A hint to the end of my research career came the first time I met the Institute Director at QAAFI in November '22. He opened our online meeting with 'Hello, your work doesn't really fit in here does it?'
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Bare-chested, riding the unicorn of the TGA announcement in February '23, Chris Appleyard, the first person to legally grow magic mushrooms in Australia (to my knowledge), called me up and offerred me a job. A stroke of good fortune for yours truly.

My role at Funky Fungus is in designer shrooms and we have approval to fruit mushrooms. This was something missing from my permission to work on shrooms at UQ. 

Chris has stocked the Funky Fungus library with all of the cultivars I've researched, and what a pleasure to finally get to see them in the flesh. Chris owned one of the properties where we first collected Psilocybe cubensis back when I began studying magic mushrooms (the Woodford specimens, specifically Delaney's Creek, if you want to see where they fit in the Australian population in the Manure Tour or posts below).

​Where's all this going? I'll keep the blog going because we have a research grant to characterise the concentration of psilocybin (and related tryptamines) of 100 genotypes in the FF library and to compare differences between growth treatments (such as blue light while pinning, degradation of psilocybin after harvest, mycelium vs mushroom, and whatever else we think of). I also thought I should explain the reasoning behind designer shrooms and why we're crossing different haplotypes.

Here's a brief outline of the process: fruit cultivars, make haploid cultures, cross against Oz haplotypes, identify cool phenotypes and genotypes that produce high or different concentrations of tryptamines. Who knew I'd get to put all the knowledge we've found to good use!

​You've heard me harp on about Penis Envy for ages. It's one of the most interesting cultivars to me because of very different alleles at the psilocybin locus compared to most other cubes (Treasure Coast is the other interesting cultivar), and P-envy is highly homozygous and will be pretty cool to cross against.

Just over two years ago I started this research journey on magic mushrooms. I feel privileged for this opportunity and to have joined a community fascinated by these Fungi.

Some new knowledge may have slipped by over two years, and now, at the end, it seems like a good time to summarise the outcomes, knowledge gaps, rejected hypotheses, and my predictions.

Psilocybe cubensis
Is naturalised in Australia. We know this because there is a large drop in its effective population size, followed by a recovery. It has very low mitochondrial diversity (we expect high diversity in a centre of origin), low diversity at psilocybin loci, but reasonably high diversity at mating loci because it has been outbreeding since its arrival.

Psilocybe cubensis probably does not infiltrate the soil, which is why it doesn't occur in soil sequencing data. Rather it colonises manure, develops its hymenium, fruits, spreads new spores that are probably eaten by livestock, and then they come out the other end. Each genotype is ephemeral, which is why temporal sampling over three years at Tallebudgera shows unrelated genotypes. I believe there is population structure by geography, but movement of cattle and manure helps long-distance dispersal.

Cubes were certainly not introduced intentionally by recreational growers. They've been here too long and there is no shared ancestry with cultivated lineages based on genetic analyses. There is more allelic diversity of P. cubensis in one piece of manure in Australia than in entire cultivated lineages. With this reservoir of diversity, someone with the right laminar flow could fix traits unseen by the community and put some 'spice' back into cultivated cubes.

The majority of cultivated P. cubensis either came from one naturalised population or one genotype, it's hard to tell (to understand this, look at some of my cube networks with knowledge that clusters in Australia all came from one parental genotype). Cultivars like Penis envy have been completely inbred to the point where there is almost no heterozygosity in their genomes. This is part of the breeding process to fix traits over time, and may explain why 'a cube is a cube' despite unexplored diversity waiting for innovation.

Taxonomists describe new species of fungi based on phenotypic differences reflected in morphology and ecology, and genetic differences, usually by comparison of conserved genes (like the ITS and LSU regions of ribosomal DNA). The challenge for taxonomists is to find a happy medium between lumping and splitting species. Species boundaries that are too broad cannot distinguish among actual differences (such as pathogenicity and geographic range), and lose meaning when too narrow and many names are applied across intraspecific variation.

The ITS region is a biological gem. It can be amplified with universal primers across the fungal kingdom, it is interspecifically variable (usually), and intraspecifically conserved (usually). I fell in love with the ITS region working on smut fungi and rust fungi, both of which are more easily amplified with ITS primers than any other genes. But the ITS is not a Cinderella fit for all Fungi, there is not enough variation among species of Ascomycota (e.g., the ITS is not variable enough to distinguish species of Fusarium), and some familiarity is needed to understand the limitations of the ITS region in different groups.

The Psilocybe subaeruginosa species complex has grown based on variation in the ITS region, phenotypic variability, and geographic range. It includes P. cyanescens, P. australiana, P. eucalypta, P. tasmaniana, P. azurescens, P. makarorae, P. weraroa, and P. allenii, the latter two described on differences in rDNA. Below is a phylogenetic hypothesis based on an alignment of the ITS region including all of the 86 sequenced genomes of P. subaeruginosa from Australia, and sequences taken from GenBank of other taxa in the complex.

​The ITS region is intraspecifically variable across the complex in Australia. This variation makes P. cyanescens, P. azurescens, and P. allenii paraphyletic in regard to P. subaeruginosa. A taxonomic solution would be to either call everything P. subaeruginosa, or potentially describe more and more species to reflect groupings. This 'splitting' option is unsatisfactory because it would not match the population analyses posted previously (essentially the relationships among ITS do not reflect population networks based on >1.5 million SNPs).

​My opinion is that P. subaeruginosa is one phenotypically variable species with a widespread distribution caused by saprotrophic invasions in the northern hemisphere. Describing more species in the complex ultimately adds more future synonyms.

Another sub season is soon upon us. The story of how populations of P. subaeruginosa are structured in Australia is quite clear, even though we're missing samples from the entire length of NSW. Populations are structured by geography, with some localised, mountain-top populations in Queensland having few opportunities for outcrossing. Populations in southern Australia are divided by a mountain range. There is evidence of distribution of P. subaeruginosa in mulch, for example, the populations from Clifton Hill and Geelong (both mulched garden beds).

It's been fantastic to speak with the collectors of these mushrooms. Based on their information, it is likely mycelium of P. subaeruginosa is persistent from year to year. This persistence keeps the same genotypes at the same locations, which is different to P. cubensis for which genotypes are ephemeral and dependent on spore distribution in manure (in my opinion). A comparable example to P. subaeruginosa is the humungous fungus among us, which is a huge clonal network of mycelium. Mycelia of P. subaeruginosa is ubiquitous in natural Australian environments, and those of you who re-visit mushrooms from season-to-season see the same genotype every time. There are opportunities for outcrossing, especially with mulch distribution and artificial movement of soils and wood chips.

Psilocybe azurescens and P. cyanescens are introduced to the northern hemisphere from Australia. They cluster nicely among our populations from Australia, although we weren't lucky enough to sample their exact provenance in this study. For context, much of the sampling is of siblings, sampled from sister basidiospores from single mushroom caps. Keep in mind that many of the genomes in this figure are offspring and that will put the genetic distance in context. The genome of P. subaeruginosa is about 50 million base pairs, and I'm looking at a tiny fraction of differences (1.5 million base pairs) to illustrate how these genomes are related. I think these data support P. subaeruginosa is one species and other related taxa are synonyms. My opinion is no indication of the future taxonomy, and many taxonomists prefer to split groups of microorganisms into different species.

There is a lot of variation at the psilocybin locus in our sampled populations of P. subaeruginosa. It's about to get interesting if we can link any of these variations to Wood Lover's Paralysis. Should have something for you in the next couple of weeks. 

The P. cubensis study is finished. It's sitting with my co-authors for their magic touch. Looking forward to sharing some new knowledge about mating in cubes with y'all as well :).

​Apologies for the delay. At last, the data are in a state to analyse. We ended up with 85 genomes funded by BioPlatforms Australia, of which 80 were Psilocybe subaeruginosa.

The data are assembled and annotated, and the below figure is a way to visualise the relationships among the sampled populations, plus three genomes from GenBank of P. azurescens and P. cyanescens. The most obvious take home at this early stage is P. azurescens and P. cyanescens are likely conspecific with P. subaeruginosa. No surprises here, this was always the hypothesis. But good to see it play out the way we expected.

These data aren't perfect yet and I'm still finalising the SNPs. More information coming, especially on the psilocybin gene cluster, soon!

Most of my time at the moment is toward publishing the analyses of P. cubensis and the story behind their introduction to Australia. We've resolved mating in cubes, which is similar to P. subaeruginosa, but with more red herrings for compatibility. I'll tease this out a tad more soon.

@Feral Beryl Bunyip, I sequenced a genome of one of the mushrooms you sent me. Give me a shout if you have dreams of what should happen with this treat :).

I've been contacted by many Australians interested in volunteering for clinical trials. The jam is I work on the evolution of mushrooms and don't run clinical trials.

Here's an opportunity for those of you who microdose psilocybin and live in Sydney, Australia.

Dr Vince Polito in the School of Psychological Sciences and his team at Macquarie University are looking for individuals who microdose (or are already planning to microdose) using psilocybin or ‘magic mushrooms’. 

Participants will visit their lab on two occasions: once after taking a microdose and once after taking a placebo. They will use an MEG scanner to explore what happens in peoples’ brains when they microdose.

Added bonus, they can reward your participation financially!

For more information and to sign up, please visit microdosingstudy.com or email microdosing.research@mq.edu.au

You can find more about Vince Polito’s research here. 

You can read more about their study here. 

Thank you Ronny, Eva, Jay, Jacob, Nicole, Billy, Sophie, and Caine for taking care of us and all the hospitality at Entheogenesis. Fun on a bun, and fantastic to meet so many of the people who have sent me spores this year.

After my talk, which you can watch here if you attended the conference, I spoke to more than 10 people about their experiences of Wood Lover's Paralysis, which occurs as a side effect when eating Psilocybe subaeruginosa, and temporarily paralyses people during a psilocybin experience. 
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The grey areas around the cause of WLP are: (i) is there variation among humans (are there more susceptible people), (ii) is it caused by metabolism when mushrooms grow on particular substrates, or (iii) is it caused by secondary metabolites produced by genotypes of P. subaeruginosa?

It felt like the majority of you have experienced WLP and with varying symptoms (from full paralysis including not being able to speak, to paralysis of limbs). We might be able to rule out that some people are immune, even though one member of our community has never experienced WLP after >30 years of practice.

The anecdotal evidence leans toward substrate or genotype. Paralysis occurred in people who had picked from wild areas and cultivated garden beds, and paralysis reoccurred when people ate smaller doses of the same batch of harvested mushrooms (interesting).

In a manuscript coming out soon (it's been reviewed, just stuck in a bottleneck), we show an additional copy of one of the genes that produce an enzyme in the metabolic pathway of psilocybin. PsiH genes convert tryptamine into 4- Hydroxytryptamine, but when you have redundant copies of a gene, they can start to innovate. Redundant genes are not under selection (their previous function is wrapped up by the original copy) and they can explore mutations over generations to take on versatile functions. If the new enzymes produced are not deleterious and confer a slight advantage, they'll stick around in the gene pool.

The first hypothesis to explore is that there are functional copies of the psiH3 gene in the population of P. subaeruginosa, and individuals with those copies produce a Wood Lover's Paralysis phenotype. Those data should be coming to me soon (it is Christmas after all) and we can all share in the excitement.

I collected a couple of Psilocybe cubensis before the season commences because these might have alleles that are suited to fruiting in versatile conditions. Could be interesting to incorporate some of these genetics in future crosses.

The cultures for genomes have grown and I will take samples to AGRF this week. We could expect genome data by Christmas, and with that we'll have a good indication of species diversity in Australia (to answer the question of whether Psilocybe subaeruginosa is more than one taxon).

We're submitting a manuscript on mating in P. subaeruginosa, and if that gets through review, I can share some neato knowledge about the psilocybin gene cluster in wood loving species. More answers on cubes coming soon as well.

Thank you to Kelly from BioPlatforms Australia, who has helped support sequencing of 100 genomes of Psilocybe subaeruginosa collected in 2022. Thank you also to the collectors of P. subaeruginosa: Caine B (and helpers), Snu, Jan T, Dave H (and team from Kosciuszko), Tim S, and anyone who has anonymously sent prints. Our project is just over one year in, with quite a lot of data considering there has been no financial support (until BioPlatforms stepped in). There are over 100 haploid cultures of Psilocybe lodged in the herbarium, and we will have sequenced 140 genomes in less than 18 months. This is an effort from the community interested in Psilocybe, I'm just the bloke at the end of the chain.

The stories on mating, biodiversity, introduction of P. cubensis, and variation at the psilocybin locus are unfolding nicely. I probably won't have finished everything before my contract finishes in November, but I'll keep working on genetics of magic mushrooms as long as I have a computer, and all these data will be made public for the community to blaze ahead. Pictured below, preparation of the 100 cultures for sequencing. Get in touch if you would like to make use of the data, otherwise they'll be public in 2 years maximum (from when I get the raw data).

Here are some new analyses with a longer-term aim to determine how long gold tops have been in Australia. Originally, in the network analyses from past posts, it appeared that populations of gold tops were structured by geography, e.g. mushrooms from Mareeba would not reproduce with mushrooms from Lismore.

On the road to determine the effective population size of Psilocybe cubensis in Australia, it now looks like there is some admixture (sexual reproduction among populations).

The below figure is a journey to determine how we should treat the populations of P. cubensis in Australia. I tested how many groups best fit the data (which is 'K' in the figure). The figure depicts how populations across Queensland and NSW would look if treated as 2–6 different groups. I settled on 6 as the number of groups in the sequenced populations. If we added new locations, no doubt we would see more geographic structure.

​The curious result is that populations from Cairns, Gold Coast and Sunshine Coast all group together. They are genetically similar. 

​This could mean that manure has been moved from southeast Queensland to northern Queensland (or vice versa). Though it is difficult to know for sure. It's also interesting that different mushrooms from the Sunshine Coast are either related to those in Cairns, or form their own segregated population.

Leave it with me, but if you have ideas on why these three locations may not be structured by geography, let's explore them together :). Thanks again to the legends who collected the mushrooms for these analyses!