Here's a taste of what will come from 32 new Psilocybe cubensis genomes freshly assembled. First of all, thank you to Andrew B, Matt W, Bocky and Dan, Rhys L, and all landholders in southeast Queensland for spores. We ended up with over 50 haploid (or so I thought) cultures of Psilocybe cubensis.

Three of the 32 genomes were probably dikaryotic, which is a reflection on how good I think I am at culturing compared to my actual culturing ability. The figure is a network based on 529,027 SNPs. Preliminary interpretation, there is probably structure by geography, the populations are not admixed.

More soon, including some answers about mating! We've resolved reproduction in Psilocybe​, now just going through the motions of publishing.

This figure compares basidiospores from P. subaeruginosa collected in SE Queensland (A & C) and Tasmania (B, by Caine B), and of P. cubensis (D). Psilocybe cubensis and P. subaeruginosa are sister taxa (they shared a most recent common ancestor). One might interpret there is not much morphological difference between the populations of P. subaeruginosa separated by a strait and two states.

Recording available here: https://vimeo.com/713129405/2c456a2298

Recommend watching the talk on Clandestine Dimethyltryptamine (DMT) laboratories encountered in Queensland by Dr Tim Currie. 

The team at Psilopedia have released genomes of 84 cultivated lines of Psilocybe cubensis. How exciting. I did what anyone with a couple of spare genomes of P. cubensis and access to high performance computing would do to determine how the Oz population was related to what people are growing overseas.

The Psilopedia genomes are based on DNA extracted from spore prints, which means there are thousands of haplotypes in the assembly. Not great for contiguity but still fantastic for analyses of genetic diversity. I annotated 80 of the Psilopedia genomes and three of our Australian haploid genomes (collected from the Gold Coast). I called SNPs from protein coding regions of sites that were present in every assembly.

The SplitsTree figure above is from over 500,000 SNPs and it shows that the Gold Coast population of P. cubensis is completely different to the popular cultivars used by the community. Another interpretation is that there is more genetic diversity in the three haploid genomes from Australia than the entire population of cultivated Golden Teacher (based on branch lengths of genetic distance). Likely, gold tops were introduced to Australia before P. cubensis was widely cultivated.

I further looked at the genetic diversity in the Golden Teacher population. This is open to interpretation and would be better with other comparative populations. I interpret this relationship as inbreeding, with little reticulation toward the tip ends and closely related individuals on longer branches. The Index of Association here is a little meaningless at the moment. In obligate outcrossers we expect lower linkage among sites. The Index of Association gets smaller when there is sexual reproduction (increased randomness of allele combinations across the dataset) and higher in clonal populations (the same alleles are always recovered together).

Stay tuned. Next week I will submit another 32 isolates of P. cubensis to sequence genomes from NSW and Queensland and determine whether the population is from one introduction, whether there is outcrossing, the age of introduction and whether there have been any escapes from people cultivating mushrooms. 

This post contains two updates, (i) a part on the hallucinogenic species in the Northern Territory, and (ii) how to set the mood for fungal reproduction.

There was some media attention after the last blog post. It was refreshing to see how many people are interested in fungi, not just magic mushrooms. Thank you to all who have volunteered time to help the project, this means a lot.

I looked for names to apply to the species in Kakadu and Litchfield National Parks in case something was a clear match. No name can be applied with 100% confidence, however, when Gaston Guzman described Psilocybe brunneocystidiata from PNG, he hypothesised it would be a close relative of P. yungensis, which was described as a wood decomposer from Bolivia. Is it a coincidence that the mushroom from the NT was sister to P. yungensis in the exact relationship the most prolific Psilocybe-taxonomist predicted for P. brunneocystidiata? We may never know for certain whether this name could be accurately applied to the taxon in Kakadu, but it is a starting point considering we don't have the fungus in hand.

I have received emails aplenty of magic mushrooms occurring in the NT, including in Kakadu. Some of these we might suspect are Panaeolus cyanescens (based on their described appearance and their niche in buffalo dung). If anyone from the Mirarr people know about traditional use of mushrooms and they have knowledge that is appropriate to share, we should get in touch.

One last point, a new species in taxonomy is one that does not have a binomial Latin name. I accept hypotheses of a clock-like rate of speciation (in which species are approximately 500,000 – 5 million years old), technically there are no 'new' species unless humans have caused selection pressure or new pathways for outbreeding/hybridisation. To say something is a 'new species' is taxonomic jargon for 'let's give this thing a name so we can improve communication'.

If you have watched some of my recent seminars (aren't you lucky), you'll have your finger on the pulse with the mating (MAT) genes annotated from the assemblies of P. subaeruginosa. By chance, the population I sampled has a perfect distribution of MAT alleles so that I can confirm our predictions about mating from the genomes.

Basidiomycota (mushrooms, smuts, and rusts) have homeodomain genes (usually two of them close together on one chromosome) and pheromone/receptor genes used for mate recognition and signalling. These genes will become useful when we study whether P. cubensis and P. subaeruginosa are native to Australia because we expect a lot of diversity at homeodomain genes in natural populations. Generally opposites attract for sexual reproduction and two haploid fungi must differ at their MAT loci for a compatible cross. If they must differ at both HD and P/R loci, mating is controlled by two loci and different MAT alleles are needed (tetrapolar mating). If fungi only need to differ at their HD locus for a compatible cross, or the HD and P/R loci are closely linked (usually by proximity), only one locus controls compatibility (bipolar mating). Tetrapolar mating is useful for fungi that cannot move long distances because it reduces the chances of inbreeding.
​
Psilocybe subaeruginosa has three P/R loci, each of which segregated in our population. Now we need to work out which of these is controlling compatibility. Here are my predictions for the crosses in the image below based on what we know from the genome. Successful mating will be 8x5 and 8x6, the rest probably will be incompatible.

Yesterday I guest lectured in Liz Aitken's Fungal Biology class at UQ (BIOL3210). Fun on a bun. One student asked a question along the lines of 'are gold tops only present in cow farms?' I answered that they are potentially everywhere but need certain conditions to fruit, this is based on their ability to distribute spores and that saprotrophic fungi could form hyphae wherever there is a source of carbohydrates they can access.

How to test this question a bit more? I jumped on to Bioplatforms Australia and downloaded the curated fungal ITS database, which includes all ITS sequences (~250 base pairs) from sampling sites around Australia (link to the data here). I expected that P. cubensis would occur in many sampled environments, even if there were no cows around. How wrong I was.

There are 3,412 BASE sample sites. Many of the fungi identified as Psilocybe in their dataset were Deconica, and when these are removed, species of Psilocybe occur at 17 of those sites (<0.5% of sampled sites!). Here's a link to a pdf of the above figure if you want more resolution.

One of the most exciting things to me is the putative hallucinogenic species of Psilocybe in the Northern Territory, recorded in Kakadu and Litchfield National Parks. These sequences, which had just under 100 repeats in the dataset, formed a monophyletic group sister to the Psilocybe yungensis clade, and not closely related to anything else. Potentially a candidate for a new taxon (if species of Psilocybe have not been described from the NT), and potentially evidence that hallucinogenic species of mushroom occur in Kakadu NP. To all those reading in the NT, why not head to Litchfield NP for the day next time you've had some rain :). Send me some spores and we can describe it if it's a new taxon.

I've added the location and number of reads to the BASE sequence data. You'll notice there are no sequences of P. cubensis or P. semilanceata in the BASE data, which I find surprising for two 'weedy' taxa in Australia. Plenty of P. subaeruginosa s. lat., with some of the those sequences near-identical to P. weraroa (which is not very informative in this complex). More sequences and locations than I had expected for the P. papuana clade. 

What does this mean? Psilocybe are present, whether as spores or mycelium, at the sample sites. The sampling method of BASE may have biased whether Psilocybe was sequenced, which may explain the relatively few sites that had Psilocybe present. And lastly, in answer to the question from mycology yesterday, I'll change my answer to maybe P. cubensis is less ubiquitous than I thought :)

I photographed these Psilocybe cubensis to illustrate what I think is herbivory (and I'm always prepared to be wrong). A chemist colleague of mine was chatting about the origin of psilocybin to deter insects, but I think that hypothesis requires testing.

​Maybe knowledge of who eats Psilocybe now can shed light on why psilocybin evolved. Whether it was a deterrent or an attractant to help spread spores is something I would give anything to know :). A complex gene pathway that has evolved convergently and been acquired horizontally indicates there is an evolutionary advantage to produce psilocybin. 

I've also wondered whether cows mistakenly hoover up stray mushrooms from long grass. I can imagine they would, and a farmer thought his cows went 'loopy' every so often. Surely humans aren't the only beneficiaries of psilocybin.

All this talk about haploid stages, I thought I should illustrate it for y'all. Mushrooms (n+n) are the site of recombination, which occurs after nuclei fuse in the basidium (karyogamy to make a diploid nucleus, 2n). Basidiospores are haploid (n), containing one recombinant nucleus.

I use a single basidiospore to grow cultures to make the genetic analyses easier with only one copy of each chromosome instead of two in a dikaryon (n+n) or diploid (2n). I've added mating genes in Basidiomycota to the figure; the homeodomain locus (HD), which usually has two genes, and the pheromone/receptor locus. Alleles of all genes at these loci must differ for a compatible cross. I'll cover this more when I've annotated the mating genes in our genomes of P. cubensis and P. subaeruginosa.

Thank you to AGRF for the high quality genomic data that we have since assembled. Each sample had 19–27 million reads (about 6–8 Gb of data). I was worried that I didn't sequence Psilocybe, however, our rDNA contigs are a perfect match for P. subaeruginosa and P. cubensis (pictured above). Phew. (The resolution of the tree is atrocious, if you're desperate to see the taxon selection, there is a pdf here).

My next worry was that I had contaminated haploid cultures with dikaryotic cultures. What a relief, everything that is meant to be haploid, was haploid. The below figure shows that the one dikaryon (essentially a diploid) we sequenced was a much worse assembly than our haploids. I'll use this information to only sequence haploids in the future. Next step is to improve these assemblies with some long reads from PacBio. Stay tuned for updates on what we pull out from these genomes in the next few weeks... with a decent annotation, we may already be able to make a case that P. cyanescens is con-specific with P. subaeruginosa​, depending on their similarity.