UvA-DARE (Digital Academic Repository) DNA markers for forensic identification of non-human biological traces Wesselink, M. Publication date 2018 Document Version Other version License Other Link to publication Citation for published version (APA): Wesselink, M. (2018). DNA markers for forensic identification of non-human biological traces. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:26 Sep 2021 Chapter 1 Molecular species identification of “Magic Mushrooms” M. Wesselink, Esther M. van Ark and I. Kuiper Abstract The use of DNA markers for the identification of fungal species has been described for diverse applications. However, the ideal marker for forensic identification of hallucinogenic species of fungi has been the cause of debate. As species identification of seized samples is required for law enforcement, and seizures of magic mushrooms often lack the morphological features required to identify the species, a better understanding of the performance of different DNA markers is necessary. Markers ITS and LSU were sequenced from authenticated specimens of several Psilocybe, Deconica and Panaeolus species, as well as from seized samples and samples sold in shops prior to the banning of these mushrooms in the Netherlands. Sequences were obtained for both markers from all samples. However due to amplicon length, ITS is expected to outperform LSU when DNA degradation of samples has occurred. Inter and intraspecies variation was calculated based on the obtained sequences, complemented with all Psilocybe, Deconica and Panaeolus ITS and LSU sequences present in GenBank. Use of these sequences revealed the presence of several incorrectly labeled or misidentified sequences, demonstrating both the pitfalls and value of such public databases. The between species variability of markers ITS1 and ITS2 is demonstrated to be far greater than that of marker LSU, caused by both differences in sequence length and composition. The vast majority of intraspecies variation detected for Psilocybe and Deconica species was due to single differences in nucleotide composition, or insertion/deletion of single nucleotides. Of the studied DNA markers, the complete ITS1-5.8S-ITS2 region was shown to be the most informative for identification at the species level, although several groups of closely related species were found that could not be distinguished due to insufficient interspecies variability. A comparable performance was noted for markers ITS1 an ITS2 separately. The interspecies variability of marker LSU was lower than of the ITS regions, rendering this region less suited for species identification. 13 Chapter 1 1. Introduction For centuries the hallucinogenic properties of certain species of plants, animal and fungi have been known to man, such species being appreciated for their value in ritual and medicinal proceedings (reviewed in [1]). In more recent times, some of these species of fungi have reached a wider audience and have become more widely used as drugs of abuse [1,2]. Although the health risks of using such “magic mushrooms” are not considered to exceed the risks of for example drinking alcohol [3,4], most countries have some form of legislation in place to prevent wide scale production, sales and consumption of hallucinogenic mushrooms. Regulation of the pure chemicals responsible for the hallucinogenic properties of most “magic mushrooms” (MM), psilocin and psilocybin is clear as these are prohibited in most UN member countries after being placed on Schedule 1 of the United Nations Convention on Psychotropic substances. Regulation of the fungus capable of producing these components is far more diffuse, as regulations can depend on the phase of the lifecycle that is encountered (spores, mycelium, sclerotia and fruiting bodies), on the species of fungus that is encountered, on the intrinsic capability of the encountered material to produce the regulated components and in some cases on a combination of the above (e.g. [5,6]). Although many fungal species in the order Agaricales have been recognized that can produce hallucinogenic components, the majority of the abused species of “magic mushrooms” belong to the fungal genera Panaeolus and Psilocybe [2]. In the recent past, the genus Psilocybe consisted of species with and without hallucinogenic capacities, with the non-hallucinogenic Psilocybe montana being the lectotype. Since the proposal of taxonomical redesignation of the genus Psilocybe, the name Psilocybe has been given to the species with hallucinogenic properties (Psilocybe semilanceata as type), whilst species without these properties have been placed in the genus Deconica [7,8]. To identify forensically relevant samples, from a scientific point of view, identification of the genus Psilocybe would be sufficient. However in many countries, correctly identifying a species as opposed to this genus, is still necessary from a legal point of view as legislation precedes this scientific advancement, and identification of the species of fungal material may be required for prosecution. In the Netherlands for example, fruiting bodies (mushrooms) of 188 species of fungi, mainly belonging to the genera Psilocybe and Panaeolus, have been forbidden by law on December 1st 2008 [5]. The import, export, trade, cultivation and possession of mushrooms of these species has thereby become illegal, calling for a robust technique to correctly identify samples of these species, to enable distinction between these and other (legal) species of mushrooms for regulatory purposes and to prosecute offenders. When complete fungi in an informative phase of their lifecycle are the subject of investigation, morphological identification of the species can be performed. However other lifeforms of fungi (such as sclerotia or mycelium) or material that has been dried, shredded or otherwise treated, are not easily identified morphologically and may require either cultivation or molecular identification. As the majority of seized samples have been dried, grinded or powdered, DNA based 14 Molecular species identification of “Magic Mushrooms” identification of forensic samples seems a potent method, as has been described repeatedly for animal and plant samples [e.g. 9-13]. In fungi, DNA markers have been applied for several distinct purposed, each imposing different requirements on the DNA markers selected. In phylogenetic studies of fungi, the internal transcribed spacers (ITS1 and/or ITS2) and the ribosomal large subunit (LSU) are used as markers, where ITS is mainly used to study relationships at the species/genus level, and LSU is generally used to study genus/family level relationships [14-17]. In many fungi, including the order Agaricales, the internal transcribed spacers display variations not only in sequence composition, but also in sequence length, which hampers meaningful alignment of these sequences, leading these sequences to be omitted from comparative analyses (e.g. [16- 18]). Studies describing the DNA based identification of fungi from a DNA barcoding or species descriptive perspective, generally rely on markers ITS2 and/or ITS1, supplemented with additional markers for certain orders or families of fungi [17-18]. In these studies, ease of alignment of sequences is of less importance than a (much) larger between species variation than within species variation, the ‘barcoding gap’. Length variations that may hamper alignment efficiency often even increase the informative value of a species identification marker by adding many unique characters. Apart from the sequence information, robust amplification and sequencing conditions throughout a large group of organisms (i.e. fungi) with ‘universal’ primers is of importance for DNA based identification efforts. Additionally the availability of reliable reference sequences is a valuable criterion, although additional databases can obviously be built for specific purposes. Several studies have focused on the DNA based identification of specific species of (hallucinogenic) fungi for forensic applications [19-22]. Identification of magic mushrooms has been reported favoring either marker ITS [19,21,22] or LSU [20,21]. However as some of the results of these studies seem contradictory, there is no consensus yet on which marker is most suitable for MM identification for forensic applications [23]. Obviously to enable the use of DNA based identification of seized fungal samples, discussions concerning the applied DNA markers are undesirable. Therefore this study aims to clarify the differences between previously published (forensic) studies, and explore whether marker ITS1 and ITS2 together, or single markers ITS1 or ITS2 are the most appropriate