Magic Mushrooms”

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.

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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.

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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 for forensic identification of magic mushrooms or whether these spacer regions should be abandoned in favor of marker LSU. To this extent, these markers were examined extensively for their amplification and sequencing ease in both reference samples and seizures of MM, the extent of intra and interspecific variation and the presence of reliable reference sequences in the public database GenBank [24].

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2. Material and methods 2.1. Sample material DNA extracts of seven specimens of Psilocybe, one specimen of Deconica and one specimen of Panaeolus were purchased from the Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands. A single Deconica tissue sample was obtained from Gent Herbarium, Belgium (Table 1). Two species of Psilocybe mushrooms were obtained from a store before December 2008, as were two species of Panaeolus mushrooms. Three Canadian accessions of Psilocybe cubensis, Deconica montana and Panaeolus sphinctrinus kept at Canadian National Mycological Herbarium (DAOM) could not be used for additional DNA extraction or sequencing, but resequencing of these samples was performed in Canada and sequences kindly communicated by Scott A. Redhead (Canadian Collection of Fungal Cultures). In a later stage of the study, ten unidentified seizures were used that were submitted to our laboratory by the National Police of the Netherlands for either species identification or identification of chemical substances. These seizures consisted of dried mushrooms, fragments of mushrooms, grinded material and sclerotia.

Table 1. Psilocybe, Deconica and Panaeolus samples used in this study

Sample name IDa GenBank accession nr

Psilocybe Ps caerulescens CBS strain 837.87 HM035072 Ps coprophilla CBS strain 417.82 HM035073 Ps cubensis CBS strain 140.85 HM035074 Ps cubensis CBS strain 590.79 HM035075 Ps cyanescens CBS strain 295.94 HM035076 Ps mexicana CBS strain 831.87 HM035077 Ps montana CBS strain 101791 HM035078 Ps montana Gent 3330 HM035079 Ps semilanceata CBS strain 101868 HM035080

Deconica D montana CBS strain 101791 HM035078 D montana Gent 3330 HM035079

Panaeolus Pa sphinctrinus CBS strain 582.79 HM035081

Market samples “Ps cubensis ” Production line 1.002 HM035082 “Ps mexicana ” Production line 9596 HM035083 “Pa cyanescens” Production line 1.007 HM035084 “Pa” Production line 1.045 HM035085 a CBS: Centraalbureau voor Schimmelcultures (Utrecht, the Netherlands); Gent: Herbarium Universitatis Gandavensis (Gent, Belgium).

16 Molecular species identification of “Magic Mushrooms”

2.2. Extraction of DNA and quantification Fungal tissue was grinded using a TissueLyser (Qiagen), after which DNA was extracted using the DNeasy Plant Mini kit (Qiagen) following the manufacturers’ protocol. Total DNA concentration was estimated using NanoDrop ND-1000 (NanoDrop Technologies).

2.3. Amplification, visualization and sequencing ITS was amplified using the fungal specific forward primer ITS1-F [25] and the universal reverse primer ITS4-R [26] (Figure 1). PCR reactions were performed in 25 μl reaction mix containing either (I) 1x reaction Buffer II, 3 mM MgCl2, 0.2 mM each dNTP, 1 unit of AmpliTaq Gold (all Applied Biosystems), 20 pmol each primer (Biolegio, the Netherlands), 5 μg BSA (New England Biolabs) and < 10 ng of template DNA or (II) 1x HotStarTaq PCR reaction mix including 1.5 mM MgCl2, 1.5 mM additional MgCl2, 0.2 mM each dNTP, 0.5 units of HotStarTaq (all Qiagen), 10 pmol each primer (Biolegio, the Netherlands) and < 10 ng of template DNA. LSU was amplified using universal fungal primers 5.8SR and LR7 [27] (Figure 1). PCR reactions were performed in 25 μl containing either (I) 1x reaction Buffer II, 2.5 mM MgCl2, 0.2 mM each dNTP, 1 unit of AmpliTaq Gold, 5 pmol each primer and <10 ng of template DNA or (II) 1x HotStarTaq PCR reaction mix including 1.5 mM MgCl2, 1 mM additional MgCl2, 0.2 mM each dNTP, 0.625 units of HotStarTaq (all Qiagen), 5 pmol each primer (Biolegio, the Netherlands) and <10 ng of template DNA. PCRs were performed on MyCycler (BioRad Laboratories, CA, USA) or GeneAmp 9700 (Applied Biosystems) thermocyclers using the following cycling parameters for ITS: (I) 10 min denaturation step at 94 °C; 32 cycles of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 2 min; 10 min elongation step at 72 °C or (II) 15 min denaturation step at 95 °C; 35 cycles of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min; 10 min elongation step at 72 °C. For amplification of LSU the following cycling parameters were used: (I) 10 min denaturation step at 94 °C; 35 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min; 10 min elongation step at 72 °C or (II) 15 min denaturation step at 95 °C; 35 cycles of 94 °C for 1 min, 55 °C for 90 s, and 72 °C for 90 s; 10 min elongation step at 72 °C.

Figure 1. Relative positions of DNA markers and primers (arrows) used in this study.

PCR success was determined by gel-electroforesis followed by ethidium bromide coloring and UV detection. Positive and negative controles were used throughout. PCR products were sequenced using the abovementioned primers ITS1-F and ITS4-R for ITS and primers 5.8SR, LR0R, LR16 and LR7 for LSU [27,28] either commercially (BaseClear B.V., Leiden, the Netherlands) or in house (purification of PCR products with ExoSAP-IT® (Affymetrix) following the manufacturer’s protocol, cycle sequencing with 0,5-2 μl of purified PCR product

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in a 10 μl sequencing reaction containing 1x BigDye sequencing buffer, 0.2x BigDye Terminator v3.1 Ready Reaction Mix (Life Technologies) and 2.5 pmol of primer, for 1 min at 96 °C; 28 cycles of 95 °C for 10 s, 50 °C for 5 s and 60 °C for 4 min in the abovementioned thermocyclers, removal of residual sequencing components with BigDye XTerminator® Purification Kit following the manufacturer’s protocol, separation of sequencing products on an AB3500 XL genetic analyzer containing 50 cm capillaries using POP-7TM polymer (all Life Technologies).

2.4. Data analysis Evaluation of sequences and assembly of forward and reverse sequences was performed with Geneious 4.7.6 [29]. Sequence data was compared with GenBank [24] through BlastN in March 2014. To compare sequences from this study to previously published sequences and sequences submitted to GenBank, a local copy was made of all ITS1, 5.8S, ITS2 and LSU sequences named Psilocybe, Deconica or Panaeolus present in GenBank on March 23rd 2014. For readability, all species considered a (non-potent) Deconica species [18,24,30-32], will be referred to as Deconica, independent of whether these sequences were originally deposited in GenBank as a Psilocybe or Deconica sequence. To further investigate a peculiarity, three ITS1, 5.8S, ITS2 sequences named Galerina clavata were additionally downloaded. Several sequences were removed from the dataset prior to analyses; a sequence containing more than 25 N’s (GU565174), ITS1-5.8S-ITS2 sequences that differed from other sequences from that genus in the 5.8S region by more than 5 nucleotides (EU870081, KF429543, JF961375 and JF961370), LSU sequences shorter than 500 nucleotides or containing only other LSU regions than the studied 5’ D1-D2 region (multiple) and one accession that differed from all other Psilocybe, Deconica and Panaeolus sequences in LSU length and sequence (FJ755221). Alignments of ITS1 and ITS2 sequences were manually edited in Geneious to accommodate length differences; the readily alignable ‘conserved’ 5’ and 3’ regions were not edited, the highly variable regions were added to the 5’ ‘conserved’ region thereby reflecting sequence length more than sequence composition. The obtained alignments were used to create maximum likelihood trees in Mega 5.2.2 [33] (using the Tamura-Nei model evolutionary distances with invariable sites [34], 1000 bootstrap replicates, without deletion of missing data/gaps) and calculate pairwise distances between sequences (Tamura-Nei model [34], with gamma distribution, pairwise removal of ambiguous positions). The distance matrices were ordered by species and genus to enable selection of intraspecies distances, interspecies but intragenus distances and intergenus distances.

18 Molecular species identification of “Magic Mushrooms”

3. Results and discussion 3.1. Amplification of ITS and LSU Markers ITS and LSU were amplified directly in known samples and seizures with the ‘universal’ primers and both conditions described. Amplicons of roughly 500 to 800 bp voor ITS and 1800 bp for LSU were obtained. Although differences have been noted in PCR performance of these markers [16,17], such differences are not expected in the studied species as these are closely related. Positive PCR results were obtained with as little as 0.5 ng total DNA as template per reaction. Whilst sample or DNA availability was not limiting in amplification of known samples, the detection of such small amounts of DNA is of great value in forensic investigations where samples are often ill conserved, powdered, or otherwise formulated leading to degradation of DNA. This is demonstrated by amplification of both markers in the investigated seizures.

3.2. ITS and LSU sequence data Using the described primers, markers ITS1, 5.8S, ITS2 and LSU (1st 900 nucleotides, variable regions D1 and D2) were sequenced for all samples (see Table 1 for accession number of sequences deposited in GenBank). The sequence of the complete ITS amplicon was obtained in a single sequencing reaction, whilst at least three sequencing reactions were needed to obtain the complete LSU sequence (see Figure 1 for primer locations). Neither long mononucleotide stretches nor other challenging internal structures were encountered. The sequences of all samples were aligned, both pairwise and as a group. The LSU region of the sequences was easily aligned for all Psilocybe, Deconica and Panaeolus species. The 5.8S region of all sequences was also readily aligned for all tested species. This is in agreement with the nature of the LSU and 5.8S regions, as these parts of the DNA are under evolutionary constraint as has been demonstrated in large scale studies of fungi [17]. Alignment of ITS1 and ITS2 of different samples of the same species was also straightforward as no large length differences were observed within a species. Alignment of ITS1 and ITS2 of samples of different species proved more troublesome, as not only nucleotide- but also length differences were present between the different species. This effect was more pronounced in ITS1 than in ITS2. However by aligning the sequences as described reflecting sequence length differences, it was possible to further investigate the differences between the different regions.

Between the two ITS1-5.8S-ITS2-LSU sequences of Psilocybe cubensis that were generated from authenticated samples (HM035074 and HM035075), the market sample sold as Psilocybe cubensis (HM035082) and multiple seized samples, no differences in sequence length or composition were detected, demonstrating absence of intraspecies variability at least within the available Psilocybe cubensis samples. The sequences of the two Deconica montana strains (HM035078 and HM035079) differed from each other by one position in ITS1 and one position in ITS2. No differences were detected between the 5.8S or LSU regions of these sequences, illustrating minor intraspecies variability in Deconica montana. Comparison of the sequence obtained from

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the authenticated Psilocybe mexicana strain (HM035077), the market sample sold as Psilocybe mexicana (HM035083) and all seized sclerotia demonstrated the presence of three variable positions in ITS1, two in ITS2 and none in both 5.8S and LSU. No intraspecies length differences were observed for these three species. Through interspecies comparison of pairs of sequences both large differences in sequence length and sequence composition were detected.

3.3. BLAST results The sequences obtained from the known samples (Table 1) were compared to all sequences present in NCBI GenBank to find the deposited sequences that were most similar at both the individual nucleotide and length level. These comparisons were hampered by the fact that only very few ITS1-LSU sequences were present in GenBank, and that blasting our long ITS1-LSU sequences therefore automatically led to a high bit-score (and low E-value) with the long sequences present, even though the actual percentage of similarity was too low to reflect sequences from the same species. By dividing our sequences into smaller portions that correspond to portions more frequently deposited in GenBank (i.e. ITS1, ITS1-5.8S-ITS2 and LSU separately), this artifact was circumvented. However comparison of these different regions led to the finding of significantly different results for the different regions of the Psilocybe cubensis, Deconica montana and Panaeolus sphinctrinus sequences.

The ITS1 region of the Psilocybe cubensis sequences HM035074 and HM035075 obtained in this study were identical in length and sequence composition to ITS1 sequences deposited in GenBank with species descriptions Psilocybe cubensis (four accessions), Deconica montana (AY129360), Panaeolus sphinctrinus (AY129348) and Galerina clavata (AY281021). Moreover the ITS1 region of the Psilocybe cubensis sequences HM035074 and HM035075 differed from two other sequences deposited in GenBank as Psilocybe cubensis (AY281023 and AY129351) both in length and sequence. Comparison of the ITS1-5.8S-ITS2 region of the Psilocybe cubensis sequences HM035074 and HM035075 to GenBank only returned sequences with identical length and composition that had been deposited as Psilocybe cubensis (two accessions). Comparison of the LSU region of these Psilocybe cubensis sequences returned a large number of hits with sequences of the same length. The accessions in the database with the most comparable sequence have been deposited as Psilocybe cubensis (three accessions) or the closely related Psilocybe subcubensis (two accessions).

Comparable results were found for the Deconica montana sequences HM035078 and HM035079. ITS1 sequences with comparable length and sequence present in GenBank have been deposited as Deconica montana (four accessions) or Psilocybe cubensis (AY129351). ITS1 accession AY129360, deposited as Deconica montana, differed in both length and sequence composition from the ITS1 region of HM035078 and HM035079. However all sequences present in GenBank matching the ITS1-5.8S-ITS2 region or the LSU region have been deposited as Deconica montana or closely related Deconica species.

20 Molecular species identification of “Magic Mushrooms”

For the ITS1 region of Panaeolus sphinctrinus sequence HM035081, all sequences with comparable length and sequence present in GenBank have been deposited as Panaeolus sphinctrinus. One ITS1 accession named Panaeolus sphinctrinus (AY129348) differed in both length and sequence from the ITS1 region of HM035081. All accessions matching the ITS1- 5.8S-ITS2 region or the LSU region have been deposited as Panaeolus sphinctrinus or closely related Panaeolus species.

These results may indicate that marker ITS1 is insufficient to correctly identify certain species of interest due to absence of a barcoding gap as identical sequences are found in (at least) four genera, as suggested by Nugent and Saville [20]. To further investigate the similarities and differences between the different markers in these genera, especially as the sequences causing this finding have been generated in two studies [20,35], all ITS1, ITS1-5.8S-ITS2 and LSU sequences of Psilocybe, Deconica and Panaeolus present in GenBank were downloaded, as were all Galerina clavata sequences for these regions. These deposited sequences, together with our sequences, were aligned as described for ITS1, ITS1-5.8S-ITS2 and LSU.

3.4. Evaluation of markers 3.4.1 ITS1

Alignment of ITS1 sequences was performed as described, enabling the distinction of several groups that displayed minimal variability in both length and DNA sequence. Because several sequences were significantly shorter than others and the sequences were not all trimmed to the same starting and ending position, the placement of these sequences in the Maximum Likelihood tree was influenced. In the Maximum Likelihood tree (Supplementary Figure S1) four groups could roughly be distinguished: the Panaeolus species, the Deconica species, a diverse Psilocybe group harboring the Galerina clavata sequences and a small group containing sequences of the two Psilocybe species Psilocybe calongei and Psilocybe magnivelaris. Although the majority of sequences was placed in the expected group, several sequences were placed in unexpected positions in this tree. Firstly one accession of Psilocybe fasciata (DQ001401) was placed in the Deconica group whereas the other accession of this Psilocybe species (AB158635) was placed in the Psilocybe group. Secondly, the Psilocybe australiana sequence (AY129366) was placed in the Deconica group where placement near Psilocybe subaeruginosa would be expected.

A third incongruity was found in that three near identical groups of species were recognized in the tree that consisted of multiple species that are not closely related; (I) the “Psilocybe cubensis subgroup”, consisting of Psilocybe cubensis, Psilocybe subcubensis, Deconica montana, Panaeolus sphinctrinus and Galerina clavata sequences placed within the Psilocybe group. (II) The “Deconica montana subgroup” within the Deconica group, consisting of Deconica montana and Psilocybe cubensis sequences. (III) The “Galarina clavata subgroup”, consisting of Galerina clavata and Psilocybe cubensis sequences. A separate comparison of the ITS1 sequences of Psilocybe cubensis, Deconica

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montana and Galerina clavata supplemented with the available Panaeolus sphinctrinus sequences (Figure 2), shows that four groups of near identical sequences can be identified that do not correspond to the four groups based on the species name of the accessions. We hypothesize that the groups with near identical sequences actually are single species groups, and that the heterogeneity of the groups is caused by misidentification, contamination or accidental swapping of samples, DNA extracts, PCR products or sequence data of AY129348 (Panaeolus sphinctrinus), AY129351 (Psilocybe cubensis) and AY129360 (Deconica montana) [20] and AY281021 (Galerina clavata) and AY281023 (Psilocybe cubensis) [35].

Figure 2. Alignment of ITS1 sequences deposited in GenBank as Psilocybe cubensis, Deconica montana, Panaeolus sphinctrinus and Galerina clavata. Grey: identical positions; green: adenine; blue: cytosine, black: guanine, red: thymine.

To test this hypothesis, samples of Panaeolus sphinctrinus DAOM 180389, Psilocybe cubensis DAOM 169061 and Deconica montana DAOM 167409 held by the Canadian Collection of Fungal Cultures were requested, from which sequences AY129348, AY129351 and AY129360 are said to have been derived. Unfortunately samples or DNA could not be transferred, however the original samples were resequenced and these sequences kindly provided by Scott A. Redhead. Comparison of these unpublished sequences to the previously deposited sequences [20] strengthens our hypothesis that the published ITS1 sequences are erroneous. We therefore removed the abovementioned sequences from our dataset, prior to estimation of the ITS1 intra and interspecies variation.

Pairwise distances were calculated between all pairs of ITS sequences; identical sequences resulting in a distance of 0. The highest difference in the dataset (0.765) was found between Psilocybe caerulipes AY129371 and Psilocybe subcubensis KC669297. All observed pairwise distances between ITS1 sequences obtained from the same Psilocybe species were below 0.17 (Figure 3A), with the vast majority of pairwise distances (95%) below 0.05. Within the genus Deconica, more variation was detected (Figure 3B) with only 55% of distances lower than 0.05. Closer inspection of the data showed that this was mainly due to the various Deconica montana and Deconica coprophila accessions differing by multiple nucleotides. For the majority of Psilocybe and Deconica species the interspecies variation proved far higher than 0.05 (Figures 3A-B), due to both differences in sequence length and composition. Although a higher interspecies than intraspecies variation is visible, a true barcoding gap is absent. Upon further inspection of

22 Molecular species identification of “Magic Mushrooms”

Figure S1 and the sequence alignments, several groups without interspecies variation were detected within the genus Psilocybe (e.g. Psilocybe cyanescens – Psilocybe subaeruginosa, Psilocybe serbica – Psilocybe moravica and Psilocybe arcana – Psilocybe behomica, Supplementary figure S1). Additionally groups were detected with low interspecies variation. These consisted of sequences with identical sequence length, but slight differences in nucleotide composition (e.g. Psilocybe cubensis – Psilocybe subcubensis and Psilocybe allenii – Psilocybe cyanescens).

Pairwise distances of ITS1 sequences Pairwise distances of ITS1 sequences 0,3 0.74 Pairwise distances of ITS1 sequences 0,3 0,3 Psilocbye intraspecies (n=142) Deconica intraspecies (n=40) Panaeolus intraspecies (n=21) A Psilocybe interspecies (n=3018) B Deconica interspecies (n=236) C Panaeolus interspecies (n=279) 0,25 0,25 0,25 between Deconica sp. and Psilocybe sp. (n=1920) between Deconica sp. and Psilocybe sp. (n=1920) between Deconica sp. and Panaeolus sp.(n=634) between Psilocybe sp. and Panaeolus sp. (n=2000) between Deconica sp. and Panaeolus sp.(n=634) between Psilocybe sp. and Panaeolus sp. (n=2000) 0,2 0,2 0,2

0,15 0,15 0,15

0,1 0,1 0,1 Frequencyof observed pairwise distance Frequencyof observed pairwise distance Frequencyof observed pairwise distance 0,05 0,05 0,05

0 0 0 <0,1 <0,2 <0,3 <0,4 <0,5 <0,6 <0,1 <0,2 <0,3 <0,4 <0,5 <0,6 <0,1 <0,2 <0,3 <0,4 <0,5 <0,6 <0,01 <0,02 <0,03 <0,04 <0,05 <0,06 <0,07 <0,08 <0,09 <0,11 <0,12 <0,13 <0,14 <0,15 <0,16 <0,17 <0,18 <0,19 <0,21 <0,22 <0,23 <0,24 <0,25 <0,26 <0,27 <0,28 <0,29 <0,31 <0,32 <0,33 <0,34 <0,35 <0,36 <0,37 <0,38 <0,39 <0,41 <0,42 <0,43 <0,44 <0,45 <0,46 <0,47 <0,48 <0,49 <0,51 <0,52 <0,53 <0,54 <0,55 <0,56 <0,57 <0,58 <0,59 <0,01 <0,02 <0,03 <0,04 <0,05 <0,06 <0,07 <0,08 <0,09 <0,11 <0,12 <0,13 <0,14 <0,15 <0,16 <0,17 <0,18 <0,19 <0,21 <0,22 <0,23 <0,24 <0,25 <0,26 <0,27 <0,28 <0,29 <0,31 <0,32 <0,33 <0,34 <0,35 <0,36 <0,37 <0,38 <0,39 <0,41 <0,42 <0,43 <0,44 <0,45 <0,46 <0,47 <0,48 <0,49 <0,51 <0,52 <0,53 <0,54 <0,55 <0,56 <0,57 <0,58 <0,59 <0,01 <0,02 <0,03 <0,04 <0,05 <0,06 <0,07 <0,08 <0,09 <0,11 <0,12 <0,13 <0,14 <0,15 <0,16 <0,17 <0,18 <0,19 <0,21 <0,22 <0,23 <0,24 <0,25 <0,26 <0,27 <0,28 <0,29 <0,31 <0,32 <0,33 <0,34 <0,35 <0,36 <0,37 <0,38 <0,39 <0,41 <0,42 <0,43 <0,44 <0,45 <0,46 <0,47 <0,48 <0,49 <0,51 <0,52 <0,53 <0,54 <0,55 <0,56 <0,57 <0,58 <0,59 Figure 3. Frequency of intraspecies (light blue), intragenus (red) and intergenus (green and purple) pairwise differences between ITS1 sequences of . A:Psilocybe, B:Deconica, C:Panaeolus.

In the genus Panaeolus, a different pattern was observed (Figure 3C). Upon inspection of S1 and the sequence alignments, two Panaeolus sphinctrinus sequences (FJ55227 and HE819397) were found to be identical to a Panaeolus rickenii sequence (JF908516), but this group of sequences differed from a group of two other Panaeolus sphinctrius sequences (DQ182503 and HM035081) and a second Panaeolus rickenii sequence (JF908523) in sequence length and composition. The same was observed for two different sequences of Panaeolus campanulatus and Panaeolus retirugis. As Panaeolus sphinctrinus is the only species for which more than two accessions are available, the available dataset was considered insufficient to calculate intra and interspecies variation. Sequencing of Panaeolus specimens is advisable to evaluate the potential for sequence based identification of this genus.

3.4.2 ITS1-5.8S-ITS2 Although less complete ITS1-5.8S-ITS2 sequences were available than ITS1 sequences (respectively 124 and 151), the overall alignment and obtained trees were in agreement with one another other. Alignment of the 5.8S region was straightforward, with only two sequences (KC66926 and KC669300) differing from all others in sequence length and only two additional variable positions in the complete dataset. Alignment of ITS2 proved less troublesome than alignment of ITS1, as less sequence length variation was observed. In the Maximum Likelihood tree (Supplementary Figure S2) the same groups could be distinguished as described for ITS1 alone.

The patterns of intraspecies sequence variation that were described when marker ITS1 was considered, were also present when both markers (Figure 4), or only marker ITS2 was considered (data not shown). The calculated pairwise distances between all pairs of ITS1-5.8S- ITS2 sequences were between 0 and 0.348 (Figure 4). As for marker ITS1, the lowest

23 Chapter 1

intraspecies variation was found within the genus Psilocybe with 95% of differences below 0.02. The highest Psilocybe intraspecies difference was 0.067 (Figure 4A). The highest intraspecies values observed for Deconica and Panaeolus were 0.094 and 0.103 respectively (Figures 4B and 4C). As described for ITS1, the histogram reflecting the frequency of observed pairwise difference values, demontrates a higher between species variation than within species variation for Psilocybe and Deconica although a true barcoding gap is absent.

0,82 Pairwise distances of ITS1-5.8S-ITS2 sequences Pairwise distances of ITS1-5.8S-ITS2 sequences 0,3 Pairwise distances of ITS1-5.8S-ITS2 sequences 0,3 0,3 Psilocbye intraspecies (n=105) Deconica intraspecies (n=33) Panaeolus intraspecies (n=16) A Psilocybe interspecies (n=2040) B Deconica interspecies (n=138) C Panaeolus interspecies (n=215) 0,25 0,25 0,25 between Deconica sp. and Psilocybe sp. (n=1254) between Deconica sp. and Psilocybe sp. (n=1254) between Deconica sp. and Panaeolus sp.(n=418) between Psilocybe sp. and Panaeolus sp. (n=1452) between Deconica sp. and Panaeolus sp.(n=418) between Psilocybe sp. and Panaeolus sp. (n=1452) 0,2 0,2 0,2 0,15 0,15 0,15

0,1 0,1 0,1

Frequency of observed pairwiseobserved Frequencyof distance Frequencyof observed pairwise distance Frequencyof observed pairwise distance 0,05 0,05 0,05

0 0 0 <0,1 <0,2 <0,3 <0,1 <0,2 <0,3 <0,1 <0,2 <0,3 <0,01 <0,02 <0,03 <0,04 <0,05 <0,06 <0,07 <0,08 <0,09 <0,11 <0,12 <0,13 <0,14 <0,15 <0,16 <0,17 <0,18 <0,19 <0,21 <0,22 <0,23 <0,24 <0,25 <0,26 <0,27 <0,28 <0,29 <0,01 <0,02 <0,03 <0,04 <0,05 <0,06 <0,07 <0,08 <0,09 <0,11 <0,12 <0,13 <0,14 <0,15 <0,16 <0,17 <0,18 <0,19 <0,21 <0,22 <0,23 <0,24 <0,25 <0,26 <0,27 <0,28 <0,29 <0,01 <0,02 <0,03 <0,04 <0,05 <0,06 <0,07 <0,08 <0,09 <0,11 <0,12 <0,13 <0,14 <0,15 <0,16 <0,17 <0,18 <0,19 <0,21 <0,22 <0,23 <0,24 <0,25 <0,26 <0,27 <0,28 <0,29 <0,005 <0,015 <0,025 <0,035 <0,045 <0,055 <0,065 <0,075 <0,085 <0,095 <0,105 <0,115 <0,125 <0,135 <0,145 <0,155 <0,165 <0,175 <0,185 <0,195 <0,205 <0,215 <0,225 <0,235 <0,245 <0,255 <0,265 <0,275 <0,285 <0,295 <0,005 <0,015 <0,025 <0,035 <0,045 <0,055 <0,065 <0,075 <0,085 <0,095 <0,105 <0,115 <0,125 <0,135 <0,145 <0,155 <0,165 <0,175 <0,185 <0,195 <0,205 <0,215 <0,225 <0,235 <0,245 <0,255 <0,265 <0,275 <0,285 <0,295 <0,005 <0,015 <0,025 <0,035 <0,045 <0,055 <0,065 <0,075 <0,085 <0,095 <0,105 <0,115 <0,125 <0,135 <0,145 <0,155 <0,165 <0,175 <0,185 <0,195 <0,205 <0,215 <0,225 <0,235 <0,245 <0,255 <0,265 <0,275 <0,285 <0,295 Figure 4. Frequency of intraspecies (light blue), intragenus (red) and intergenus (green and purple) pairwise differences between ITS1-5.8S-ITS2 sequences of . A:Psilocybe, B:Deconica, C:Panaeolus.

Further inspection of Figure S2 and sequence alignments showed that accessions that were (near) identical in the ITS1 region, were (near) identical in the ITS2 region and sequences differing in sequence length in the ITS1 region were easily distinguished in the ITS2 region. The species with a moderate ITS1 intraspecies variation (e.g. D. coprophila, D. montana) also displayed a large ITS2 intraspecies variation. The species that showed no or little interspecies variation (e.g. Psilocybe cyanescens - Psilocybe subaeruginosa and Psilocybe cubensis and Psilocybe subcubensis) did not display interspecies variation for ITS2 either. The species that could not be identified based on their ITS1 sequence due to large intraspecies variation (e.g. several Panaeolus species), could also not be identified based on their ITS2 sequences. As described for ITS1, further investigation of such species groups should be performed to determine the true nature of these findings.

3.4.3 LSU Alignment and grouping of the 129 LSU sequences was easily performed as no significant length differences were present. Independent of the chosen distance model and tree building method (data not shown), three main groups could be distinguished; the Psilocybe species, the Deconica species, the Paneaeolus species (Maximum Likelihood tree shown in Supplementary Figure S3). The groups are in agreement with the ITS1 and ITS1-5.8S-ITS2 trees (Supplementary Figures S1 and S2). Where a small fourth Psilocybe group was recognized for ITS1 and ITS1-5.8S-ITS2, no LSU sequences are available for these species, preventing further comparison.

Within the LSU tree, three incongruities were observed. One accession of Psilocybe silvatica (AF042618) was placed in the Deconica group whereas the other accession of this MM species (AY129383) was placed in the Psilocybe group. Secondly the only Panaelous uliginosus (AY129384)

24 Molecular species identification of “Magic Mushrooms”

accession was placed within the Psilocybe group. Also a Psilocybe cyanescens accession (EU029946) was placed in the Panaeolus group, between three Panaeolus cyanescens sequences. Several groups were also discovered that consisted of more than one species that did not show any sequence differences. Moreover other accessions carrying the same species names were present in different groups. Whether these groups consist of wrongly named sequences or of closely related species displaying less between than within species variation could not be determined without resequencing multiple samples. Unfortunately, due to strict regulations governing the transport and possession of the species of interest, obtaining these samples for additional investigation is nearly impossible, even when collection curators are willing to contribute material. However, as marker LSU is often considered uninformative for species identification due to lack of a sufficient barcoding gap [17], we hypothesize that LSU accessions AF042618 (Psilocybe silvatica), AY129384 (Panaeolus uliginosus) and EU029946 (Psilocybe cyanescens) may have been wrongly identified, that contamination occurred during laboratory procedures or that sequence mix-up has occurred. These questioned sequences were removed the abovementioned sequences from our dataset, prior to estimation of the LSU intra and interspecies variation. The histogram reflecting the observed pairwise difference values of LSU sequences (Figure 5), shows a higher interspecies than intraspecies variation, however even less of barcoding gap is observed than for ITS1 and ITS1-5.8S-ITS2.

0.65 Pairwise distances of LSU sequences 0.42 Pairwise distances of LSU sequences 0.42 Pairwise distances of LSU sequences 0,3 0,3 0,3 Psilocbye intraspecies (n=89) Deconica intraspecies (n=50) Panaeolus intraspecies (n=12) A Psilocybe interspecies (n=1808) B Deconica interspecies (n=621) C Panaeolus interspecies (n=133) 0,25 0,25 0,25 between Deconica sp. and Psilocybe sp. (n=2366) between Deconica sp. and Psilocybe sp. (n=2366) between Deconica sp. and Panaeolus sp.(n=646) between Psilocybe sp. and Panaeolus sp. (n=1071) between Deconica sp. and Panaeolus sp.(n=646) between Psilocybe sp. and Panaeolus sp. (n=1071) 0,2 0,2 0,2 0,15 0,15 0,15

0,1 0,1 0,1

Frequencyof observed pairwise distance Frequencyof observed pairwise distance Frequencyof observed pairwise distance 0,05 0,05 0,05 0 0 0 <0,1 <0,1 <0,1 <0,01 <0,02 <0,03 <0,04 <0,05 <0,06 <0,07 <0,08 <0,09 <0,11 <0,12 <0,01 <0,02 <0,03 <0,04 <0,05 <0,06 <0,07 <0,08 <0,09 <0,11 <0,12 <0,01 <0,02 <0,03 <0,04 <0,05 <0,06 <0,07 <0,08 <0,09 <0,11 <0,12 <0,002 <0,004 <0,006 <0,008 <0,012 <0,014 <0,016 <0,018 <0,022 <0,024 <0,026 <0,028 <0,032 <0,034 <0,036 <0,038 <0,042 <0,044 <0,046 <0,048 <0,052 <0,054 <0,056 <0,058 <0,062 <0,064 <0,066 <0,068 <0,072 <0,074 <0,076 <0,078 <0,082 <0,084 <0,086 <0,088 <0,092 <0,094 <0,096 <0,098 <0,102 <0,104 <0,106 <0,108 <0,112 <0,114 <0,116 <0,118 <0,002 <0,004 <0,006 <0,008 <0,012 <0,014 <0,016 <0,018 <0,022 <0,024 <0,026 <0,028 <0,032 <0,034 <0,036 <0,038 <0,042 <0,044 <0,046 <0,048 <0,052 <0,054 <0,056 <0,058 <0,062 <0,064 <0,066 <0,068 <0,072 <0,074 <0,076 <0,078 <0,082 <0,084 <0,086 <0,088 <0,092 <0,094 <0,096 <0,098 <0,102 <0,104 <0,106 <0,108 <0,112 <0,114 <0,116 <0,118 <0,002 <0,004 <0,006 <0,008 <0,012 <0,014 <0,016 <0,018 <0,022 <0,024 <0,026 <0,028 <0,032 <0,034 <0,036 <0,038 <0,042 <0,044 <0,046 <0,048 <0,052 <0,054 <0,056 <0,058 <0,062 <0,064 <0,066 <0,068 <0,072 <0,074 <0,076 <0,078 <0,082 <0,084 <0,086 <0,088 <0,092 <0,094 <0,096 <0,098 <0,102 <0,104 <0,106 <0,108 <0,112 <0,114 <0,116 <0,118 Figure 5. Frequency of intraspecies (light blue), intragenus (red) and intergenus (green and purple) pairwise differences between LSU sequences of . A:Psilocybe, B:Deconica, C:Panaeolus.

The calculated pairwise distances between all LSU sequence pairs ranged between 0 and 0.089 (Figure 5). Differences between LSU on one hand and and ITS1 and ITS1-5.8S-ITS2 on the other, were that frequency of identical sequences obtained within a species was higher than for the other markers, 65%, 42% and 42% for Psilocybe, Deconica and Panaeolus respectively (Figure 5). Also the pattern observed for Panaeolus as a whole was more comparable to the patterns of Psilocybe and Deconica. Upon further evaluation of the Figure S3 and the sequence alignments, the within species variation of Psilocybe semilanceata and Panaeolus sphinctrinus were due to one single sequence differing from the other available sequences (AY129380 and FH755227 respectively). As shown in Figure S3 these two accessions were placed in other subgroups than the other sequences of these species and therefore require further evaluation.

25 Chapter 1

3.4.4 Comparison of markers If all sequences that in our hypothesis are incorrectly named, are omitted from the comparisons, only single species or related species groups with near identical sequences are found for all studied regions within the genera Psilocybe and Deconica. The resulting inter- and intraspecific variation for ITS1, ITS1-5.8S-ITS2 and LSU in this selection of accessions present in GenBank is in agreement with the within species variation presented by others studying the usability of ITS as a marker for barcoding and species identification [16,17,36,37]. Additionally, the authors of the study rejecting the use of ITS in favor of LSU for the forensic identification of magic mushrooms [20] based their conclusion on the sequences that we consider erroneous after resequencing of these samples. Extrapolation of these results for fungi in general and for the genera Psilocybe and Deconica in specific to the genus Panaeolus would imply that markers ITS1 and ITS2 are suitable to identify at least groups of closely related species. However the (unvalidated) Panaeolus sequences currently present in GenBank do not support this statement as sequences of several unrelated species share identical sequences whilst sequences bearing the same species name have significantly different sequences.

The presence of incorrectly identified or described sequences in GenBank has been demonstrated for fungi [37,38] and other species [11], as has the presence of imprecise sequence editing of fungal sequences (e.g. [39]). In the future alternative databases, including the Barcode of Life database [40], and UNITE [37] with more stringent quality control mechanisms for sample identification, and trace file availability have been suggested to improve the feasibility of DNA based identification. Although these efforts will certainly improve the overall reliability of DNA databases for species identification, human errors including contamination and accidental swapping of samples or sequences will unfortunately remain potential causes of incorrect sequences being submitted to databases. Additionally, at present GenBank contains such a wealth of information that ignoring such databases for this reason would be a waste of resources, even though such errors may be hard to detect when only minute numbers of sequences of a certain taxonomic group are available. The interpretation of comparisons with databases therefore remains a crucial step when molecular markers and public databases are used to identify unknown samples, especially when the results are to be used in forensic investigations. Accurate and transparent records of such comparisons should be kept, including the value assigned to the obtained results such as previously described for the DNA based identification of plants [13,41]. Additionally many laboratories will maintain an additional collection of sequences from known samples that may be used to validate public databases or obtained results when using such databased for forensic purposes.

26 Molecular species identification of “Magic Mushrooms”

4. Conclusions Comparison of the sequences obtained in this study and sequences present in GenBank illustrates advantages and disadvantages of the studied DNA regions for answering different forensic questions. Both ITS and LSU amplicons were obtained from all samples in this study, including police seizures consisting of dried and powdered samples. Due to amplicon length, amplification of marker ITS is expected to still be feasible when amplification of LSU fails. Additionally, due to the length of the informative region, the LSU sequences used in this study can only be obtained through multiple sequencing reactions whilst the complete ITS amplicon is readily sequenced in a single reaction, which gives rise to higher costs and more opportunities for error.

Of the studied markers, ITS1-5.8S-ITS2 is shown to be the most informative region for identification at the species level, outperforming the separate ITS1 and ITS2 regions and far superior to LSU. The sequences described in this study illustrate that previous disqualification of marker ITS1 for the forensic identification of magic mushrooms [20] is based on at least three erroneous sequences and marker ITS1 does not perform differently in the genera Psilocybe and Deconica than in other species of fungi. The length of both markers ITS1 and ITS2 was constant within different accessions of a species, whereas variation of up to 50 bp was observed in ITS1 length between species of Psilocybe. Although alignment of such sequences is challenging and could be considered arbitrary, if the sequence of an unknown sample is identical to a reference sequence in both length and sequence, length differences do not hamper the identification process, but add to the variation thereby increasing the certainty with which a sample can be identified. Several groups of closely related species have been identified that, based on the current sequences available, cannot be distinguished due to insufficient interspecies variation. Only by the addition of ITS, LSU and other marker sequences obtained from authenticated reference samples, ideally performed by multiple laboratories with stringent quality control measures, will help discern whether these groups of species can be distinguished by molecular markers.

In cases where the ITS1 and ITS2 sequences of an unknown sample do not match any known sequences, species identification is impossible, and the presence of the conserved 5.8S region in the obtained ITS1-5.8S-ITS2 sequence may enable genus identification through alignment to sequences of related species. Marker LSU is equally well suited for genus identification, and is sufficiently variable to distinguish between groups of closely related fungi. Through alignment of LSU sequences, an unknown sample may readily be placed in the Deconica or the Psilocybe genus, enabling categorization of non-potent or hallucinogenic species. However, as only few informative nucleotides have been identified in this marker that may be used to discriminate between closely related species, usage of only marker LSU will lead to failure to identify closely related species in more cases than when marker ITS is used, especially when only a smaller

27 Chapter 1

section of this marker has been sequenced. If the legal requirement is not species identification but categorization of a sample as Psilocybe sp. as opposed to Deconica sp., marker LSU outperforms marker ITS.

At present nucleotide databases like GenBank contain a wealth of information but those who use this information to classify or identify unknown samples should be well aware of the absence of quality control of sequences submitted to such databases and the potential pitfalls accompanying this fact. To use such public databases for forensic investigations a thorough understanding of the deposited sequences is required and in some cases it may be beneficial to generate valid reference sequences instead of or in addition to publicly available sequences.

Acknowledgements We thank Scott A. Redhead (Canadian Collection of Fungal Cultures) for resequencing of DAOM 169061, DAOM 167409 and DAOM 180389.

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30 Molecular species identification of “Magic Mushrooms”

Following pages:

Supplementary Figure S1: Maximum Likelihood tree of 151 Psilocybe, Deconica, Panaeolus and Galerina clavata ITS1 sequences extracted from GenBank. Sequences clustering in unexpected genera are highlighted. Pages 32 & 33

Supplementary Figure S2: Maximum Likelihood tree of 124 Psilocybe, Deconica, Panaeolus and Galerina clavata ITS1-5.8S-ITS2 sequences extracted from GenBank. Sequences clustering in unexpected genera are highlighted. Pages 34 & 35

Supplementary Figure S3: Maximum Likelihood tree of 129 Psilocybe, Deconica, and Panaeolus and LSU sequences extracted from GenBank. Sequences clustering in unexpected genera are highlighted. Pages 36 & 37

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Deconica

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Psilocybe (incl. Galerina clavata)

Deconica

Panaeolus

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Deconica

Panaeolus

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Panaeolus

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Psilocybe

Panaeolus

Deconica

Supplementary Figure S3: Maximum Likelihood tree of 129 Psilocybe, Deconica, and Panaeolus and LSU sequences extracted from GenBank. Sequences clustering in unexpected genera are highlighted.