Unexpected applications of basic research: moss forensics

Different TV shows have popularized some details of the scientific methodology used in forensics in order to unravel the circumstances of a death. Regardless of the accuracy of your favorite TV series, sources of relevant forensic information are truly diverse and multidisciplinary: projectile physics, rate of chemical reactions or the knowledge of ecological processes, among many others, may contribute to narrow the spectrum of possibilities in the reconstruction of a crime scene or to properly identify a corpse. During the past years, some publications have stressed the advantageous use of bryophytes (mosses and allied plants) in forensics, mainly based on two different sources of evidence: the identification of small fragments and the estimation of the Post Mortem Interval.

It is easy to understand that any substance or material associated to a corpse may be informative in a criminal investigation. Botanical items such as pollen grains or plant fragments have been traditionally used to learn about the whereabouts of a human body or different items that can be thus linked to a particular location. Bryophytes are an advantageous addition to these sources if we consider their characteristics: 1) they are ubiquitous in the field and small, being their fragments suitable to remain in the soil, dirt and debris attached to shoes, clothes or vehicles; 2) Bryophyte species show wide distributions, but are often microhabitat specialists, potentially narrowing a location or itinerary; 3) Even microscopic fragments can be useful, since ordinarily this is the scale used for identification by specialists. A tiny moss leaf of less than a millimeter long may be distinctive enough for an accurate identification; Finally, 4) as a consequence of their ability to survive intermittent periods of dehydration or adverse conditions, their resistance to decay and their unpalatability for most herbivores, bryophyte DNA is very likely to be successfully extracted and studied.

Arguably, the first report of bryophyte fragments used in court took place more than ten years ago1. The clothes and the vehicle of the suspects of a homicide contained fragments of the species Brachythecium albicans, Calliergonella lindbergii, and Ceratodon purpureus. The three moss species were also found in the crime site, in a forest, which already could be understood as a potential link. Additionally, since B. albicans and C. lindbergii reproduce mostly clonally, a DNA fingerprinting analysis could provide a stronger connection between the fragments found on the suspects and the wild populations of the site. The study concluded that a fragment of B. albicans found in the car was clearly originated in the crime site, and a piece of C. lindbergii material found on a suspect’s clothes likely shared the same origin.

Figure 1. Calliergonella lindbergii. | Credit: Barry Stewart
Figure 1. Calliergonella lindbergii. | Credit: Barry Stewart

The suitability of bryophytes as forensic evidence was also tested by the same research group2 paying attention to the possibility of fragment recovery and the persistence of DNA in a sample subject to extreme environmental conditions. In the first experiment, the shoes (hiking and rubber boots) of a group of 16 people were brushed in order to study the debris after a 20-hour trekking period on a forested area followed by a 4-hour walk on gravel and paved roads. Almost 30% of the studied shoes had some bryophyte fragment attached, belonging to 5 different species that could be routinely identified with a compound microscope. The persistence of bryophyte DNA was tested in a different experiment, in which nine samples of different mosses were kept in paper bags in an unheated shed, during 18 months in Southern Finland. Although the temperatures fluctuated between -28°C and 25°C and some of the samples were partially moulded by the end of the experiment, the DNA could be extracted normally and easily genotyped using different microsatellites.

Figure 2. This is probably going too far. | Credit: Andrew Bowden
Figure 2. This is probably going too far. | Credit: Andrew Bowden

Probably one of the most extremes (but well known) examples of bryophyte fragment identification linked to a human corpse lies on the field of archaeology instead of forensics. In 1991 a frozen body that lived 5,200 years ago was found in the Italian Alps. Also known as the Tyrolean Iceman, or “Ötzi”, this naturally mummified man has been subject to extensive research and became a valuable source of information on the Alpine populations of his time, from genetics to clothing. The examination of Ötzi’s digestive track provided many fragments of bryophytes, belonging to six different species that have also contributed to the reconstruction of his lifestyle and last days3. Since mosses have no nutritional value, it is assumed that the presence of these mosses in his intestines are the result of other kind of use: Ötzi probably used Neckera complanata (a common moss in the area) and other mosses to wrap his food provisions during his unsuccessful Alpine trek. Another interesting subject of discussion was the presence within intestinal samples of a species linked to marble substrates and other lime-rich rocks: Hymenostylium recurvirostrum. This species is absent in the site where the Iceman was recovered, and it is only found in very discrete localities within the surrounding areas, suggesting an active trekking activity during his last days.

Figure 3. Leave fragment of Anomodon viticulosus found in the digestive track of Ötzi; even minute bryophyte samples can potentially be identified. | Credit: Dickson et al. (2009)
Figure 3. Leave fragment of Anomodon viticulosus found in the digestive track of Ötzi; even minute bryophyte samples can potentially be identified. | Credit: Dickson et al. (2009)

Bryophytes are also a potential source of information for a minimum extreme of the Post Mortem Interval (PMI), this is, the time period elapsed since the moment of the death till the present. Several biological phenomena have been used to narrow the PMI estimation, most of them related to the decomposition of the corpse. For instance, the life cycle timing of different carrion insects are precise enough to be used as proxies of the PMI. For relatively old corpses, the growth of plants can be used as a source of information, but some bryophytes have two particular advantages over their vascular relatives: they can grow on bare bone itself and the age of some colonies can be estimated rather accurately. The most suitable mosses for this task share a monopodial growth form; their tufts will show a number of annual segments that represent the number of years passed from the colonization. Obviously, mosses can only colonize a bone after all the soft tissues have decomposed and the skeletonization process is complete. Even if the age of the colony can be deduced clearly from the study of the moss, this would only represent the extreme lower limit of the PMI, and the previous taphonomical process needs also to be considered.

Figure 4. Annual segments of some monopodial mosses allow estimating the age of the colony. | Credit: Cardoso et al. 2010
Figure 4. Annual segments of some monopodial mosses allow estimating the age of the colony. | Credit: Cardoso et al. (2010)

This method for PMI estimation has been used at least in two different occasions. The first one took place in 2008, after the discovery of a skeletonized human body in Northern Portugal4. Only one missing person had been reported in the area, his disappearance going back six years. For the PMI estimation, several variables were taken into account, such as the degree of disarticulation of the skeleton, the bleaching process of some exposed bones, and the presence of bryophyte colonies on the bones and clothes. The remains hosted colonies of three different moss species: Bryum capillare, Hypnum cupressiforme and Campylopus introflexus, and the ages of the oldest colonies were above 3 years (plus the lapse of the skeletonization process: 4 months to 3 years). Together with the rest of the evidence, the PMI estimation was compatible with the 6-year frame, and the identity of the body could be assigned with a very high certainty.

Figure 5. Colony of Bryum sp. growing on a tibia of the body found in Northern Portugal. | Credit: Cardoso et al. (2010)
Figure 5. Colony of Bryum sp. growing on a tibia of the body found in Northern Portugal. | Credit: Cardoso et al. (2010)

More recently, in November of 2010, the remains of a woman were found in a forested area of Perugia, Central Italy5. The base of the cranium showed an extensive colony of the moss Leptodictyum riparium and it was used as evidence for the PMI estimation. Bryophyte specialists examined the sample, confirmed that the species was showing an annual segmented growth in the area (the possibility of two annual growth pulses needs to be considered case by case) and estimated the age of the colony in 24-30 months after the skeletonization. The PMI and the characteristics of the body were cross-referenced with the records of missing people and their medical and dental histories and the window was narrowed down to a single individual: a missing woman disappeared in 2007.

Bryological forensics is a recent, unexpected application of more than 200 years of accumulated knowledge on the diversity and ecology of mosses and allies. Although basic research, with the only purpose of expanding our current knowledge, should be considered as a worthy objective itself, we have to keep in mind that a common trait of applied research is that it cannot be anticipated a priori, and that is another powerful justification to continue exploring the unknown.

References

  1. Korpelainen, H. & Virtanen, V. 2003. DNA fingerprinting of mosses. Journal of Forensic Sciences 48, 804–807
  2. Virtanen, V., Korpelainen, H. & Kostamo, K. 2007. Forensic botany: usability of bryophyte material in forensic studies. Forensic Science International 172, 161–163
  3. Dickson, J. H., W. Hofbauer, R. Porley, A. Schmidl, W. Kofler & K. Oeggl. 2009. Six mosses from the Tyrolean Iceman’s alimentary tract and their significance for his ethnobotany and the events of his last days. Vegetation History and Archaeobotany 18, 13–22
  4. Cardoso, H. F. V. A. Santos, R. Dias, C. Garcia, M. Pinto, C. Sérgio & T. Magalhães. 2010. Establishing a minimum postmortem interval of human remains in an advanced state of skeletonization using the growth rate of bryophytes and plant roots. International Journal of Legal Medicine 124, 451–456
  5. Lancia M., Conforti F., Aleffi M., Caccianiga M., Bacci M. & Rossi R. (2013). The Use of Leptodyctium riparium (Hedw.)Warnst in the Estimation of Minimum Postmortem Interval, Journal of Forensic Sciences, 58 S239-S242. DOI:

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