This post is inspired by an overlapping blog post on the Psymbiotika Lab website here and a journalist bud, Pat, who writes for Double Blind magazine. 

​Do either of these dogmas sound familiar:

• Psilocin binding to 5HT2A receptors dictates the trip. Dose, set, and setting are the variables that cause differences in user experiences.
• A cube is a cube.

To some extent they go hand-in-hand, although the former is more extreme. 

To what degree do we understand serotonin pathways in humans? Are the brain and neural networks simple or complex? Are biological systems simple or complex? Do we understand the full pathways and mechanisms of prescribed serotonin agonists like SSRIs? Do we understand how non-psychoactive receptor agonists impact binding of hallucinogens, and do we even understand agonism of psilocin at 5HT2A?

I would argue we have a long way to go before we set dogmas on psychoactive substances and their actions to humans. We have a piece, currently in preparation, discussing how psilocybin skipped preclinical research and that we need to get basic knowledge to help advance all research on psilocybin.

Here’s a little figure I whipped up on LucidChart to illustrate where you land with how open you are to hypotheses beyond the two dogmas above.

We are testing a hypothesis that differences in the ratios of psilocybin and its analogs will impact the psychoactive experience. These ratios vary among different genotypes of P. cubensis when bred for allelic differences at the psilocybin locus. Ultimately we will either support or reject that allelic diversity at the genes that produce psilocybin impacts an experience.

Until there is clear evidence that supports or rejects whether a cube is a cube, it could be fun to keep an open mind. Cubes under cultivation have low founding diversity and have been highly inbred. There are some easy comparisons to make if you want to test for yourself if a cube is a cube. We have shown (figures in a couple of places below) that the following populations of cultivars differ in their alleles at the genes that produce psilocybin: Penis Envy, Golden Teacher, Treasure Coast, and Luminous. Compare different members of those populations to make up your own mind on whether a cube is a cube in the meantime… but hopefully genuine data coming to you soon.
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Links to references in the figure (and a couple of others)
Psychoactive effects cannot be differentiated by users (ref here)
Analogs of psilocybin bind to serotonin receptors just as well as (if not better than) psilocin (refs here and here)
Analogs of psilocybin are non-hallucinogenic but norpsilocin may have antidepressant activity (ref here)
Naturally derived psilocybin outperforms synthetic psilocybin in terms of longevity of neural plasticity in mice (ref here)
And here’s a link to the piece on diversity at psilocybin alleles in cubes (here)

I’ve pondered the evolutionary origins of psilocybin for longer than I’ve researched magic mushrooms. Framing the case for designer shrooms landed me with a satisfactory answer, and I can stop thinking about it. I recently shared these thoughts in a talk to the Queensland Mycological Society (available here, but the sound could be better) and thought I should put them on the blog. These ideas are not new; some are explored by Meyer and Slot (available here) and by Walton (available here), who writes about amatoxins.

​Evolutionary Timeline: Psilocybin has been around for a long time, at least as long as Psilocybe, which is 20–90 million years old according to a clock-like rate of evolution. The most recent common ancestor of Psilocybe has a mean age of 66 million years (estimated by Bradshaw et al., 2024). Magic mushrooms have diversified into similar niches worldwide, each with different symbionts in their ecosystems. As mentioned for designer shrooms, the genetic pathways to produce psilocybin have been refined over 20+ million years to suit particular niches, and this may lead to variations in user experiences between different species of magic mushrooms, but this hypothesis requires testing.

​Innovation of psilocybin in evolution: I end up on the hypothesis that psilocybin was innovated to either protect, attract, or control metazoans that ate mushrooms (nematodes, insects, gastropods) across different niches. The exact reason psilocybin evolved in the common ancestor remains unclear and can never be truly known. Instead, let’s examine its current potential evolutionary innovations.

Protection: Psilocybin likely was/is used as a deterrent over evolutionary time. Serotonin controls appetite in all metazoans that eat fungi (see here). Magic mushrooms might trick their predators into early satiation, thus saving their fruiting bodies from grisly mastication. Meyer and Slot discuss this hypothesis. Psilocybin could also protect through disorientation, which overlaps with control. To support a hypothesis of protection in extant species of magic mushroom, metazoans would need to be observed rejecting more after an original taste, and it would be beneficial to know which serotonin pathway was triggered.

Attraction: One of my co-authors, Jan T, has photographed slugs and insects consuming spores inside lamellae of Psilocybe subaeruginosa, likely aiding in dispersal. Animals that provide dung niches, like cows dropping a substrate for P. cubensis, might help spread spores in ideal environments. Walton (link in paragraph one) discusses similar symbioses in relation to amatoxins. Walton makes the case of an evolutionary dead end if an organism harms the provider of their niche. Cows likely eat P. cubensis, whether accidentally in deep grass or possibly intentionally if mushrooms are palatable. Cubes could cut out the middle man (grass) by having their spores directly eaten by their primary niche provider. In some niches (particularly for taxa in dung), Psilocybe may well have benefitted from attracting spore dispersers. To support a hypothesis of attraction, metazoans would need to preferentially choose Psilocybe over comparable food, further, it would be necessary to determine whether spores remain viable after digestion (which we know is true for P. cubensis), and whether attraction is linked to any serotonin-based reward.

Control and manipulation: Psilocybin might play a role in controlling its predators with an example I liken to 3D-chess. Amateur mycologist and absolute legend, Dave Bennett, contacted me when I was working at UQ and described an experiment in which they fed slugs magic mushrooms. Slugs with a magic mushroom diet switched from nocturnal to diurnal feeding. Psilocybin may affect photoreception, which is controlled by serotonin in arthropods (and made famous by the parasites that manipulate amphipods, read about it here). In this case, is psilocybin manipulating its predators to be active during the day to increase their chances of predation by birds or bigger animals? 3D-chess indeed. To support a hypothesis of manipulation, a behavioural change must be observed that can be linked to benefit the mushroom.

Conclusion: Psilocybin was/is likely innovated by magic mushrooms as a protectant, attractant, and manipulator, all in the name of sexual reproduction and spore dispersal. We will never know with certainty why the common ancestor of Psilocybe produced this secondary metabolite. Instead, we should consider psilocybin as a compound continuously innovated by magic mushrooms in various niches. Mammals likely had no symbiosis with the common ancestor of psilocybin-producing mushrooms, but evolutionary innovation allows old compounds to take on new roles over time and space, and magic mushrooms formed new symbioses based on a dung niche and serotonin receptors of mammals. The profound and life-changing effects of psilocybin on humans is an evolutionary stroke of good fortune, but I’ve come around to the idea that selection may have innovated it closer to our mammalian home.

Clear Statement: The evolutionary origin of psilocybin had nothing to do with mammals. However, if we treat psilocybin as a dynamic compound innovated by fungi to exploit different niches, then mammals certainly provided new opportunities for niche exploration, spore distribution, and protection, attraction, or control through their serotonin receptors. The difficulty is testing these hypotheses ethically and legally, I’m just glad we’re part of a community that does the science despite the hurdles in place to develop new knowledge.

When the TGA down-scheduled psilocybin, I felt hopeful. I envisioned Australia leading the charge to alleviate a global mental health crisis. Some psychiatrists have expressed concerns that the TGA announcement may have been premature. Importantly, the announcement has advanced the discussion and helped to further erode a stigma that psilocybin is a dangerous drug without potential.

Strict clinical use is not a solution
Psilocybin shows potential to treat mental health disorders affecting up to 40% of the population, however, clinical trials face significant challenges. One key issue is to demonstrate meaningful performance over placebos — participants often discern whether they have received the treatment or a control. This is a challenge for all psychedelics, and outperforming a placebo applies to all medications. Current research does not conclusively support psilocybin as a prescribed treatment and there is ongoing debate within the psychiatric community on how to accurately measure its benefits for mental health (discussed in a review here).

The population interpreted the TGA decision as tacit approval that psilocybin is a promising treatment for depression. I know first-hand because I was contacted by many to be included in trials, even though I work on the evolution of fungi. The TGA announcement swelled demand from desperate people who cannot be treated in a short time frame. This highlights a need to evaluate legislation and operational procedures for psilocybin. Under the current trajectory, even those who fit the criteria for clinical use may be financially excluded given the estimated prices of treatment (at $15–25,000).

The second problem is that benefits of psilocybin are not limited to those with mental health disorders, and a therapeutic-only model unfairly excludes many. People seeking profound and life-changing experiences from psilocybin, which are agreed clinical outcomes, are still penalised harshly for possession of a compound that can only be prescribed if they meet conditions of a mental health disorder.

What about safety?
Harm from psilocybin use typically stems from impaired function during an experience, contraindications with serotonin receptor medications, discomfort or unpleasant thoughts, and it should be avoided by those with a predisposition to schizophrenia (some reviews on the dangers exist here, and here). Legal penalties for possession are perhaps the strongest deterrent for people not on antidepressants or without a genetic link to schizophrenia.

While acknowledging the potential risks of impairment during a psilocybin experience, we must recognise that safety concerns alone should not dictate legality. Many legal activities carry inherent risks, from extreme sports to alcohol consumption, yet society manages these risks through education, regulation, and responsible use guidelines. Similarly, promoting safe usage practices and informed decision-making around psilocybin can mitigate potential harms and allow individuals to responsibly explore its therapeutic potential.

Psilocybin is arguably safer than many substances we encounter daily. It is non-addictive, has no known lethal dose, and has been used tens to hundreds of millions of times without incident (based on grey-market data, there are more than 1 million doses taken in California per month). The impairment lasts up to four hours and is best experienced privately and in a stress-free environment, hence an emphasis on set and setting in the clinical space. Human culture has found ways to mitigate risks in many activities considered risky, and we can surely solve responsible use of psilocybin through education and restricted access.

What Safe Use Might Look Like
Given that psilocybin is non-addictive, has not caused any direct deaths in its countless uses, and we know how to mitigate the primary risks, is it time to reconsider legislation around psilocybin? These authors consider psilocybin as a drug with low potential for abuse and low risk of addiction, equivalent of a schedule 4 medicine. What would be the outcome if it was not considered a medicine at all (similar to a restricted substance, alcohol)?

Changing psychedelic legislation is complex and requires creative solutions. If someone has done their research, believes they might benefit, and wishes to use psilocybin responsibly, should they be penalised under laws created when psilocybin was misunderstood and stigmatised?

Under a model where people are not prosecuted for psilocybin use, they could operate under a social contract of responsible use, whether for self-improvement or therapeutic benefit. This approach would reduce legal penalties, allow use beyond mental health conditions, eliminate clinical expenses for users, and still be applicable for clinical use for those in need. This could happen if psilocybin was not treated as a medicine but as a restricted compound, similar to addictive and carcinogenic compounds such as alcohol and nicotine.

Conflicts of interest and bias
Conflicts of interest can bias this conversation. Psychiatrists advocate for controlled, clinical use and their training and expertise is the requirement to assess and prescribe psilocybin to those who fit their criteria. Psilocybin is still available in psychedelic assisted therapy if it is a restricted rather than controlled compound.

Those outside the clinical model argue that psilocybin has been safely used for decades without therapeutic oversight. The psychedelic community has developed guidelines and knowledge for responsible use, available to anyone at minimal expense and outside clinical settings.

There are concerns about biases in the production and distribution of psilocybin. Those who have developed doses would benefit from keeping psilocybin as a scheduled substance, as it keeps competitors out and prevents access to market by mushroom growers who can produce psilocybin very cheaply (and who would benefit from an open supply chain).

If you’ve made it this deep, you may know my opinion already: I think psilocybin should equitably be available to those who have done independent research and believe they would benefit (giving them the same standing as tens to hundreds of millions of people who have tried psilocybin illegally). Our new company researches the phenotypic outcomes of genetic diversity and provides psilocybin for research use. We benefit if psilocybin is heavily legislated and excludes others from competing. However, the humanitarian outcomes of changing legislation around psilocybin and my personal beliefs are contrary to my commercial interest at this point in time.

Where we need research
We need foundational knowledge on psilocybin and other tryptamines found in magic mushrooms. This knowledge should come from cellular models and invertebrate animal models (the original targets of psilocybin). Human clinical trials seem premature without a thorough understanding at the cellular level.

There seems to be a trend in clinical literature that psilocybin is only safe with therapy. The non-clinical community would posit that the psilocybin experience itself is therapeutic. It would be insightful to see a controlled experiment comparing outcomes with and without psychotherapy following psilocybin use. The psychedelic assisted therapy model has adopted therapy without meaningful comparison to a control (see section on conflicts of interest).
​
Areas we must discuss as a community
Clinical use has helped reduce the stigma around magic mushrooms, but it cannot be the only safe avenue for psilocybin use. We need a conversation about the safe and legal use of magic mushrooms outside clinical settings to meet the growing demand. Psilocybin will be used by people no matter how it is scheduled, and given its availability in magic mushrooms that are ubiquitous in Australia, and its ease of production, the black market demand will remain and be met. A discussion should address therapeutic access and public safety concerns without exacerbating black market activity and aim to:

1. Increase safety (through set and setting, mushroom identification, purity).
2. Promote responsible use.
3. Decrease disproportionate legal penalties.

People should not be prosecuted as criminals if they are aware of the risks and choose to use psilocybin to improve their mental health or for recreational benefits. The risks and safety of psilocybin have been demonstrated by its extensive use over the past 50 years. Even though these data are not peer-reviewed, they are meaningful and have advanced knowledge on psilocybin more than a scientific community that must operate under ethical rules and within the legislation of scheduled compounds.

Taxonomy is the bedrock of communication about life. If other facets of communication changed, it'd be pretty frustrating, e.g., "from here on, hello will mean mustard". 

I witnessed the saltiness of taxonomic change first hand in South Africa. Africans had spent their lives calling their iconic, thorny trees Acacia. Acacia even means thorny. That didn't suit taxonomists and all the African acacia were renamed to Vachellia. The non-thorny Aussie trees, of which there were >1,000 species compared to 100-ish in Africa, took the name. Ja, I can see why they'd be salty.

In other areas of science, we support or reject hypotheses and move on. Taxonomy is descriptive, hypotheses can be loosely applied (we hypothesise this is a monophyletic group of taxa), but it's a stretch. When two people describe the same piece of art or music, they may differ in their opinion. The same is true for taxonomy. Applying taxonomic ranks to organisms is arbitrary and cases can be made to split or lump any rank at a shared common ancestor. Ultimately, determining gene flow among populations paints a clear picture of species boundaries, but who has the time to do this for millions of fungi.

I find it interesting that we become attached to taxonomic names, even when there is evidence to support that they should be called something else. I think we are more emotional because of the vagueness in species/taxon delimitation. In the smut and rust section of this blog, you can read about the taxonomic name for corn smut and why it should be called something other than Ustilago (but jeeze, you'd have to be bored). Clearly even I become emotional about taxonomy.

Taxonomic names, as long as they were validly described, are always there for communication and can be used eternally (as long as people know what organism you mean). Just 'cause some fella is saying all the northern hemisphere wood-loving shrooms are Psilocybe subaeruginosa doesn't mean you have to forgo these names. Keep on using them... what do you think African people call acacia (hint: not Vachellia)? The only change here is that we know Australia is the centre of origin of P. subaeruginosa and it has spread to the northern hemisphere where multiple new names have been applied to one taxon.

​The figure illustrates genomic relationships based on core and accessory genes between sister species. For magic mushrooms, the sisters are cubes and subs. Note they are highly separated by core genes and there is almost no overlap of accessory (non-core) genes. Colletotrichum is the other example, and it has been split like a banana in a dessert bar. Note that the core and accessory don't clearly separate species. Likely these are all the same taxon and taxonomists have described clones as different species (which could be acceptable in some instances).

After this explanation, if you still prefer species complexes and lots of names for the same taxon, why not spend your time and resources examining the boundaries of recombination to demonstrate speciation in process?

Set and setting, and psilocin binding to 5HT2A receptors are the two domgas of how magic mushrooms work. To the point it feels that's all we think matters to dictate a psilocybin experience from magic mushrooms.

Let's think back a couple of million years to before mammals were on the scene.

Under a clock-like rate of speciation, species are between 500K to 5 million years old. Genera are somewhat older, and Basidiomycota are an anomaly compared to other organisms. Genera of Basidiomycota are usually 20–100 million years old. (Check out this paper if you want to read more: ​https://academic.oup.com/mbe/article/32/4/835/1078218).

Psilocybe could be anywhere from 20 to 100 million years old. We assume that the most recent common ancestor of all species of Psilocybe could produce psilocybin (because all except one produce it), and likely psilocybin is the same age as Psilocybe.

All species of Psilocybe, extant and extinct, have forged their way in niches of leaf litter, wood, grass, moss, and dung. We don't know the exact purpose of psilocybin, whether to repel, control, or attract, but we can be certain it targets metazoans with serotonin receptors. Whatever metazoans dominate the different niches of Psilocybe, we can be sure over an evolutionary time scale the genes that produce psilocybin in 200 or so species are refined to best target species of slug, arthropod, nematode, or whatever was eating it for whatever reason in whatever niche. This has happened time and again with co-evolved relationships, especially those with metabolites governing the symbiosis.

If you accept that there are different predators across 200 species of Psilocybe, you may be on the way to understanding why I think different magic mushrooms give different psychedelic experiences. Different effects from different species given an evolutionary time scale is hopefully palatable, but to say that allelic variation in one species changes an experience is more difficult to swallow, especially with the dogmas of how trips work.

We showed that populations of P. subaeruginosa maintain genetic diversity in the alleles that produce psilocybin rather than just one becoming dominant (balancing selection to be fancy). This is probably why WLP can be in some, but not all genotypes and is maintained in populations. What the blazes is the benefit of all this diversity? Diversity to the extent that psilocybin alleles are barely shared by closely related populations and there is reason to suspect recombination within the locus itself that would ensure that the same alleles in the pathway are not linked (or not always inherited together).

Long story short, our work on subs shows an evolutionary process of maintaining allelic diversity in the psilocybin pathway (or balancing selection).

Cubes have a solid 5–6 alleles at the psilocybin locus in the entire population of cultivated mushrooms and a bunch more in naturalised populations. I've grown most of these and am in the process of testing the tryptamine landscape in homozygous genotypes. I've crossed different alleles to see what happens when the psilocybin locus is heterozygous.

I've never been more convinced that there are phenotypic impacts from genotypic diversity, and I hope to hold hard evidence soon. Rest assured, if the time comes, the people will be able to decide if there are differences themselves. In the meantime, do me a favour next sub season: don't combine harvests from different patches. Experience them separately and share whether there is a difference (given a consistent set and setting). Expect there to be different psilocybin alleles at different mushroom sites, unless the mushrooms are harvested from mulch/woodchips that have been spread artificially (wild harvests will guaranteed be different alleles).

More on all this soon.

Full piece at Current Biology here:
https://www.sciencedirect.com/science/article/pii/S0960982223014604?dgcid=author.
​And just in case you hadn't seen that our sub work is publicly available, just not peer reviewed right here: https://www.authorea.com/users/700719/articles/687526-wood-loving-magic-mushrooms-from-australia-are-saprotrophic-invaders-in-the-northern-hemisphere.

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?'
​
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. 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. subaeruginosa paraphyletic in regard to P. cyanescens, P. azurescens, and P. allenii. 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.