this is a running list that I’ve just now decided to start. don’t ask me why.
“Each one looked very much like the other…and some looked even more like each other that they did like themselves.” -Norton Juster, The Phantom Tollbooth
“Mycelium is a body without a body plan.” -Merlin Sheldrake, Entangled Life
“If it doesn’t have an ITS sequence, it doesn’t exist.” -Tom Bruns, paraphrased from a presentation at an MSA meeting one year
A lot of progress has been made recently in understanding the genetic basis of psilocybin biosynthesis. Much of this work is being pioneered by Dirk Hoffmeister’s lab, with a significant contribution by Jason Slot’s lab demonstrating the independent acquisition of psilocybin biosynthesis by several species through horizontal gene transfer. My own research has reproduced and refined these results and added new information on the convergent evolution of psilocybin biosynthesis in the genus Inocybe. We now know that psilocybin is synthesized by fungi using four enzymes that catalyze reactions to convert tryptophan to psilocybin (Fricke et al. 2017, Reynolds et al. 2018, Awan et al. 2018). We also know that these four enzymes occur physically next to each other in a cluster (Fricke et al. 2017, Reynolds et al. 2018, Awan et al. 2018), and that this cluster is likely subtelomeric (Awan et al. 2018). This cluster has been acquired by distantly related species through horizontal gene transfer (Reynolds et al. 2018, Awan et al. 2018) and at least in one species through convergent evolution (Awan et al. 2018). We also now know that in addition to psilocybin, at least some species of Psilocybe also manufacture a class of compounds known as ß-carbolines (Blei et al. 2020), chemicals that function to inhibit the enzyme monoamine oxidase (MAO) that normally deactivates psilocybin and related chemicals. This is analogous to the psychedelic cocktail ayahuasca, which combines plants that have ß-carbolines to inhibit the activity of MAO, and plants that have dimethyltryptamine (DMT) that act in the brain similarly to psilocybin (McKenna et al. 1984). So, at least some Psilocybe spp. appear to be like miniature, naturally-occurring nuggets of ayahusaca. And that blue staining reaction so familiar in psilocybin-containing mushrooms? It is formed when cells are broken or senesce, releasing enzymes that link psilocin molecules into short chains (Lenz et al. 2019). These chains of psilocin not only create a chromophore that reflects blue wavelengths of light, but they may serve a protective purpose: like the common plant anti-herbivory flavonoids and polyphenolic tannins, these chains of psilocin may release reactive oxygen in mycophagous insect guts, burning holes in them (Lenz et al. 2019). Like I said, we’ve learned a lot about psilocybin recently. But where did this all start? What is the origin of psilocybin and the organisms that contain them? Although it’s very difficult (or impossible) to reconstruct the past, we can make inferences that at least put some bounds on what is reasonable and what is not. A few questions that were recently posed to me were: how old is Psilocybe? How old is Psilocybe cubensis? And did the acquisition of psilocybin cause them to diversify rapidly? To answer these questions, we need to know how all of the species of Psilocybe are related to each other. And we also need to know some approximate age of things near to them, like using fossils to put some absolute minimum ages on a phylogeny. Unfortunately, the fungal fossil record sucks, so it’s pretty difficult to use fossils to put dates on phylogenies. But we can use secondary dates, i.e. those that are derived from larger studies in which fossil evidence is used. The danger is that using secondary dates can amplify inaccuracies to the point where they are completely meaningless. But, nonetheless, they can be useful to provide some idea about the scale of the time period. Now, there are ~300 species of Psilocybe worldwide, but only a handful have been included in molecular phylogenies (a prerequisite for putting dates on evolutionary trees). The most recent systematic study was completed in 2013 (Ramírez-Cruz et al. 2013). So I borrowed the published phylogeny from this study and ingested it into the ‘ape’ package (Paradis & Schliep 2019) in the statistical software R (R Core Team 2019). I then transformed the branch lengths (the lines connecting the species to each other) to reflect time with a statistical tool called penalized likelihood (I’ll spare you the gritty details), using the divergence time between Psilocybe and the earliest diverging species in the phylogeny to date the origin. This date came from the recent review of divergence times in He et al. (2019). I also calculated a lineages-through-time plot (the red line), which can show departures from a steady birth and death of new lineages, indicating shifts in diversification that may correlated with certain events (e.g., the origin of psilocybin biosynthesis). Now it should be said that patterns like this are probably unreliable, as recently slammed by mathematicians. But it’s fun to look, anyway!
First, the approximate age of Psilocybe (the psychedelic ones) is ~28 million years. This is a ballpark estimate, but I think the important take home is it is measure in millions of years, not thousands or hundreds of millions. They certainly pre-date humans…
Second, there doesn’t appear to be any change in the origin of new lineages in Psilocybe following its origin (presumably coincident with psilocybin biosynthesis). However, it should be emphasized that only a small fraction of the known species of Psilocybe are included here, and this may not be an accurate representation of the entire genus. I think it would be a fascinating investigation to include all species of Psilocybe and get whole genome data from them, wouldn’t it?
The students in BIOL1625 lab course at the University of Utah added some ITS sequences of porcini that were not included in the BIOL5425 class results, so I’ve updated the tree with these new data. I used MAFFT (L-INS-i) again, divided the multiple sequence alignment into ITS1, 5.8S, and ITS2 regions, used partition finder in IQ-TREE with the edge-unlinked partition model to identify the best partitioning scheme and model (-sp & -m MF+MERGE), then ran a ML analysis with the best partitioning scheme and edge-unlinked partition model and ultrafast bootstrapping (1000 replicates) with resampling of partitions and sites (-bb 1000 -bsam GENESITE). The tree is rooted with Buchwaldoboletus hemichrysus again and I’ve collapsed nodes to simplify the display. All sequences derived from citizen scientist contributions (beginning with the MO#) are preserved in black and have both the Mushroom Observer ID (MO######) and the Utah fungarium accession (UTM######) in the label. I also color-coded the species labels according to geography: North & Central America (blue), Asia (green), Europe (red), Madagascar (yellow), and Australia & New Zealand (turquoise). Boletus edulis is magenta because it spans North America and Eurasia as it is currently circumscribed.
This is the second installment of the expanding porcini ITS tree thanks to specimens provided by citizen scientists that were sent in response to our solicitation on MushroomObserver (MO). For this dataset, I had my students in Mycology (BIOL 5425) at the University of Utah extract DNA, PCR amplify, and Sanger sequence the ITS region from 23 specimens from MO (plus a few others). The reference matrix is a curated dataset of single representative sequences for each species of porcini in existence that I am aware of (including a handful of undescribed species). A number of these sequences/specimens are still unpublished. I used MAFFT L-INS-i to align them. The tree is the best ML tree using a partitioned analysis in IQ-tree and ultrafast bootstraps (sign. at 95%). I rooted with a sequence of Buchwaldoboletus hemichrysus. The MO sequences are highlighted in red and are labeled with the MO number, followed by the UT-M accession number, followed by the putative identification.
porcini_all5425.mafft.fas.partitions.best_scheme.nex.treefile.collapsed
There are a few exciting results to highlight:
One additional observation I want to make is that the typical 3% species-level divergence metric commonly used to delimit “Operational Taxonomic Units” (OTUs) would vastly underestimate the number of species of porcini. The graph below shows the range of pairwise divergences for a selection of species in the tree above. The bottom “whisker” indicates the minimum pairwise distance between species. The red line indicates the typical 3% divergence cutoff used to delimit OTUs. Most species have minimum pairwise divergences far below the 3% threshold (and many also below a “stringent” 1% threshold). 
I want to acknowledge the important expertise and efforts of the citizen scientists who contributed material for this study (and I hope will continue to contribute material!), my students in BIOL 5425 who generated the sequence data, and especially my PhD student Alex Bradshaw for managing the student activity and doing the bulk of the work. I also want to acknowledge Jean-Marc Moncalvo, who is my collaborator on the Sarawak and Vietnam material, Sven Buerki who contributed the enormously important specimen of Boletus phaeocephalus, and QMM in Fort Dauphin, Madagascar, for facilitating fieldwork that resulted in the undescribed Boletus that falls near B. albobrunnescens (admittedly, this may be an artifact of sampling bias, which is being revisited with whole genome sequencing…).
So, I’ve been slowly accumulating ITS barcodes for samples of Boletus edulis s.s. from around the northern hemisphere (mostly in North America). There have been some proposals in both Europe and N. America to distinguish subspecies of Boletus edulis (e.g., B. edulis var. clavipes, B. edulis var. persoonii, etc.), and some of these have even been raised to the rank of species (B. pinetorum, B. rubriceps). While some of these proposals are based entirely on morphological features, others have claimed support from molecular evidence. In both cases, I have been skeptical of these taxonomic divisions for two reasons: 1) Boletus edulis is extremely variable morphologically, and 2) sampling has been very incomplete, with vast areas of its range without representation. In the former, morphological features that are consistent between collections could be due to ecological context such as host or abiotic factors such as sunlight, temperature, humidity, and pH. This variation might also result from heterogeneous distributions of genes and alleles that are the basis of these different phenotypes within a panmictic population, such as we see in other organisms including humans. Thus, it is critical to sample individuals that are representative of the complete range of a putative species, to ensure patterns of geographic structuring are not misinterpreted as non-overlapping when samples come only from the extremes of their distribution. Non-representative sampling like this can be particularly problematic for DNA barcoding, because the total variation used to diagnose the limits of monophyletic clades is dependent on the variation observed in the samples. So, if the samples come from two extreme ends of a continuum with no samples representing intermediate locations, they will appear to be mutually exclusive, leading to misdiagnosis as reproductively isolated lineages, i.e. species.
The ITS clade representing 220 phased (i.e. heterozygotes were split into corresponding haplotypes based on sequencing clones) sequences of Boletus edulis s.s. has some structuring in it, but it is not clear-cut and highly polytomic. For instance, some sequences of specimens are clustered geographically, such as the group recently segregated as B. rubriceps from the southern Rocky Mountains. But, further sampling has revealed 1) southern Rocky mountain B. edulis/B. rubriceps are not morphologically homogeneous, and 2) heterozygous individuals that carry haplotypes represented in the southern Rocky mountains AND the west coast of North America. Both observations strongly suggest either current gene flow between those populations or incomplete lineage sorting of ITS copies. So, rather than being independent and reproductively isolated species, there is either ongoing gene flow within a panmictic population that is structured geographically, or the population has speciated recently without enough time passing for ancestral multiple ITS lineages to sort among them. Because the ITS region is homogenized through concerted evolution, it seems that the explanation requiring multiple ancestral copies sorting among descendant lineages is less parsimonious. But, the mechanisms of concerted evolution are not well understood, so I can’t rule it out (I would love for some discussion about this!).
Below is a haplotype network constructed from the same set of sequences used for the phylogenetic analysis above. I used the HaploNet function in the R package ‘pegas’ (Paradis 2017) and plotted the pie charts according to composition of geographic representation. The legend indicates which region the haplotype is from: AK= Alaska, ENA= Eastern North America (east of the Rockies), EU = Europe, MX = Mexico, NZ = New Zealand, RM = Rocky Mountains (incl. CO, NM, UT, and MT), WC = West Coast (CA, OR, WA & BC). A few interesting patterns are apparent: 1) one haplotype (the most common in GenBank) is present in Europe and the western parts of North America (Alaska, West Coast, and the Rockies); 2) Eastern North America is distinct from all others except for one divergent sample that is from a late-season collection with an odd morphology under European conifers in western NY (so probably an import and not biogeographically honest); 3) samples corresponding to B. rubriceps have haplotypes derived from or shared with haplotypes in Europe, West Coast, and Alaska and appears to not be reproductively isolated. The New Zealand haplotype is shared with European haplotypes, confirming suspicions that it is an import with European trees. Although I consider this to still be preliminary, I interpret this structure as indicating ongoing, or until very recently, gene flow between populations in the Rocky Mountains, West Coast, Alaska, and Europe (via Alaska?), with reproductive isolation seen only in the eastern North American population (i.e. Boletus edulis var. clavipes should be recognized at the species rank, viz. Boletus clavipes). Given the diversity of haplotypes seen in Alaska, I would venture to guess that this is the crossroads of porcini migration, possibly representing an ancestral population that gave rise to expanding population to/from Eurasia via the Bering Land bridge ca. 12,000 years ago.
This is a work in progress, but here are 38 currently accepted species of porcini s.s. (i.e. not including “Alloboletus” sensu Dentinger et al. 2010). This will soon be updated following data from sequencing of type specimens…
Boletus aereus Bull. (=Boletus mamorensis Redeuilh)
Boletus atkinsonii Peck
Boletus austroedulis Halling & N.A. Fechner
Boletus bainiugan Dentinger
Boletus barrowsii Thiers & A.H. Sm.
Boletus botryoides B. Feng, Y.Y. Cui, J.P. Xu & Zhu L. Yang
Boletus castanopsidis Hongo
Boletus edulis Bull. (=Boletus chippewaensis A.H. Sm. & Thiers, =B. rubriceps Arora & Simonini)
Boletus fagacicola B. Feng, Y.Y. Cui, J.P. Xu & Zhu L. Yang
Boletus fibrillosus Thiers
Boletus frustulosus Peck
Boletus gigas Berk. (suspect — may be a Leccinum instead!)
Boletus himalayensis S. Jabeen, S. Sarwar & A. N. Khalid
Boletus hiratsukae Nagasawa
Boletus indoedulis D. Chakr., K. Das, Baghela, S. Adhikari & Halling
Boletus leptocephalus Peck
Boletus luteoloincrustatus R. Flores & Simonini
Boletus meiweiniuganjun Dentinger
Boletus monilifer B. Feng, Y.Y. Cui, J.P. Xu & Zhu L. Yang
Boletus mottiae Thiers
Boletus multipunctus Peck
Boletus nobilissimus Both & R. Reidel
Boletus occidentalis B. Ortiz & T.J. Baroni
Boletus phaeocephalus Pat. & Baker sensu Corner
Boletus pinophilus Pilát & Dermek
Boletus quercophilus Halling & G.M. Mueller
Boletus regineus D. Arora & Simonini
Boletus reticulatus Schaeff. (= B. aestivalis (Paulet) Fr.)
Boletus reticuloceps (M. Zang, M.S. Yuan, & M.Q. Gong) Q.B. Wang & Y.J. Yao
Boletus rex-veris D. Arora & Simonini
Boletus shiyong Dentinger
Boletus sinoedulis B. Feng, Y.Y. Cui, J.P. Xu & Zhu L. Yang
Boletus subalpinus (Trappe & Thiers) Nuhn, Manfr. Binder, A.F.S. Taylor, Halling & Hibbett
Boletus subcaerulescens (E.A. Dick & Snell) Both, Bessette, & A.R. Bessette (=B. edulis subsp. aurantioruber E.A. Dick & Snell)
Boletus subreticulatus Corner
Boletus umbrinipileus B. Feng, Y.Y. Cui, J.P. Xu & Zhu L. Yang
Boletus variipes Peck (=Boletus insuetus A.H. Sm. & Thiers)
Boletus viscidiceps B. Feng, Y.Y. Cui, J.P. Xu & Zhu L. Yang
For a while now, I have been on the hunt for new, newly recognized, and long forgotten species of porcini mushrooms. Porcini have been a kind of weird, academic obsession of mine since I started my graduate research way back in 2001 (and I also love to eat them!). Since then I have been slowly accumulating specimens and sequences over the years, but have been reticent with publicizing the information due to the incompleteness of my study. But, science is a decidedly incremental process (especially for me), and sometimes the information needs to be shared to make advances. Also, as much as I’d like to spend most of my time foraging for boletes, I can’t be everywhere at all times, and my career choice has (somewhat ironically) kept me from dedicating more time to fieldwork. So, I’ve turned to friends and colleagues to help source porcini worldwide to include in my expanding dataset. Dan Molter (a.k.a. “shroomydan“) and I have created a citizen science project on MushroomObserver, soliciting specimens of porcini from the broader nonprofessional community. As a way to reciprocate their generosity and efforts, I aim to continuously update a phylogenetic tree based on ITS sequences of these specimens.This is the first installment.
This tree is based on a dataset of 180 hand-curated ITS sequences representing the core Boletus sensu stricto clade (sensu Dentinger et al. 2010; i.e. not including the “alloboletus” group typified by Boletus separans), both from unpublished sequences generated in-house and published sequences captured from GenBank. I built the alignment with the L-INS-i algorithm in MAFFT, partitioned the alignment into ITS1, 5.8S, and ITS2 segments, and used IQTree to find the best ML tree with automatic model selection and ultrafast bootstrapping. I’ve collapsed clades and relabelled them according to my opinion on the current species names that should be applied (with some agnosticism in a few cases). This is subject to change in the near future following a type specimen sequencing study I have been pursuing, but that’s a story for another day. Labels of sequences from MushroomObserver specimens are retained in red and include the original identification supplied by the collector. The species names are color-coded according to geography: red = East Asia, blue = North & Central America, green = Europe, yellow = South Asia, and purple = Australia. In total, I recognize 39 (give or take 2-3) good species of Boletus s.s.
Many thanks to my undergraduate student, Jimmy Arnold for help with this project.
porcini_ITS_13Mar2018_partitions.txt.best_scheme.nex.treefile
This will, I hope, make a lot more sense in the coming months…
I’ve done a bit of fieldwork in Sarawak (Borneo) and one year had the great fortune of partaking in pre-celebratory festivities leading up to Gawai, the celebration of indigenous culture in Sarawak. This recipe comes from the Headmaster of the longhouse at the mouth of the Batang Ai river. This was originally posted on sarawakfungi.org, but that site is no longer being maintained so I’m reposting it here.
1. Cook 2 kg of sticky rice with two volumes of water (i.e., twice as much water as rice), then allow it to cool to room temperature.
2. Mix in 1 kg of yeast (special yeast cake found in shops in Sarawak).
3. Wait one week.
4. Bring 2-3 kg of sugar (depending on taste) to boil in 20-30 L of water, cool to room temp, then add to the rice mash.
5. Add 0.5 kg yeast (same special yeast cake as before), mix and let ferment for at least one week.
6. Serve in small glasses at room temperature.
Well, here we have some fermenting sugar cane juice from a cachaça maker in Brazil.
And here is the total genomic DNA (and RNA smear at the bottom) from the above juice.
So, I loaded it into one of these portable sequencing contraptions from Oxford Nanopore.
And generated not very much sequence data, probably because the flow cell and library kit was very old. But still, some interesting preliminary results using WIMP…
dikaryon 