Plants and fungi forensics - plants as forensic indicators (week 8, lesson 1/1)

The use of plants and fungi in criminal investigations

They can be evidence

They can be the perpetrator by poison

They can set a time and place

Whole plants and fungi, pollen, algae

Areas of plants that can be used in forensics

There's different areas within the plants and fungi to be looked at which require specialists like anatomy, physiology, ecology, taxonomy (classify to species level) and geography (where/what environment growing in)

Kingdoms plantae and fungi

Plants and Fungi are useful in

forensics because:

• Abundant-400000 + species

• Very diverse, grow in different habitats

• Environmental distribution

> certain types of soils, habitats, grow at certain times - all help to pin point

• Species specific feature

• Dispersal adaptations

> change throughout year, if have spores/fresh leaf on clothing it's likely it happened in spring

Can pin point time and environment as forensic indicators with these factors about plants

What are plants and fungi - how their structures & contents act as forensic indicators

Eukaryotic and (mostly) multicellular but have major structural, cellular, physiological and ecological differences.

- Have lots of abundant DNA and varying types from different organelles that can be used

- Roots can grow but stay under soil, where lots of crimes happen in or around. Can use roots to determine season when crime occurred

- Have times of year reproductive structures appear at certain time of year so finding a body can pin point down to months or even weeks

- Many not all fungi produce fruiting bodies - again very time specific and habitat specific

- Cell wall keeps cell and DNA more in tact than animal cell so better preserves it for forensic use

Historical Forensic Botany in legal cases

Been used long time in criminal cases, started 1932 in America - small child 20 months old kidnapped & murdered, left ransom note but no fingerprints. Couldn't pin to number one suspect without fingerprint but found evidence at his house that was similar paper and pens to ransom note. Forensic botanist called in concluding the ladder at crime scene was made from his attic.

Goes much further back to being used as lie detectors. Trial by ordeal used in Middle Ages to detect murder, conspiracy & theft. Cerbera tanghin was a poison that disrupts heartbeat and way in which you fell was the determiner. Calabar bean physostigma venenosum used and if vomitted then innocent but digested guilty.

Plants as forensic indicators

Very sensitive - change with altitude, seasons.... Plants are ubiquitous in

the environment and

physiologically sensitive

to environmental

change helping to

pinpoint time as well as

location of an event

Plant anatomy - study or single cells and tissues e.g. stomach contents

Plant taxonomy - species identification e.g. trace evidence

Plant ecology - relationship between plants and environment e.g. clandestine graves

Generalised plant life cycles

Plants growth is seasonal so structures such as leaves, and reproductive features such as

fruit and seeds can help to pinpoint time as well as location.

Some years may get fruit some may not so helps pin point. Seeds evolved to be dispersed (animals, wind, us, AND perpetrators) and dehydrated to ~5% water content so survive very well so can be found on clothing and shoes. Bryophytes and ferns produce (smaller than seeds so no one notices) but very hard to see so hard to collect as evidence.

Fungi as forensic indicators

Spatial and temporal evidence - will be in certain place and can be quite specific to where they grow, grow at certain times a year, also are decomposed they break things down back to carbon at very specific rate, we eat them so used for stomach contents analysis but also poisonous (e.g. death cap mushroom), mushrooms have also been used as murder weapon to poison. Can grow on buildings and in houses, as decomposers can be used as PMI indicators. Location of bodies and buried corpses as criminal will surface seeds but fungi break down bodies so different nutrients come to surface and change soil composition. Can grow in woodlands, open fields so get idea of where crime occurred.

Generalised fungal lifecycle

More simple than plant but still useful for temporal and spatial evidence. 80% plants are in fungi symbiosis with mycelium. Spores are tiny for fungi (smaller than pollen, perpetrator would never be able to remove all of them) so go for pin pointing time and place.

The role of fungi in the environment-

heterotrophs and decomposers

Part of the necrobiome (postmortem community that includes bacteria, fungi, arthropods, nematodes, other cadaver-associated organisms). Can help establish place and time of death. Some fungi break down corpses more than others so gives idea of species specific role. Take specific amounts of time to decay things so gives time estimation for death.

Which parts of a plant for fungi are used as evidence

Wood, paper, different timbers make part of evidence. Parts of plants that make products as well as plants themselves.

Legal applications

Very rare to have plants as main piece of evidence in case. Evidence must be collected very carefully as they're everywhere and can be contaminated so must keep evidence chain in tact. Evidence must be collected on behalf of people who might be collecting evidence later in different discipline. Need to know what's growing around so through photographs. Type of soil is important. Other characteristics like colour, smell, are plants wilting. To collect evidence must be done at crime scene soon as possible (soil, water, evidence from bodies) as can transfer at later date and be classed as evidence. Collect control background samples. Things packed properly as plants can still breakdown. Things described & labelled well. Water and soil (surface & beneath) samples taken from different surfaces.

All this helps to reconstruct crime scene.

Wildlife crime, crimes against animals, murder, agriculture crime

Whole plants: presence

Plants are highly trafficked more than animals. Cycads, succulents, orchids, illegal logging (usually hard wood like timber and teak) Very few people know what rare plants look like so hard to know when they've been trafficked. Anti-poaching people in areas to help detect. It's a multi-million dollar industry. Illegal trade in contraband too like cannabis farms, drug shipments, often associated with organised crime. Of 37,000 species on critically endangered list, 30,000 are plants.

Whole plants: damaged

Two ways they can be helpful. If there's a struggle or somethings happened then plants will break so can often see site of struggle or entry/exit route of crime scene. Generate suspicion. Criminal damage (driving through fields to escape or on purpose to damage). Valuable crops are guarded. Seed stocks are very valuable.

Accidental or suspicious death

Elderly man dead at foot of steep hillside, PM determined death not caused by fall and didn't have significant blunt force trauma but had heart disease and hypothermia. At scene a survey was taking place, he was clutching leaves when found and found on his clothes and these where the same at scene. Looked at point he lost balance, point of fall and point of impact. Concluded it was an accidental death, he had cardiac event that caused him to fall. He died a few days later from hypothermia. Coroner ruled accidental death

Whole plants: growth

Changes in colour, density and phenology can be observed over time. Change in species assemblage and growth orientation caused by root destruction & soil disturbance. If dig a grave plants will be overturned but then new plants will grow and habitat will change allowing us to place time.

Deuteromycetes are first colonisers of microbiome, next type of fungi ascomycetes break down nutrients broken down from body in first place, basidiomycetes break down bones. Release carbon to soil above which is why plant community changes above. This helps with dating things. Helps if find grave after few years but not thousands of years.

Light and plants

Reflect light back depending on how much they're photosynthesising which depends on how much nitrogen they have which depends on how much is in soil. If body in ground then have lots of nitrogen released. Plants will be very dark green so amount of red and far red light to emit will be specific. Can use this to pin point location of graves.

Example: clandestine graves

Begonia species and pig limbs placed underneath. After so many months, amount of growth with pig limbs have dense amount of growth as plants essentially been fertilised. Depends on humidity, soil pH and oxygenation. Gives indication of something that may have been buried under site. Only evidence at recent time rather than later.

Example: Clandestine graves in forest canopies

Cases where mass graves have been found, forests can be enormous however so can be hard to find. Drones can be used. Changes in nutrients to trees like amount of nitrogen. Can calibrate drone with hyperspectral imaging to detect signatures and fluorescence spectra have the potential to provide valuable information to detect foliar anomalies that are influenced by human decomposition. So then don't have to use soil. Will get huge change in case of clandestine graves.

Example: Establishing link between victim and crime scene

Feb 2011 partially skeletonized remains of a girl that had disappeared in the previous November was discovered in a field in a in northern Italy. Worked out age and gender and mapped vegetation in surrounding area, only a few metres around where Skelton found, nothing had grown under Skelton, so likely to be scene of crime. Because farmland could work out species and how long had been growing for. Looking at plat distribution knew it was primary crime scene and calculated time since deposition. Looked at missing people reports. This evidence useful in short term so able to pin point time as only feb-nov. Managed to identify victim.

Whole plants: Bryophytes

Moss, Liverwort & hornwort, produce spores. Very dehydration tolerant, very green in winter but brown in summer. Can get place and time. Very little and unnoticed.

Example: suicide or murder?

Young woman in Italy found lying in ground had fallen from three stories above. Was roof of shopping centre but no access to public so no one should've been up there, had to decide fallen or pushed. No CCTV footage of that area. Took moss from bottom of her shoes and from all along likely path. Staff had access. Had to go on carpet first, along walkway then railings and compared to moss on her shoes. Able to trace only one person had gone. They found moss right on railings. If pushed she would've fell over railings, so this was ruled as a suicide.

Plant roots

80% are in symbiosis with fungi. Bidirectional flow of nutrients. Water can go up and down and around plants. Can extract DNA and determine species, work out local climatic conditions. Can tell this from structure and condition of roots. Root hairs grow further and further, main roots have rings on them, can grow through and around bodies

Example: Estimating PMI

Fully clothed skeletal remains found. Roots growing through remains so been there some time. Moss growing on remains too. Grow 2 different ways, first way grow for around a year like Xmas tree then grow out, have very defined growth pattern basically so can determine how long moss has been on bones which is good indication of PMI. Able to place time of death between roots and moss, able to use DNA records to confirm who victims was.

Fungal spores

Fungal species occupy restricted ecological niches, limited geographic despite the many species and spatial distributions. Spores residual for extended period (temporal restrictions to when release spores). Indicate which plants are growing and which plants were growing, tells what environment and geography is.

Example: Wildlife crime

Illegal to disturb badger setts. Crime where one disturbed and looked at if soil was same from badger sett as shovel from suspect.

White veined truffle fungus Choiromyces melandriformis, ascospores with distinctive peg-like ornamentation, rare in UK. Mutualistic associations with roots of deciduous trees, e.g. Fagus

sylvatica or Quercus robur, oak trees

Badgers like truffles and both soil from badger sett soil and shovel covered in spores from this fungus. Badgers had foraged for the truffles and taken back to their sett. RSPCA had first conviction.

Plant Fragments: Bark

Tree bark contains lignin, resistant and

persistent in environment. Produce tough outer layer. Different types of barks on different trees. 2010 20yo man body found in woodland bed been shot in head and chest. Had eye witness said man had been handcuffed to tree before tree then cuffs taken away. This was a mafia informer so police didn't know if could trust evidence. Found lots of splinters on victim from same tree, allowing confirmation of eye witness statement.

Plant Fragments: Seeds and fruit

Seeds and fruit are ubiquitous in the environment, have specific dispersal mechanisms and patterns and seasonal

variation in distribution. Large size compared to spores and pollen. Can fall near and far from parent plant. Don't decay and can extract DNA from them. May be noticed by perpetrator as larger and removed. Not available at all times all times of year.

Transfer of Evidence

Seeds can be revo

Example: Linking to perpetrator

1995 Texas, sleeping toddler abducted and

sexually assaulted. Evidence from fingerprints, DNA and plant material from crime scene and suspects shoes. Found on shoes of perpetrator had seeds from particular plant and in room where toddler sleeping. Had taxonomist on case who could identify species but don't always have this specialist available.

Seeds and Fruit: Diet

Digestive processes

• Stomach retains ingested foods

> Enzymatic digestion of proteins

> Maceration of foods via churning

• Digestion in small intestine

• Gastric transit subject to numerous variables

> Diet, health, lifestyle choice, snacking/ grazing

Plant materials are part of our diet but we are unable to digest plant cellulose. It passes through our intestines before being expelled as waste. Can often tell what someone's last meal was, when and where.

Stomach contents analysis - when?

• If undigested stomach contents are present, then death occurred 0 to 2 hours after last meal

• If the stomach is empty but food is found in the small intestine, then death occurred at least 4-6 hours after last meal

• If the small intestines is empty and wastes are found in large intestine, then death probably occurred 12 or more hours after last meal

> very good to determine when last eaten or placing them at particular food place

• Death= pyloric sphincter muscle closes down

• Degradation & position of food = rough estimate of time since last meal

• Focuses enquiries more directly

• Individual variability in intestine

Example: Stomach contents pinpointing time of death

Man had hash browns, eggs and toast. Found dead home next day. He was his wife's 6th husband. He was taking her to court over money. Was hit over head with spade, shot three times & burned with stun gun. Found where he'd eaten the hash browns didn't have onions in them but were cooked on same griddle. Determined that breakfast was his last meal and his ex wife and new husband no longer had alibi and was convicted.

Plant and fungal identification and

analysis: anatomy and physiology

Someone needs to be analyse these samples, must be specialised, have databases, ID keys, electron microscope, databases as plant material can be difficult to identify or be degraded. Can do this forensic botany in a royal botanic gardens in Kew.

Example: black mould

Toddler died from severe respiratory condition found to be caused by black mould. Evidence was collected from bathrooms and rooms in house. House completely infested with mould, had to analyse mould in house and that lodged in toddlers lung and found to be a match, coroner ruled respiratory arrest was due to long exposure to black mould in house.

Stable isotope analysis

• Compares ratios of common elements within samples to indicate origin (C, H, O, N)

• Absorbed & incorporated into molecular

structure of tissues

• Signatures vary spatially & temporally - identify origin

• Used frequently in forensics − Analysis of explosives, food, wildlife, drugs, persons, plants

Example: Timber testing

Illegal logging very profitable worth billions and highly involved with organised crime. Some species shouldn't be logged at all, evidence can come from wood or wood products. Can see if your mahogany wood table came from a reputable source or was unethically sourced from a forest.

Phytochemistry: Toxins and poisons

Plants and fungi contain large amount of secondary metabolites that are defence against predation. Long history of these plant photochemical used in murder, suicide and for illicit drug use. We have many receptors so can interact with a lot of these in own bodies.

Photochemical analysis

Range of

molecular and

biochemical

techniques are

routinely used

Lots of different ways to analyse these techniques

Example: Medicinal plants and narcotics

Medicinal plants can be exploited as a

treatment specific dosage and as deadly

poisonous substances to kill in higher dose. Dose is important.

Example: Determining cause of death

41 yo man dead after drinking homemade liquor, didn't find anything so had to do specialist analysis of what was in his blood, had large amount of Gelsemium elegans in him which are poisonous but found lots Lonicera japonica around him too which is edible and looks very similar so he likely mistakened the plants.

Australian 3 murder

• Erin Patterson charged with 3 counts of murder and 5 counts of attempted murder.

• Claimed to have bought dried mushrooms

purchased at an Asian grocery months earlier.

• A food dehydrator was recovered by police from found a local tip, found evidence of spores and mushrooms on it

DNA analysis for plant and fungal identification

Chloroplast DNA in plants, ___ DNA in fungi

Metabarcoding

While barcoding focuses on a species, metabarcoding focuses on a whole community. Used for mixed and environmental samples however reference

DNA must be available.

Mitochondrial gene CO1 preferred in animals. Mitochondrial genome evolves slowly in plants, chloroplast and nuclear genomes are preferred. 2009 standardised as 2 region combination rbcl recoverability, matK resolution.

Allows to look at lots of species at once, as look at DNA altogether

Metabarcoding pipeline

1. Sample collection

2. DNA extraction

3. Library preparation and PCR amplification of barcoding locus

4. Sequencing of DNA barcodes

5. Data analysis

6. Phylogenetic tree

7. Species identification

8. DNA barcodes library

> Different barcoding search engines online can use to compare with

Example: Metabarcoding and plant poisons

Plant species contain different classes of

poisons and poisoning either accidental

or intentional often required emergency

use of specific antidotes. Difficult to

visually identify plant from vomit, faecal

and stomach contents analysis.

Not routinely used but promising for future. Can be used for plants, fungi and animals.

Example: Metabarcoding and fungal

decomposition, indicator species.

4 pig carcasses and soil collected at different PM intervals. Interspatisl repeat marker used to detect fungi. Looked at different species of fungi growing and living around pig at different intervals. Mapped pig necrobiome over time so could tell spatially what fungi would grow in what area. And what fungi would grow and the community change over time. May be able to determine PMI in future if this is repeated over time by growing up knowledge on necrobiome.

Summary

• Presence of plants and fungi + relationship to activity should be taken into account.

• Valuable circumstantial information to direct forensic enquiry:

-Cause/time since death

-Trace evidence to associate people and place

• Traditional ID approaches developed further through genetic and chemical analysis

• Requires a diverse and open approach- anything can

be evidence

• Plants are circumstantial indicators - Important not

to overstate value

• Research + casework provides data and precedent to

support interpretation