Forensic Botany: Utilizing Plant Evidence in Crime Scene Analysis

Contents

General Brief Introduction to Botany

Botany, also sometimes called plant science or plant biology, is a subdivision of biology that studies plant anatomy (structure), physiology, biochemical processes, and how environmental factors affect plant growth and development. This fascinating field delves into various plant types, from tiny mosses to towering trees, and examines their evolutionary history and ecological importance. Researchers in botany investigate essential processes such as photosynthesis, respiration, and reproduction, which are critical for sustaining life on Earth. Their work enhances our understanding of plant life and contributes to agriculture, horticulture, and conservation efforts, thus playing a crucial role in addressing global challenges such as food security, climate change, and biodiversity loss. Through the exploration of genetic diversity and plant interactions with other organisms, botany continues to uncover the intricate connections that sustain ecosystems and support human well-being.

Thus, Botany includes mainly three things:

Plant Anatomy: Structures of Plants, including roots, stems, leaves, flowers, and various tissues that contribute to the plant’s growth and development.
Plant Physiology: Functions of Plants, including processes such as photosynthesis, respiration, and nutrient uptake, that play crucial roles in plant growth and development.
Factors affecting plant growth and development: Environmental factors play a crucial role in influencing the growth and development of plants, including light, temperature, water availability, soil composition, and atmospheric conditions, all of which can significantly impact overall plant health and productivity.

The term “botany” likely comes from the Greek words botanikos (related to plants) and botane (plant or herb). The Greek philosopher Aristotle gathered information about plants, but his student Theophrastus [371-286 B.C.] took over his teacher’s library and created more detailed ways to classify plants. He is often called the “Father of Botany.”

Botany Vs Forensic Botany

While Botany refers to the scientific study of plant life, growth, and development, Forensic Botany can be defined as the specialized field that explores plants and various plant parts, along with their derivatives, to understand their significance in forensic contexts. This discipline examines how elements such as leaves, pollen, seeds, and even wood can serve as vital pieces of evidence in criminal investigations. Forensic Botany not only identifies plant species but also analyzes their ecological and geographical characteristics to provide crucial insights into crime scenes. Such analysis can reveal timeframes, locations, and even connections between suspects and victims based on the presence or absence of specific plant materials. Furthermore, the forensic aspects of this field require a comprehensive understanding of the criteria necessary for botanical evidence to be accepted in our judicial system. It is imperative to establish a robust methodology that ensures the reliability and validity of this type of evidence, thereby aiding law enforcement agencies and contributing to the pursuit of justice in legal court matters.

Forensic Botany includes many subdisciplines, for example, Palynology (the study of pollen and spores), Dendrochronology (the study of growth rings of tree stems and roots), Lichenology (the study of lichen communities), Mycology (the analysis and the identification of Fungi), Bryology (the study of Bryophytes). Particularly, bryophytes are useful for forensic investigations because many of them occur in every type of environment, even those unable to host vascular plants and other organisms.

Forensic Botany must be given the same standards as all other pieces of scientific evidence.
The methods and test results used to analyze the given evidence must be approved and accepted by the scientific community.
Botanical evidence is only acceptable in a court of law and useful to a case if it can be clearly established that the relation between the evidence and its area of origin is natural and not coincidental.

Basic Plant Morphology

**Diagram showing plant tissue systems illustration (Designed by Freepik)**

Roots anchor plants in the soil and absorb nutrients and water needed by the rest of the plant.

Stems support the upper part of the plant and act as a transport system for nutrients, water, sugar, and starches. Photosynthesis can occur in the stems of some plants, such as cacti, celery, asparagus, and bananas.

Leaves are the parts of the plant where photosynthesis usually occurs—where food for the plant is made. The green pigment chlorophyll captures light energy and uses it to convert water and carbon dioxide into plant food and oxygen.

Flowers are the reproductive part of plants. They often have showy petals and fragrances to attract pollinators such as birds, bees, and other insects. Most flowers have four main parts: petals, stamen (anther and filament), pistil (stigma, style and ovary), and sepals. After flowers are pollinated and fertilized, they produce seeds in the ovary of the flower.

**Diagram showing flower of mature sporophyte illustration (Designed by Freepik)**

Fruits are the fleshy substances that usually surround seeds. They protect the seeds and attract animals to eat them. This helps in seed dispersal. Seeds contain plant material that can develop into another plant. This plant material is called an embryo. Seeds are covered with a protective seed coat and have one or two cotyledons. Cotyledons are the food for the baby plant until it can make its own food from light and are often the first embryonic leaves of the plant.

Plant Evidence

Before collecting the evidence, we need to look for the kind of plant evidence that can aid in the investigation. The advantage of using plants as evidence is that plant cells are made up of cellulose, a complex carbohydrate that does not decay quickly. Pollen grains and spores have walls made up of sporopollenin, which can resist decaying materials.

Example of botanical evidence: –

Roots
Seeds
Fruits
Leaves
Pollen
Lichens
Bryophytes
Algae (especially diatoms)

Plants have unique bodily components and structural makeup, including ecological requirements particular to specific species, which provides valuable forensic evidence.

Searching for Plant Evidence

Crime scene investigation is based upon the theory of Locard’s Principle that “every contact leaves a trace”. In any kind of crime scene investigation, it is necessary to recognize and identify evidence and how they can establish a linkage between the scene, individual, suspect and victim. Crime scene investigation can be further considered as the first and most critical step of any forensic investigation. If evidence is not recognized or is improperly collected or packaged, it may be inadmissible in court.

Three crucial steps must be adhered to at a crime scene for all physical evidence, including botanical samples: recognition of the evidence, appropriate collection methods, and adequate preservation of the evidence for later testing and use at trial.

Only after recognition and documentation should an immediate attempt be made to protect the evidence/samples from further degradation and contamination. Proper packaging of botanical samples should be done only after drying to prevent further bacterial growth and excess moisture.

Physical Evidence

Finally, this evidence should be stored in such a manner that a proper chain of custody is maintained. Common search places for Botanical evidence include:

a. Suspect’s clothing, including pockets, cuffs or textiles/clothes.

b. Suspect’s shoes, shoelaces, within the soles of shoe or any footwear.

c. Suspect’s or deceased victim’s hairs.

d. Body orifice of a deceased or live victim.

e. Internal and external parts of vehicles used for committing the crime, mainly undercarriage, chassis, wheel wells, trenches on and around the forward-facing bumper and heater.

f. Annual rings of plants rising over, thoroughly, or near a body, skeletal leftovers, a graveyard, or an item at a crime scene, the rings can be measured and given a time period.

Collection and Packaging of Botanical Evidence

Botanical evidence can be found indoors, outdoors or even in underwater poisoning cases and even in illegal drug trafficking cases too! Thus, their collection method varies according to the type of crime scene in which they are found. The precise results depend on the primary stage of crime scene examination, which is the correct collection of evidence and its appropriate conservation. Since inappropriate collection techniques and carelessness result in the contamination of the evidence.

The table below shows essential equipment for the collection and packaging of Botanical evidence:

Equipment	Application
Digital SLR camera	Photographic documentation of sample plants on the scene of a crime
Hand pruners, knives, or scissors	Cutting and sampling of leaf and twigs from woody trees or shrubs
Garden spade or archaeological trowel	A sampling of a whole herbaceous plant with its roots
Collecting bags, paper grocery bags, or cardboard boxes	Keeping all the above-listed evidences
Wax-coated paper	Collection of fleshy and sticky fruit samples and other wet botanical evidence to avoid its adherence with paper while drying
Bound notebook, graphite pencils, or indelible ink pens	Note-making
Plant presses	Collection and temporary storage of plant samples
Tamper-evident tape	Sealing of the collection bags
Over-the-shoulder (field) bag	Keeping all the above-listed equipment together

Reference – Manual of Crime Scene Investigation, Chapter – Collection of botanical evidence. Pg no – 123

Important Points

Vacuuming (if required) should be done first to collect minute traces of botanical evidence that all the plant fragments have been collected. In other words, vacuuming should be done only if all the other evidence has been collected.

For a collection of tree rings, first, it needs to be photographed, and if it is too large to be collected, then the trunk should be cut off a few inches below and above any damage. This cut section should be collected in a cardboard box. Also, any sample leaves, fruits, or flowers of the same tree should be collected to aid in the identification of the tree.

Plant evidence should be stored in a cool, dry area. Since plants are living organisms, they need to be killed and dried for best preservation. Drying will kill the plant and stop its biological processes. Drying is best if the plants are kept flat with cardboard or blotter paper on both sides and a weight placed on top. The box should have a few mothballs or a no-pest strip included to repel any insects that may eat dried plants.

Plant evidence collection during Autopsy

Plant matter in stomach contents would most likely be collected during autopsy by the medical examiner. A medical examiner typically scoops out the plant matter found inside a dissected stomach at autopsy. A standard preservative of 10% formalin is used to denature enzymes and kill bacteria. (Heather Miller, Chapter 9, page 141) However, this formalin-treated sample cannot be used for DNA typing as formalin may inhibit the PCR amplification process.

Plant evidence collection from faecal matter and vomit

Digestive tract contents of the human body, such as stomach wash, intestinal content, vomitus, and faecal matter, are collected for detection of the presence of ingested plant materials, drugs, poisons, pollen/spores, diatoms, etc. Fresh or sometimes dried vomitus or faecal matter can be obtained directly from the crime scene or during post-mortem examination. Seeds found in the excreta matter and from the vomit matter should be collected by sterile forceps and examined under a light microscope.

Documentation of botanical evidence

For plant material to be valuable as evidence, it is critical to record several types of information as a crime scene is processed. Documentation of transient or transfer types of evidence is very important since they may be subject to alteration over time. Most plant evidence, with the exception of pollen, may deteriorate, dry, mould, or be otherwise altered from the form in which it was first discovered. In addition to general environmental conditions, other relevant information about scene conditions should also be recorded.

Descriptions of the types of information that should be documented, preferably on a botany field data sheet, are as follows:

Height of plant
Habitat – From where the evidence or sample was found in its particular habitat. Is the site forested, shrubby, an open field, a roadside ditch, or a home lawn? Is it a landscape planting, an industrial site, etc.?
Evidence location – Where was this evidence collected at the scene: on the body or vehicle or elsewhere? Where on the body or vehicle was it found?
Scene Location
Time of collection: Note the time when each sample was collected.
Date: The date of collection must be included in the log
Other details like fruit colour, flower colour, fruit shape.

At different points in a crime scene	Type of Evidence that can be found
Entry point	Broken branches, compressed grass or underbrush, wood chips left on a carpet that matches those at the point of entry, paints, body fluids or other stains, prints, clothing, and personal effects.
The path travelled by the perpetrator	Path travelled by the perpetrator
Evidence on or in the victim	Leaves, pollen, algae, plant/clothing fibres, marijuana or other drugs, tobacco or other cigarette components (e.g., paper, wooden matches), food stains, or stomach contents. Be aware that the surface of the body, all body orifices, under the body, and in the hands or under the fingernails should be searched for evidence.
Point of exit	May include all of the items mentioned for point of entry, as well as latent prints, bloodstains, soil, dust, and glass, which may be useful as physical evidence

Application of Forensic Botany to various criminal cases along with case studies

(i) Botanical evidence solved death investigation, whether it is homicidal or suicidal in Taiwan

Case Study: – The body of a young woman was found lying in a gutter in an urban area of Taiwan. On further investigation, the same woman was visible on a surveillance system tape, but after a truck passed by her, she was no longer visible in the range of view of the surveillance tape. The investigators thought it was a case of hit and run case, and the perpetrators hid her body in the gutter to hide the accident. Upon investigating her body, some plant material of berries and stems was found, which belonged to a plant mainly of the Solanaceae genus. The investigators, upon thorough investigation, found some potted plants on the edge of a railing above the gutter. Those potted plants were Solanum nigrum and showed a remarking resemblance with that of botanical evidence discovered from the victim’s hair.

Based on climate and location, wind velocity would not be that strong enough to break the stems. Also, the potted plants were kept at a height of 3.5 metres, so a normal person who is walking down the streets could not reach such a height. Thus, the investigators made a probable scenario that the female jumped from the top of the building and made strong contact with the branches and roots, which were transferred to her hair and clothing. Later on, the relatives of the deceased victim told the police that the woman was suffering from depression and had attempted suicide earlier, too.

(ii) Bryophytes aid in solving death investigation

Bryophytes occur in every environment, and their fragments can easily remain attached to shoes and other clothing, which makes them useful in forensic investigations. Bryophytes include Liverworts, Hornworts, and mosses. They are also small, and their fragments suitably remain in the soil, dirt, and debris. Therefore, they easily attach to shoes, clothes, or vehicles.

Case study – S.B., a Caucasian girl who was 22 years old and she was a student of the Faculty of Medicine at University of Siena, Tuscany, Italy. The girl was under drug therapy because she was diagnosed with depression. During her interviews, she denied committing suicidal intentions. After a few months of therapy, the girl committed suicide by jumping from the terrace of a shopping mall in the centre of Siena. The crime scene was the terrace of a four-storey building housing a shopping centre, the bus station of the city and the University for Foreigners of the University of Siena. Investigations revealed that the terrace was not inaccessible to normal users of the building, and service stairs were reserved only for the purpose of maintenance of the building. Later on, Bryologists found a few samples of mosses from the service stairs and examined those mosses with those found from the shoes of the victim. Two species of mosses were found, namely Tortula muralis and Bryum capillare. The presence of removed bryophytes on the terrace and the presence of fresh bryophytes on the stairs allowed us to reconstruct the accident: most likely, the girl had walked up to the service stairs alone, dirtying her shoes of mosses, she climbed the terrace and jumped down.

(iii) Diatoms and Algae in Solving Forensic Cases

(Reference – https://journals.sbmu.ac.ir/ijmtfm/article/view/14047)

Limnology is the study of freshwater bodies. It is particularly concerned with the presence of diatoms in crime scene tests and casualties. Diatoms are specially used in drowning cases. Diatoms are unicellular and photosynthesizing algae with a siliceous skeleton found in fresh and marine waters and soils, including major moist environments. Diatoms are classified based on their shape, colour, and pattern.

This diatom test is a very important examination to determine whether the drowning was antemortem or post-mortem in nature. Antemortem drowning occurs when an individual is still alive at the time of submersion and drowns due to the inability to breathe underwater. Post-mortem drowning happens when the person is already deceased before entering the water. At the time of recovery of the body, the following control samples should be collected from water bodies: –

A sample of drowning water was collected from the scene.
Sample collection using a spatula or short surface corner and samples over stones.

Diatoms can be extracted from water and body tissues and organs through acid digestion. They are found in almost all water bodies. The basic principle of the diatom test in drowning is based on the inference that diatoms are present in the medium where drowning took place. The inhaled water enters the alveolar spaces of the lungs and penetrates from the alveoli into the blood circulation; thus, diatoms get deposited in internal body organs like the brain, kidney, sternum, femur, and other organs.

Case Study – There was a drowning case with a total of 17 victims, out of which there were 15 males and 2 females. This drowning case test was conducted in the Serology Division of State Forensic Science Laboratory Shimla Hills, Junga, Himachal Pradesh, India, and all the cases reported here are of freshwater drowning except one drowning in a pool. A water sample from the putative site of drowning as well as hard bones (sternum, clavicle, femur) and soft tissue (liver, spleen, kidney) after the post-mortem of the dead body was sent as a crime exhibit to the Laboratory for detection of diatoms. 12 cases revealed the presence of diatoms (death due to drowning), while 5 were found to be negative (death other than drowning).

(iv) Palynological samples and their significance in forensic investigations

Palynology, the study of microscopic and not visually obvious plant entities such as plant spores, fungal spores, pollen, and other microscopic entities, is the most vulnerable source of botanical evidence for any type of crime. There are important attributes that make pollen valuable forensic evidence.

They are microscopic in size.
Pollen and spores are produced in vast numbers.
Pollen and spores can be identified as a plant taxon.
They are highly resistant to decay.
Various pollen mechanism dispersal.

Pollens are trapped and found in soil, dust, leaves, moss, and other plant fragments. Some cloth fabrics, particularly wool, are capable of sticking with plant fragments, including spores and pollens, and picking them up. Thus, the presence of palynological evidence can also distinguish between a primary crime scene and a secondary crime scene.

Pollen dust obtained from a suspect could indicate whether a suspect has visited a particular crime scene or not. The suspect doesn’t need to commit any wrongdoing.

Interpreting pollen assemblage – The association of pollen types and their relative frequency in an assemblage may be unique for a given location, a region, a larger geographical area, or even a country. These pollen assemblages provide what is often called a “pollen print” for a given location. That pollen print then becomes the “control sample” for that location against which other samples, or the pollen from forensic samples, may be compared. The total number of pollen and spores identified in a sample is called a “pollen assemblage,” or palynomorph assemblage.

A forensic palynologist follows Locard’s exchange principle, which states that “whenever two objects come in contact, there is always a transfer of material” that will give a pollen fingerprint. Therefore, the first task of the forensic palynologist is to try to find a match between the pollen in a forensic sample and that from a known geographical region. Palynologists should have knowledge of pollen dispersal, developmental responses, and phenology, which influences the solving of case problems.

Case Study

(Reference – https://biodiversity.utexas.edu/news/entry/pollen)

The first known case of using pollen to aid with forensics investigations was from a crime that occurred over 60 years ago in Austria. A man went missing near Vienna, but the police could not locate his body. Their main suspect in the case would not confess to killing the missing man, so the police decided to bring in a palynologist to examine the mud on the suspect’s boots. The palynologist found that the pollen of the suspect’s boots contained spruce, willow, and alder pollen, along with 20-million-year-old fossil hickory pollen. This hickory pollen could have only come from one specific area, which contained exposed sediment from the Miocene age. (Narrowing down suspect). This allowed investigators to home in on a location 20km north of Vienna. When the police brought this evidence to the suspect, he ultimately confessed and led the authorities to the missing man’s body, which was found in the same area that the palynologist identified through the pollen.

Since this first case, pollen has been used in homicide, missing persons, narcotic, and terrorism cases throughout many countries, including the United States.

(vi) Botanical DNA samples analysis in Forensic investigations

( Reference – https://link.springer.com/referenceworkentry/10.1007/978-981-16-4318-7_59#:~:text=Plant%20identification%20can%20aid%20in,as%20determining%20cause%20of%20death.)

When a sample cannot be positively identified based on morphological or anatomical analyses because diagnostic characteristics are absent or only small fragments are found, it may be possible to identify a species using DNA sequences. DNA sequencing of the same segment from different samples allows samples to be compared for similarities and differences. Taxonomic identification of a particular species or higher taxonomic level is a crucial first step in analyzing botanical evidence to aid in forensic investigations.

Plant DNA-based forensics facilitates the identification of a population of origin, individualization of plants, geographic origin, testing of narcotic plants, patented cultivars, poisoning plants, tracking of genetically modified plants, and investigating the origin of drug trafficking using random/anonymous markers or microsatellites of DNA.

Seeds and fruits, and even roots, can provide good quality DNA for successful genetic analyses, and thus, they are collected depending on their quality and availability on the crime scene. The tendency of degradation of DNA over time, the success rates of these samples may be quite low. Therefore, dead or dried leaves are avoided during collection if the plant is dead.

Case Study – Cynodon dactylon DNA connects the suspect with the crime scene.

[Reference – Handbook of DNA profiling page number – 875]

An elderly woman was found strangled and dead in her bedroom, and police found a shoeprint next to the corpse. A suspect was interrogated, and his shoes were recovered. Upon finding a match with the shoe prints, the suspect was taken into custody.
A fresh plant fragment was discovered associated with one of the shoes. The victim lived in a neighbourhood surrounded by many different types of vegetation, such as trees, ferns, lawns, wild grass, and various medical herbs. DNA was isolated from the fragment and sequenced to determine the species of the botanical evidence.
Using ITS sequencing primers, The plant fragment was identified as a type of grass, Cynodon dactylon, commonly known as a decorative garden lawn. Based on this information, the area surrounding the crime scene was searched again, and the same type of grass was found only on the neighbour’s property.
It was concluded that the attacker ran away after committing the crime, crossing the neighbour’s garden, where the grass got stuck with the shoe.

Summary

Forensic Botany is the application of solving crime cases using botanical evidence.
Classification of unidentified plant species and identification of botanical products that can aid in investigation.
Identification of botanical evidence in trace amounts and discovery of the geographical origin of the plants in question are necessary to solve serious crimes such as kidnapping and murder.
Forensic significance of botanica evidence.
determine circumstances and cause of death.
estimate time frames.
Prove or disprove an alibi.
determine whether there were multiple crime scenes.
establish when death could have taken place.

Various branches of botany are mentioned below: