Forensic Mycology: Fungi at the Service of Investigation

What is Forensic Mycology?

Forensic mycology is a scientific discipline that combines knowledge of mycology, legal medicine, and forensic sciences to use fungi as evidence in criminal, legal, and archaeological investigations. This interdisciplinary approach provides invaluable tools to clarify complex situations where other techniques may be insufficient.

It is a specialized forensic branch focused on the analysis of fungi - colonies, spores, growth patterns, and biochemical characteristics - to extract relevant information in legal contexts. This discipline allows establishing connections between people, objects, and places, as well as estimating intervals post-mortem or determining environmental conditions in death cases.

Although less known than other areas such as genetics or ballistics, its usefulness has been demonstrated in numerous international cases.

In this article, we explore in depth how fungi can become hidden protagonists of the scene revealing crucial clues. Throughout the text, we present real cases where forensic mycology has been decisive.

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Applications of Mycology in Criminal Investigation

Determination of Post-Mortem Interval (PMI)

One of the most important uses of forensic mycology is estimating the time elapsed since death or post-mortem interval (PMI). Fungal colonization patterns on a cadaver follow predictable sequences that are invaluable to forensic investigators. When a body begins its decomposition process, different species of fungi colonize it at specific times, creating a biological sequence that functions as a natural temporal marker.

Various studies have established correlations between the time elapsed since death and the presence of certain fungal species:

• First phase (0-7 days): Predominance of fast-growing fungi such as Mucor spp., Rhizopus spp., Penicillium spp. and Aspergillus spp.
• Second phase (7-30 days): Appearance of Fusarium spp., Alternaria spp. and certain Ascomycetes
• Third phase (1-3 months): Development of Coprinopsis spp., Coprinus spp. and other Basidiomycetes
• Advanced phase (>3 months): Presence of fungi specialized in the degradation of keratin and collagen such as Onygena spp. and Doratomyces spp.

Forensic pathologists regularly identify various fungal colonies on cadavers in different states of decomposition. Detailed analysis of these colonies, their composition, and state of development allows establishing chronologies of death with remarkable precision.

Another fascinating aspect of this application involves the study of keratinolytic hyphae. Specialized fungi of genera such as Chrysosporium, Microsporum, and Acremonium possess the ability to degrade keratin present in human hair, forming characteristic microscopic channels. The pattern, extension, and depth of these channels provide detailed information about the antiquity of the death, allowing investigators to determine with remarkable precision when death occurred.

Trace Evidence

Fungi constitute a valuable source of trace evidence in forensic investigations thanks to their geographic and seasonal specificity. The spores and fungal communities present on clothing, footwear, or vehicles function as biological fingerprints that can connect a suspect to the crime scene. When transiting through different areas, people inadvertently collect microorganisms characteristic of those environments.

Fungal spores possess a remarkable adhesion capacity to various surfaces such as textiles, leather, and plastics, remaining fixed for prolonged periods without significant alterations. The particular distribution of fungal species allows investigators to establish precise associations between specific locations or times of the year, providing crucial temporal and spatial context.

This probative value increases significantly when rare species or those with limited geographic distribution are identified. Certain fungi grow exclusively in association with particular plants or under very specific environmental conditions, becoming highly discriminatory evidence. When investigators find these distinctive spores on belongings of a suspect who denies having been at the corresponding place, they obtain scientific evidence that can be decisive in judicial proceedings.

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Applications in Forensic Medicine and Toxicology

Medical-legal expertise in healthcare negligence

Forensic mycology plays a relevant role in medical-legal expertise when investigating possible cases of healthcare negligence. The presence of certain fungi in clinical environments can be indicative of serious failures in biosafety, ventilation, or sterilization protocols.

For example, nosocomial infections caused by Aspergillus fumigatus or Candida auris have been documented in hospitals with structural deficiencies or contaminated air conditioning systems. In these cases, forensic experts specialized in mycology analyze environmental samples (air, surfaces, surgical instruments) and biological samples (infected patient tissues) to establish a causal link between the hospital infrastructure and the infection.

These findings can be key to determining legal responsibilities in malpractice lawsuits, especially when it is demonstrated that fungal exposure was preventable with adequate hygiene measures or rigorous maintenance of the facilities.

Detection of mycotoxins in poisonings

Another crucial application of forensic mycology in the toxicological field is the identification of mycotoxins in cases of poisoning, whether accidental, criminal, or suicidal. Mycotoxins are toxic compounds produced by filamentous fungi such as Aspergillus, Fusarium, or Penicillium, and can be found in poorly stored foods, animal feed, or even contaminated natural supplements.

In forensic toxicology, the analysis of blood, urine, liver, or other tissues can reveal the presence of toxins such as:

• Aflatoxins (produced by Aspergillus flavus), highly hepatotoxic and carcinogenic.
• Ochratoxin A, which primarily affects the kidneys.
• Trichothecenes, capable of causing gastrointestinal, neurological, and dermatological symptoms.

Additionally, in cases of ingestion of poisonous mushrooms, such as Amanita phalloides, forensic experts look for compounds like amatoxin, responsible for fulminant liver failure. The timely detection of these substances in victims can help determine the cause of death, the route of exposure, and whether there was criminal intent, which is essential in judicial contexts.

Environmental and Public Health Cases

Forensic mycology extends its application to the field of environmental litigation and public health cases related to toxic fungi. This branch has gained relevance with the increase in respiratory problems in damp buildings and lawsuits for exposure to mycotoxins in various environments. The genus Stachybotrys, commonly known as "black mold", is the protagonist of numerous legal cases for lung damage.

This fungus thrives in construction materials with cellulose exposed to constant humidity, multiplying especially after floods or prolonged leaks in buildings. Experts in forensic mycology analyze these environments through strategically distributed environmental cultures to determine concentrations and fungal species present.

Spore microscopy in dust samples reveals the fungal load to which occupants have been exposed, while advanced biochemical studies identify and quantify specific mycotoxins such as satratoxins produced by Stachybotrys chartarum. This scientific evidence is crucial both for resolving legal disputes over responsibilities in cases of "sick buildings" and for establishing remediation and prevention standards.

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Applications of Mycology in Archaeological Investigations

Lichenometry

In archaeological and forensic contexts of ancient burials, forensic mycology plays a key role in historical reconstruction. Lichens act as natural biological clocks: when bone remains have been exposed for long periods to the elements, they can be colonized by lichens of genera such as Caloplaca or Lecanora.

By measuring the diameter of these colonies and knowing their annual growth rate, it is possible to estimate with considerable precision the minimum time of exposure of the bones to the environment, which provides fundamental data about the time and conditions of burial.

Taphonomic Analyses

Taphonomy in forensic mycology studies the processes of fungal colonization that affect organisms after death, providing valuable information about the post-mortem interval, burial conditions, and possible manipulations of the remains. Decomposing bodies develop predictable patterns of fungal succession that can be scientifically documented and used as temporal bioindicators.

These studies allow forensic investigators to reconstruct events after death by identifying specific fungal species associated with different stages of decomposition and environmental conditions.

The Taphos-M project (2009-2012) represents a milestone in this discipline, where Spanish researchers analyzed fungal colonization in pig cadavers under controlled conditions. Using specific methodologies, they identified 30 genera of fungi and established correlations between fungal species and variables such as post-mortem time, burial depth, and soil conditions. Their findings were successfully applied in real forensic cases in northern Spain (2015-2017), transforming fungal taphonomic analyses from experimental studies to fundamental tools in contemporary criminal investigations.

"Grave Mushrooms"

Certain fungi, known as "grave mushrooms," can indicate the presence of ancient burials. Species such as Hebeloma aminophilum and H. vinosophyllum have been documented growing over ancient burials. The presence of these specific fungi can signal anomalies in the soil where there was decomposing organic matter. These biological markers are particularly useful in archaeology for locating undocumented burials or historical mass graves.

Forensic mycologists collaborate with archaeologists to interpret these fungal indicators, creating distribution maps that can guide excavations and contribute to historical investigations of great relevance.

Scientific literature has recorded several notable cases where burial indicator fungi, known as "grave mushrooms," have been fundamental in forensic investigations:

1. Holland Case (1999): Forensic archaeologists at the University of Amsterdam documented the presence of Hebeloma vinosophyllum growing in circular formation over what was later confirmed as a mass grave from World War II. The growth pattern of the fungus led to the discovery of human remains that had remained undocumented for more than 50 years.
2. Miklós Tóth Investigation (2013): In Hungary, this mycologist documented the abundant growth of Hebeloma aminophilum over a wooded area where human remains buried approximately 15 years earlier were subsequently found. The case confirmed the selectivity of this species for soils enriched with nitrogenous compounds derived from human decomposition.
3. "Orchard Grave" Case (New Zealand, 2008): Forensic investigators used the unusual presence of Hebeloma syrjense to locate a clandestine grave in an apple orchard. The anomalous distribution of this fungus, which grew in a distinctive rectangular pattern, led to the discovery of a body buried 7 years earlier.
4. Hawksworth and Wiltshire Work (2015): In a case in England, these researchers documented how Hebeloma vinosophyllum grew over a rural burial site, helping to identify the exact location of human remains in a previously extensive search area. The case is particularly notable because the mushrooms appeared during an exceptional rainy season, revealing a burial that had remained hidden for almost a decade.

These cases demonstrate the value of "grave mushrooms" as biological indicators in forensic investigations, especially when conventional search methods are insufficient or when dealing with ancient burials where other biological markers are no longer detectable.

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How are mycological evidences analyzed?

Sample Collection

The collection process must be carried out with extreme care to avoid contamination. Sterile swabs, spatulas, or contact plates are used to sample surfaces such as cadavers, vegetation, or vehicles. It is common to use transparent adhesive tape over suspicious areas to extract spores.

The laboratory examines clothing and footwear, as spores can be recovered from them using techniques similar to those used in forensic palynology. Swabs of soft tissues or body fluids are made for culture, and small portions of any mushroom found are dried.

Culture and Morphological Identification

Culture techniques are fundamental for forensic mycological analysis. Samples are seeded in media such as Sabouraud agar or other selective media (DTM, malt extract), incubating at temperatures of 25-30°C.

The resulting mycelial growth is examined macroscopically (color, texture, structure) and microscopically (shape of hyphae, sporangia, conidia) using specific stains. Specialized taxonomic keys are used for the identification of environmental fungi.

Molecular Techniques

Given the difficulty of identifying many fungal species solely by morphology, molecular techniques are increasingly important. Fungal DNA is extracted from cultures or directly from biological samples. ITS ribosomal regions (such as ITS1-5.8S-ITS2) or specific genes are amplified via PCR.

Sequencing of these fragments and their comparison with databases (such as GenBank or UNITE) allows identifying the species with great precision. Recently, massive metabarcoding (massive sequencing of fungal amplicons) is used to characterize complete fungal communities in environmental samples.

Chemical Analysis

In cases of poisonings or use of hallucinogens, specific chemical tests are performed. Metabolites are extracted from tissues or from the mushrooms themselves. They are analyzed using techniques such as gas chromatography (GC-MS) or high-performance liquid chromatography (HPLC, HPTLC). The detection of specific compounds such as ergotamines or amatoxins (α/β-amanitin) can confirm particular poisonings.

Interpretation of Results

The interpretation of mycological results in forensic context is crucial. Species profiles or fungi isolated in evidences (such as a victim's body) are compared with those of the scenario or evidence related to suspects.

The concordance of rare species can link places or people with crime scenes. Differences in species abundances can provide relevant clues.

The specific ecology of each species informs the interpretation: if a fungus grows exclusively in autumn, it can be deduced that the event occurred in that season.

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Challenges and Future of Forensic Mycology

Current Challenges

Despite its considerable potential, forensic mycology currently faces several significant challenges. There is a notable shortage of specialists with dual training in mycology and forensic sciences, limiting the practical application of this discipline in criminal investigations. There is also an urgent need for standardization in protocols for collection and analysis of fungal samples, which makes it difficult to compare results between different laboratories and investigations.

Databases on geographic distribution and ecology of fungal species require constant expansion and updating to increase their forensic utility. Additionally, there is limited knowledge about fungal succession patterns in different environmental and climatic conditions, a crucial factor for precision in estimating the post-mortem interval and other forensic determinations.

Future Perspectives

The horizon of forensic mycology appears particularly promising thanks to various technological and scientific advances in development. Molecular techniques for fungal identification are evolving towards faster, more precise, and economically accessible methods, including portable sequencing technologies applicable directly at crime scenes.

The international scientific community is actively working on creating global reference databases that document specific regional fungal profiles, incorporating integrated ecological, genetic, and morphological data. Advances in artificial intelligence and machine learning are allowing the development of expert systems for the interpretation of complex mycological data and the recognition of significant patterns in forensic investigations.

There is also a growing integration of forensic mycology into standard criminal investigation protocols, with specialized training programs emerging in academic and forensic institutions in various countries. This multidisciplinary evolution positions forensic mycology as an increasingly valuable tool in the arsenal of modern techniques for solving complex criminal cases.

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References

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