lived.

Forensic pollen samples from burials should be collected with the same degree of care mentioned earlier. Whenever possible, 10-20 grams of soil should be collected using contamination free-implements and the sample should be sealed in air-tight plastic bags. If the soil is damp, procedures mentioned above should be taken to prevent further pollen damage from soil microbes. Collection of forensic pollen samples during an autopsy should emphasize non-contamination between samples. As with soil samples, fecal or stomach samples should consist of 10-20 grams of material whenever possible.

A pollen examination of materials collected from the stomach and/or the intestinal tract of a victim might provide a variety of clues not evident from other types of studies. As mentioned earlier, ambient pollen is constantly settling out of the atmosphere. Therefore, it is possible that some of those pollen grains may have settled on foods and drinks consumed by the victim. If so, the pollen trapped in the stomach and intestines may provide clues about where the individual had been just prior to death. Also, pollen that is often accidentally incorporated into many of the manufactured foods we eat could offer clues about the diet the individual ate during the last few days, or even weeks before death.

Various pollen studies (Kelso, 1976; Williams-Dean, 1978) reveal that different types of pollen remain trapped in the human digestive track for different periods of time. Broccoli (Brassica) pollen, for example, is very small and has a reticulate-type of surface ornamentation; factors that permit it to become easily trapped in the folds of the stomach and intestine. Mesquite (Prosopis) pollen, on the other hand, is larger, has a smooth surface, and is football shaped; factors that speed its movement through the human digestive track. Experimental tests have shown that after eating a meal that includes broccoli, there will be an initial high concentration of Brassica pollen in the feces of that person about two days later. After that, the percentage of Brassica pollen in feces continues to remain high for another week or so before it begins to decline. However, tests show that traces of Brassica pollen are still detectable in feces produced up to a month after initial ingestion. Like broccoli, the percentage of mesquite pollen shows an initial rise in feces produced 2-3 days after ingestion, but unlike broccoli, all traces of mesquite pollen disappear after about 10 days.

Miscellaneous Materials. There are many other materials that can be used for pollen forensic studies. As mentioned earlier, pollen is one of the most common pollutants in our atmosphere; it settles out of the atmosphere and becomes trapped on almost any surface and can be incorporated into almost any deposit.

Examples of other kinds of materials suitable for pollen forensic studies are almost endless. Some additional examples include: 1) honey--to verify purported floral sources and specific geographical locales; 2) dried fruits-- imported apricots, prunes, or raisins to determine their country of origin; 3) tea--to determine its country of origin; 4) raw sugar--to authenticate its geographical origin; 5) coffee--to validate the country of origin by examining pollen trapped in the weave of burlap bags used to package the coffee beans; 6) sisal--to confirm that imported sisal rope actually comes from the purported country of origin; 7) coins and paper money--to validate if ancient coins or paper money are from their country of origin or are fakes, or in some cases if it has been in contact with marijuana pollen; 8) tobacco--to determine whether it is of foreign or domestic sources; 9) antique furniture--to confirm the possible age and origin of antique furniture purported to come from a specific geographical location; and 10) air filters--to indicate locales where private airplanes, trucks, or cars have visited.

Control Samples. Whenever one collects pollen forensic samples, it is also essential to collect "control samples." Control samples are specimens of surface dirt from the region where a crime was committed or from a region where a crime is believed to have been committed. The control samples are used by forensic palynologists to form a "baseline" of data about the expected pollen assemblage at a given locale. Once the baseline pollen data are determined from the control samples, then the pollen recovered from forensic specimens can be compared against the control data to see if both match.

An example of why control samples are important is illustrated by the following. If a crime is committed in a wooded locale where the dominant plants are birch, alder, pine, and maple, then control samples of surface dirt collected from that region should reveal a pollen record containing various percentages of those major pollen types and traces of other minor pollen components. Later, if the muddy shoes of a suspect, or mud from a suspect's car is collected and examined, then the pollen assemblage in those forensic samples might match the pollen assemblage found in the control samples. However, without knowing what pollen types, and in what percentages, one should expect to find at the scene of the crime, it is difficult to argue in court that pollen found on a suspect's shoes, or car, actually link that person with the scene of the crime.

Knowing how many control samples to collect from the scene of a crime is difficult. The more control samples one collects, and examines, the more pollen information one has about the locale where the crime was committed. Because the pollen in each control sample may vary slightly in the types and percentages found, their combined record offers a potential range of pollen variation for the scene of a crime. This knowledge makes matching the pollen results from forensic samples with actual locations more certain. This is especially true when it can be shown that the forensic sample data fall within the range of expected pollen variation. Not having control samples to examine, or having only one sample, weakens the certainty of forensic pollen data when they contain pollen assemblages similar to, yet not exactly like, those of the single control sample.

Cost and time factors must also be considered. A prudent policy might be to collect as many control samples as one has time to collect, yet be selective when it comes time to analyze them. Ideally, if a number of control samples are collected at the scene of a crime, and the precise location and description of where each was collected are given to a forensic palynologist, then the fewest number and the most appropriate control samples can be selected for study.

When collecting control samples, one should consider the following criteria. First, collect a control sample as close to the exact spot where a crime occurred as possible, if that is known. Second, collect additional control samples from areas near the scene of the crime. If the surrounding area within one kilometer radius consists of similar vegetation, then one or two control samples from each of the four cardinal directions, about 500 meters away from the scene of the crime, should be sufficient. If the area within a one kilometer radius contains different types of vegetation (i.e., open grasslands, agricultural fields, wooded areas, etc.), then one or two control samples should be collected from each different vegetational zone.

The ideal way to collect control samples is to use the "pinch" method (Adams and Mehringer, 1975). This can be done by selecting an area about 50 to 100 meters square and walking back and forth collecting pinches of dirt throughout the area. As each pinch of dirt is collected, it should be combined into a single, sterile, plastic bag and then sealed. The reason all the pinches of dirt from each sample area are combined is to prevent the possibility of over-representation of a single pollen type. Tests using the pinch method (Adams and Mehringer, 1975) reveal that in most cases more than eight pinches of dirt are needed for each control sample before their combined dirt yields a reliable pollen assemblage of the regional flora. Generally, collection of 10-20 pinches of dirt per control sampling location is sufficient to ensure an accurate sample. As with the collection of forensic samples, one should either wear clean, surgical gloves while collecting, or carefully wash one's hands with detergent prior to, and between, collecting each control sample. Following this procedure will prevent potential pollen contamination of samples as they are being collected.

Extraction

One of the most important parts of a forensic pollen study is the extraction process. The laboratory extraction procedure is a destructive process. In order to concentrate the pollen in forensic samples it is generally necessary to dissolve or destroy all types of non-pollen detritus. This means in most cases pollen forensic samples will not be available later for other types of forensic testing. If a sample is to be used for multiple forensic tests, then the other tests should be conducted first and the pollen test should be performed last. On the other hand, when such multiple testing procedures are used on a single sample, there is a greater chance for contamination to occur.

Personnel. The pollen extraction process is as important as the collection or analysis phase. The key to proper sample collection is to have trained personnel collect samples and then ensure that the samples remain contamination-free until they are processed in a laboratory. The same procedure is essential during the pollen extraction phase. Extractions of pollen from forensic samples should be conducted by a competent forensic palynologist familiar with the problems of working with small amounts of sample material. The palynologist's previous forensic experience, professional reputation, and the reputation of the facility used for laboratory extraction are all important factors to be considered. In court these criteria may become an issue and may be used to discredit pollen evidence.

Laboratory. A forensic pollen laboratory should be contamination free and should be tested at regular intervals to ensure it remains free of ambient pollen and spore contamination. All glassware and other equipment should be thoroughly cleaned and only distilled water should be used for each stage of the extraction procedure. Extraction procedures that ensure the maximum recovery of pollen and the minimal chance of pollen loss should be the only ones attempted. In some cases initial checks should be conducted using staining and wet mounts to determine if any of the pollen and spores in a forensic sample still have an intine or cytoplasm. As mentioned earlier, these criteria are sometimes used to determine fresh from fossil pollen. This type of pre-extraction examination may be useful in determining whether or not a sample may have been contaminated by recent pollen.

Carefully written records should be kept for each sample and precise notations of procedures and wet mount observations for each step of the extraction process should be recorded in a log book. It is important to remember that each step of an extraction process and each observation may need to be justified, explained, and defended in a court of law.

Extraction Procedures. Extraction of pollen from forensic samples requires skill, patience, and experience. There are many pollen extraction procedures known and utilized by palynologists. Some of these procedures have proven useful in forensic work, others have not. To a great degree, extraction procedures must be modified and adapted to the type of material being examined, and to the amount of material in a forensic pollen sample. When there is ample material, normal extraction procedures utilizing standard procedures such as acetolysis, Schulze solution, nitric acid, hydrofluoric acid, hydrochloric acid, heavy density separation, and other techniques work well. However, in most cases very little sample material is available for analysis. Thus, the chances of losing pollen while using standard-sized test tubes are too great a risk and should be avoided. In such cases, all extraction work should be done in very small, micro-sized, glass test tubes (1-5 ml) or in the concave depression of a hanging- drop, glass slide. We have found that we can complete all processing, even acetolysis, in a hanging-drop slide if needed.

When cellophane tape has been used to collect a forensic pollen sample it can be stuck (sticky side up) to a microscope slide using double-sided cellophane tape. The original exposed surface, with the pollen still attached, can then be stained and examined without any further processing. Sometimes, it is better to use a solvent to remove the pollen from the cellophane tape samples and then process the removed material in a micro-sized test tube. Trial and error and previous experience is the best guide to when to use one of these techniques over the other.

Other Types of Small Particle Samples

Often palynologists and other forensic specialists (e.g., hair and fiber experts, soil geologists, botanists, etc.) will work together on sample analyses. For example, a single dirt sample may contain palynomorphs mixed with diatoms, other algal fragments, nannoplankton, radiolaria, plant cuticle and trichome fragments, as well as pieces of hair or fiber. Sometimes the pollen data from a dirt sample, combined with soil data as to clay and composition structure, help to verify the precise geographical origin of a soil sample. If other types of small particles are important to the overall forensic study of a case, then the pollen extraction procedures one uses should not cause the destruction of these other elements.

ANALYSIS AND TESTIMONY

Analysis

One of the primary concerns in forensic studies is the potential for misidentification of pollen and the subsequent misinterpretation of the evidence. There are millions of species of pollen and spore producing plants in the world. No one palynologist can become an authority on all pollen and spore types and rarely does a pollen facility have modern pollen reference collections containing more than a few thousand pollen and spore taxa. Furthermore, it is rare for a palynologist to have a working knowledge of the precise pollen flora or pollen assemblages typically found in more than a few regions of the world. This is why forensic palynologists often present results in terms of "probable" rather than "precise" certainty when discussing some samples.

Testimony

Most pollen results and interpretations are circumstantial rather than precise (Mildenhall, 1990). Its usefulness is based on its ability to associate a suspect, or object, with the scene of a crime and by implication show the suspect, or object, may have been involved in the crime. This is one of the reasons pollen data have not been utilized more widely as evidence in court.

When reporting the results of forensic samples, a palynologist might say the pollen evidence "probably" indicates a specific geographical locale. Under cross examination, however, the palynologist might have to admit that without extensive testing of many control samples, it is impossible to state, beyond doubt, that a specific pollen assemblage is absolutely unique. It is always possible that some other locale or some other region, with similar floral components, might also have a similar pollen assemblage.

Pollen evidence is not like DNA fingerprinting where one might be able to state the probability of two samples being identical is one in a million or more. Although each pollen assemblage is unique in its own way, it is difficult to illustrate this point without using complex mathematical calculations, conducting additional time- consuming analyses of many pollen control samples, and relying on computer- generated programs to show statistical probability. These points should be mentioned and explained in court, but the strength of the pollen evidence should focus on its ability to provide a higher-than-average probability that the results link the pollen sample and suspect to the scene of the crime or link a sample with a specific geographical location.

One hopes to find pollen forensic samples that may contain some type of distinctive "trademark," such as pollen or spores from some unique plant only found in a restricted geographical region. In the forensic case mentioned at the beginning of this article, the presence of an ancient hickory pollen grain, found only in one locale along the Danube River valley, became the "trademark" that made the assignment of a precise geographical location possible.

Liability

One of the unfortunate aspects of working with forensic samples in the United States is the possibility of being sued as a result of presenting testimony in court. In U. S. courts, when pollen forensic studies play an important role in the eventual conviction of a suspect, there exists a potential for an expert witness, in this case a palynologist, to be sued. Such occurrences are rare, but do occur and palynologists involved in forensic studies in the United States should keep this in mind. On the other hand, being sued because of forensic evidence presented as testimony is not an issue in some other countries.

The potential for being sued is less common for palynologists in the United States who conduct their forensic studies for federal, state, or municipal law enforcement agencies than when they work for private consultants or for a private legal firm. Also, when forensic palynologists in the U. S. work for a state or federal agency, such as a state university or the USDA, and are being paid a small fee as an "expert witness," the potential for being sued is reduced. In most cases, even if sued, a forensic palynologist can generally rely on the institution or agency he represents to provide legal assistance in his defense.

We feel all forensic palynologists should seek legal advice on personal liability from their own municipal, state, provincial, or federal court systems. We also recommend them to obtain a clear understanding of their potential liability from the agency for whom they are working. In this way, all parties will understand the degree of liability involved in the release of forensic evidence and will know what to expect as a result of testifying in court.

Sadly, there is one other liability that occasionally occurs when testifying as a forensic palynologist. Occasionally, a convicted defendant may become so enraged that he/she will threaten the forensic palynologist with harm. Not long ago, a forensic palynologist working on a case for a state police force was threatened with death, by the defendant, because the pollen analysis helped convict that person of a crime.

Status Of Forensic Palynology

Forensic palynology appears to be a technique few people know about and a science few seem to utilize. We have searched through the literature published in the United States, we have contacted U.S. pollen specialists, and we have conducted a survey of law enforcement and forensic laboratories throughout the United States. The result of this combined study indicates very little is known in the U.S. about forensic palynology and that very few palynologists have attempted to use the technique.

Except for a brief mention on one episode of the television series Hawaii Five-O, we are not aware of any other mention of pollen being used in the plot of a television program in a forensic capacity. In that single TV episode of Hawaii Five-O, a group of thieves in Hawaii were tracked to their "hideout" by an examination of the pollen trapped in the air filter of a stolen car they abandoned near the scene of a crime. According to the plot of the story, the trapped pollen represented a unique combination of types common to only one geographical area on the Hawaiian island of Oahu; the location of the thieves' "hideout."

As part of our survey, we mailed written questionnaires to police departments and forensic laboratories in each of the 44 largest metropolitan cities in the United States. We also sent them to the Federal Bureau of Investigation and the Office of U.S. Customs. Of the 46 surveys we mailed, 30 completed replies were returned; a response rate of 65%. An analysis of the responses revealed only 6% knew that pollen could be used as a forensic tool and only 3% said they "thought" they remembered a criminal case in which pollen forensic work had been attempted. However, no mention was noted as to whether or not the pollen evidence proved useful.

Our survey of professional palynologists in the United States revealed only two of them had ever conducted forensic work during the past 20 years. In both cases the palynologist noted that the forensic work had been limited to a single study or several specific studies. To our knowledge, aside from these few examples, the Palynology Laboratory at Texas A&M University is the only place where forensic pollen studies are conducted on a fairly regular basis. There are, however, many examples of how forensic pollen studies have been used in felony cases in New Zealand (Mildenhall, 1990), the world's leading country in the use of forensic palynology.

Listed below are a few examples from actual cases where forensic palynology was used successfully to provide vital information in a civil or criminal suit.

United States

Case 1. In the early 1960s a beekeeper in Arizona placed his bee hives in a blooming field of alfalfa. Within a few weeks almost all the bees in each hive were dead. The beekeeper believed his bees had been poisoned by aerial insecticides that drifted into the alfalfa field while nearby cotton fields were sprayed. The beekeeper sued the cotton farmer and crop duster for damages. The defendants filed a counter suit claiming that the bees had not died from aerial spraying, but instead had died from foraging on contaminated nectar and pollen in the sprayed cotton fields. If true, they claimed, it was the bees' fault, not theirs, they died.

Chemical and forensic pollen studies were conducted in an effort to determine which was correct. A forensic palynologist was hired as a consultant to examine a number of dead bees recovered from the alfalfa field. His study revealed the bees were covered with pollen from a number of different nectar plants that grew near the hives as well as cotton pollen. The pollen data, combined with chemical tests, indicated the bees had most probably died from foraging on blooms in the contaminated cotton field. Thus, the death of the bees was determined to be an indirect result, not a direct result, from the aerial spraying.

In a similar case in Pennsylvania, a beekeeper sued because he believed his honey was contaminated with insecticides from a nearby bean field. The beekeeper argued that his bees returned to their hives with the insecticides after foraging in the nearby contaminated bean fields. A forensic palynologist was hired to examine the pollen loads recovered from bees returning to the beekeeper's hives. The results of the forensic pollen studies showed that the beekeeper was incorrect in his assumptions. None of the 122 pollen loads examined contained any bean (Phaseolus sp.) pollen. Instead, they showed that the bees had been foraging on a variety of other nectar-producing flowers in the vicinity of the hives.

Case 2. An agricultural agent in Michigan suspected a local beekeeper of illegally importing bee hives into the state without having them inspected and certified. Using a search warrant, the agricultural agent removed honey samples and pollen loads from each of the suspected hives and sent the samples to a forensic palynologist for examination. The forensic pollen study proved the agricultural agent's suspicions were correct. The examined honey and pollen loads contained a number of pollen types common to both Michigan and the southeastern United States. However, the samples also contained some pollen types from plants that are common components in the deciduous forests of the southeastern United States, but are not known to grow in Michigan.

Case 3. The Office of the United States Inspector General became suspicious that some of the honey purchased by the United States Department of Agriculture under the federal subsidy program did not meet required domestic standards. To qualify for participation in the U. S. honey subsidy program the honey must be produced in the United States. Samples collected from USDA purchased honey were sent to a forensic palynologist for analysis. The pollen assemblages from most of the 75 honey samples examined matched expected pollen frequencies found in a number of different geographical locales within the United States. However, 6% of the examined samples contained tropical pollen types common to major honey-producing regions in Yucatan, Mexico, rather than the United States.

Case 4. A one gram sample of marijuana collected from a drug dealer in Austin, Texas, was submitted for forensic pollen studies. The question was whether the marijuana had been grown locally, or was it imported? If imported, could we identify the source region where the marijuana had been grown?

Our forensic pollen studies revealed some important answers, but we were not able to pinpoint the source area as precisely as we had hoped. Our analysis revealed the sample was dominated by two taxa: pollen from plants in the ragweed group (Ambrosia sp.), and marijuana (Cannabis sativa). Other types present in lesser percentages included several pollen genera in the goosefoot/pigweed families (Chenopodiaceae and Amaranthus sp.), several different types of grass pollen, willow (Salix sp.) pollen, wild buckwheat (Eriogonum sp.) pollen, several types of composite pollen in the sunflower group, a few pine (Pinus sp.) pollen grains of the haploxylon type that compared very favorably with reference examples of pinyon pine, and several pollen grains of honeysuckle (Lonicera sp). The pollen concentration tests showed there was an average of 105,228 pollen grains per gram of material; not an unexpected high amount considering both ragweed and marijuana plants are very prolific pollen producers.

Based on the pollen data from this forensic sample, we could say more about where the sample was not from than where it was probably grown. There was very little arboreal pollen, except for a few pine grains, suggesting the sample was not from a wooded region of North America. An absence of key airborne pollen types of the Myrtaceae plant family (i.e., Eucalyptus, Melaleuca) ruled out areas of coastal and southern California and regions of southern Florida. An absence of common arboreal pollen types from eastern North American trees (i.e., Quercus, Carya, Fagus, Juglans, Ulmus, diploxylon Pinus, Juniperus), ruled out those areas as a probable source. Also, there were no pollen types in the sample that were foreign to the United States; suggesting it did not come from an overseas source, an area of Canada, or a tropical region of Mexico or Central America. Therefore, we suggest the probable east-west limit for the source area was within a region extending from central Texas west to about Las Vegas, Nevada. The probable north-south limit extends from around the Mexican border north into the Great Plains, but not including any forested regions of the Rocky Mountains.

This case study is an excellent example of why we say that it is not always possible to pinpoint geographical regions with a great deal of precision. Had the marijuana sample had a few additional pollen types, or a pollen type with a fairly restricted geographical range, we could have offered a better answer as to the exact source of this sample. Even so, the data derived from this sample did answer the original question asked. The forensic pollen analysis did show that the marijuana probably came from a locally grown source, or a source in the American Southwest, rather than being imported from Mexico, from some other foreign country, or from a locally grown source in the southeastern United States or California.

New Zealand.

Case 1. A large quantity of hashish (Cannabis sp.) resin was found in the possession of a suspect arrested in one of New Zealand's port cities. A chemical test of the various resin samples revealed a composition unlike the hashish generally recovered by law enforcement agents. This raised the question as to whether the new hashish samples reflected a new importation type from a possible new foreign source, or was a new type of hashish being produced somewhere in New Zealand. Three different samples of the confiscated hashish were sent to a New Zealand forensic palynologist for examination.

A study of the three hashish samples revealed a pollen assemblage linking them to a production region in tropical Southeast Asia or Indomalaya. The pollen analysis also proved that all three of the samples came from the same imported "block" of hashish, and that none of it had come from plants grown in New Zealand.

Case 2. A suspect was arrested in a parking lot on suspicion of cultivating and growing marijuana plants. A police dog was able to trace the suspect's route from the parking lot back to a clearing in the nearby forest where a large number of marijuana plants were growing. The suspect denied any knowledge of the plants, was not carrying any illegal drugs, and stated that he had been in the forest looking for a small shrub to plant in his home garden.

At the time of his arrest, the suspect was carrying a small shrub with soil still attached to the roots and a soil testing kit with soil still attached. Both were seized by the police for testing. Soil from the shrub collected by the suspect, soil attached to the soil testing kit, and control samples of surface dirt collected in nearby forests and the forest clearing where the marijuana plants were being grown were all sent to a New Zealand forensic palynologist for tests.

The results revealed that the pollen assemblage found in the dirt from the soil testing kit and the pollen found in control surface samples collected from the clearing where marijuana plants were being grown matched. The pollen found in the soil from the small shrub indicated it had been collected in a different area of the forest.

Although only circumstantial, the pollen results showed that the soil testing kit had been used in the clearing where the marijuana plants were being grown. Thus, it showed the suspect had visited the clearing, even though he denied any knowledge of the clearing when arrested.

Case 3. A suspect on a motorcycle was being chased by police after he committed a crime. The suspect abandoned the motorcycle when it became stuck in mud and made his escape on foot by climbing a muddy hillside. Later, the suspect tried to reclaim the motorcycle at police headquarters saying that it had been stolen from his house on the night the crime was committed. When questioned, the suspect denied he had ever been in the area where the motorcycle was abandoned and said he knew nothing of the nearby muddy hillside where the suspect had escaped on foot.

A police search of the suspect's belongings revealed a pair of muddy boots. The suspect, who worked on a farm, maintained the mud came from the farm and from nearby muddy areas. The muddy boots were sent to a forensic palynologist for examination.

A series of control samples of surface dirt were collected from the place where the motorcycle was abandoned, from the nearby muddy hillside, and from areas on and near the farm where the suspect worked. All control pollen samples were examined and their data were compared with the pollen assemblages from the mud on the suspect's boots. The pollen evidence provided convincing evidence linking the mud on the boots with the muddy hillside where the motorcycle had been abandoned. Further proof came from the pollen assemblages found in the control samples collected on the farm, and nearby muddy areas where the suspect said he worked. Each of those control samples contained a pollen assemblage very different from the pollen assemblage recovered from the boot mud.

Case 4. In New Zealand the thin layer of fur, or "velvet" as it is called, that grows on a deer's antlers is harvested annually from domestic deer. The deer velvet is then bought for resale in drugstores in Asia where it is valued for its suspected "magic" properties. In the early 1980s over $20,000 of deer velvet was stolen from the antlers of domestic deer penned in a New Zealand stockyard. Also stolen, at the same time, were several wool sacks from a nearby woolshed; probably used to hold the stolen deer velvet. Later, a suspect who had a large quantity of deer velvet in his possession was arrested. The suspect maintained that the deer velvet had been taken from the antlers of free-ranging, wild deer he had hunted in forest and scrubland regions of New Zealand (Skinner et al., 1988).

Six control samples of surface dirt collected in, and around, the stockyard pens were sent to a forensic palynologist for examination. Two samples of dirt collected from the wool sacks found in the suspect's possession, and one control sample of dirt from a wool sack stored in the woolshed at the stockyard were also examined. Finally, three samples of dirt adhering to the base of the suspect's freshly cut deer antlers (that were still in the velvet) were also sent for pollen testing.

In court, the pollen data were used to show that pollen recovered from samples in the suspect's possession clearly linked those items with stockyard area. Furthermore, none of the examined dirt samples collected from the cut antlers in the suspect's possession contained a pollen assemblage indicating they came from deer living in a forest or scrubland area of New Zealand (Mildenhall, 1982, 1988).

Summary

The full potential of forensic palynology remains untapped and ignored in most countries. Today, the country of New Zealand leads the world in its use of forensic palynology and in its acceptance of pollen evidence in civil and criminal court cases. Such is not the case in the United States. Based on our survey, we discovered that few municipal, state, or federal agencies in the United States seem to be aware of how pollen data can be used to resolve questions related to legal or criminal matters.

We were not able to determine the current status or use of forensic palynology in countries other than New Zealand or the United States. However, judging from the absence of published literature on the subject in leading journals and books published in most of the European, North American, and South American countries, we suspect the science of forensic palynology is little known and/or little used in these other regions.

Earlier, we outlined the many ways forensic palynology can be used in situations where other types of evidence may be too circumstantial to use in court or other types of evidence may not exist. We also noted that for every successful attempt to use forensic pollen samples, there will be many other circumstances and many other cases where pollen information will prove to be inconclusive or of no value as evidence. Nevertheless, when investigators fail to consider using forensic palynology, they may be overlooking a valuable source of evidence.

There are drawbacks for pollen analysts who may consider examining forensic samples. First, there are few pollen analysts in the world who have forensic experience or who are trained in the techniques of forensic palynology. There are even fewer who might be willing to work on forensic pollen projects. Of primary concern in some countries, such as the United States, are the limits of personal liability if asked to testify in court cases. Also of concern is the time needed to research the background and conduct forensic analyses and how that work might interfere with other types of pollen research the palynologist already has under investigation.

Second, sample collection remains a major problem. Forensic pollen samples collected improperly or contaminated after collection are of minimal value to the forensic palynologist. This is why we recommend that law enforcement agencies seek the advice of a skilled forensic palynologist before as well as after forensic samples are collected. No forensic palynologist wants to waste time examining samples and conducting analyses of materials that later prove to be of little value because of improper collection or contamination.

Third, many palynologists may not be equipped to conduct forensic work. Contamination-free laboratory facilities equipped with special types of equipment are needed and only the best, optical microscopes should be used for conducting analytical work. Also, because there are generally hundreds or thousands of potential plant taxa that could exist in any given environment, each of which could be a potential pollen or spore contributor to the pollen assemblage, precise identification of all taxa in a forensic pollen sample is often time consuming. Unknown pollen and spore types must be checked against extensive reference collections and taxonomic keys. Although most palynologists have access to pollen and spore reference collections, not all palynologists have collections that encompass a broad range of pollen types found in many different regions of the world. Finally, not all palynologists have access to scanning electron microscopes which in some critical circumstances are needed to determine precise identifications.

And fourth, funding for forensic work can sometimes be a major concern. Like any other type of pollen research, funds are needed to pay for expendable supplies, replenish laboratory chemicals, and for the time spent by a forensic laboratory technician and the pollen analyst. Although some forensic palynologists are employed by state, provincial, or federal agencies, and may be able to conduct a few forensic studies without charge, most palynologists cannot. In most cases palynology laboratories are under funded and often rely on a charge for conducting outside research.

Any agency seeking a place to have its forensic pollen work examined also needs to consider potential problems it may encounter. First, it is essential to find an experienced forensic palynologist who is competent and who is respected for the accuracy of his research. Without this, the creditability of that person's analysis or court testimony may be challenged. Second, it is important to find a palynologist who is willing to conduct forensic analyses and who can meet essential deadlines for sample analysis. Third, agencies anticipating the need for collecting forensic pollen samples should enlist the help of a forensic specialist before collecting samples. This will ensure that proper sampling is conducted and that correct procedures for maintaining contamination-free samples are followed. Fourth, funding to pay for forensic pollen work should be anticipated and expected. And five, if court testimony will be needed, the agency requesting the analyses needs to know if the forensic palynologist they hire will be willing to testify in court, and they need to explain what limits of liability the forensic palynologist should expect as a result of his testimony.

Forensic palynology is in its infancy. It remains untried in many regions of the world, is under used in other regions, and is not yet accepted or recognized as being valuable evidence in most court systems. We believe the 1990s will become a "trial" period for forensic palynology; a period when some agencies will become willing to try forensic pollen samples in cases they examine and will test the strength of forensic pollen evidence in the courts. Hopefully, by the end of this century forensic palynology will become another valuable tool in the arsenal of techniques used by most law enforcement agencies.

Acknowledgments

There are many people who have helped us on this article. These include forensic scientists of the U.S. Customs Agency, various policemen and detectives, and forensic specialists in county Medical Examiner's offices who asked important questions about pollen evidence and made us explore new collection and laboratory techniques. We are also indebted to James King, Richard Hevly, and Alfred Traverse for telling us about some of their early forensic studies. We also thank Ray Christopher, David, and Susan Jarzen for their careful reading of the original manuscript and for the many useful suggestions they offered. Finally, Karen Taylor of the Department of Anthropology is acknowledged and thanked for her careful proofing of each version of the manuscript.

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