A proposed phylogenetic history of Ergot: the Claviceps species complex through time
Scientists (Kamila Píchová, et al) compiled genetic and chemical data to better understand and shed light on grass-parasitizing fungi Claviceps species complex, more commonly known as Ergot. Through their work they propose that the species originated in South America infecting a common grass ancestor around 70M years ago and split into sections around 47M and 27M years ago. Wherever grains were grown for food, Ergot, in its many varieties, was found to have significant historical impact or medicinal use.
Project Proposal and Author Note
Here, through combining molecular and chemical data, the scientists desire to provide a better definition of species within and separation of the Claviceps genus from its relatives. The genus is well known for parasitizing all sorts of grass seeds (Fig 1). The name is derived from the Latin noun clava meaning club, and the suffix -ceps meaning head; thus Claviceps purpurea (Rye Ergot) means purple club head fungus. Generally speaking, people have used the term Ergot, to describe almost every sclerotia forming fungi that affects grass seeds. The scientist propose to, through molecular biology and chemical analysis, show with higher resolution than has been seen before: the Claviceps family tree and fully defined "by combining phylogenetic information for five genes of a large set of species with data on the host and geographical range, morphology, and spectrum of secondary metabolites." They also not that this is thought of as one of the the first studies intending to map the distribution of ergochrome toxins across the genus Claviceps; going so far as to build out "a new analytical method enabling simultaneous screening of ergochromes and [Ergot Alkaloids]s" which are the two major entheogenic, and sometimes toxic, compounds present in almost all Claviceps species; of which erchochromes will get their own article on in the future.
Another dive into genetics and entheogens! I believe that to best understand an organism from a molecular biology perspective (my background), that morphological, chemical, and genetic data, each alone, cannot provide a full enough picture of any organism; much less entheogenic ones! Fungi are of particular interest in that they tend to be the exception to numerous biological definitions. We have previous talked about symbiotic fungi and how they produce entheogenic compounds, we have also review phylogenetic breakdowns or entheogeic organisms; here is presented a phylogeneitc breakdown of the parasitic fungi genus Claviceps. Typically sharing a fundamental life cycle within the species (Fig 2), a parasite is fundamentally tied to their host organism, they present unique lifeforms to study and require more than just simple barcoding to really categorize. "Since Claviceps are parasitic fungi with a close relationship with their host plants, their evolution is influenced by interactions with the new hosts, either by the spread to a new continent or the radiation of the host plants." Thus understanding the global distribution of the species and its flow over time plays a role in understanding how that mirrors agriculture and human expanse; how humanity has learned to live in the same ecosystems as this powerful entheogenic organism.
Ergot Alkaloids: the Thin Line Between Medicine & Toxin
The most important factor to understand about Ergot Alkaloids (EAs) is their affinity profiles and the similarities of their effects. The backbone that is the amino acid tryptophan again emerge as the important functional structure of all EAs; which in turn leads to their effect on 5-HT2 receptors similar to other tryptamines like DMT, psilocin, and serotonin. EAs are deeply diverse group of metabolites and are typically divided into four classes (Tbl 1): simple clavines (ergoline), amides of lysergic acid (ergine) amides, and highly complex ergopeptines (ergotamine) and ergopeptams (ergocristam).
Historically, poisonings in humans occured, termed ergotism, or the disease that comes from ergot-grain consumption, typically show up as clenching and tightening of muscles, gastrointestinal stress, mania, gangrene, and psychosis (Fig 3). There have been documented outbreaks of ergotism in India in the '70s and in Ethiopia in '78 and '01. There have even been modern theories proposing the Salem Witch Trials might have been caused by weather conditions that might induce a bout of ergot infection [Caporeal 1976, Matossian 1982] (Fig 3). They have been shown to have effect on cattle as well, and are argued to have anti-grazing effects on most pests. The scientists noted that "[f]eeding ergot-contaminated grains can result in...necrosis, reduced feed intake, reduced growth and reproductive disorders ...Paspalum staggers caused by C. paspali or Bermuda grass staggers caused by C. cynodontis" depending on the infected-substrate used as feed-stock.
Medically, the first documented use by European civilization is from 1582, possibly older if a documented German prescription from 1474 is authentic (Anonymous, 1474; Appendix B). If true, this script is one of the first pieces of evidence of the application of any fungus for medicinal purposes in Europe. In modern times, ergonovine (ergometrine) is still used to prevent postpartum haemorrhage after childbirth; while ergotamine and its derivatives are used to treat migraines. Gaining more traction in the 21st century entheogen based psychotherapeutics have recently been considered; being developed as semisynthetic lysergic acid diethylamide (LSD), since LSD is generated from the same ergot alkaloid source material.
There are other compounds besides EAs that have been found to be medically viable, one being the yellow-pigmented ergochromes which include secalonic acids (SAs), ergoflavine and ergochrysines, have been shown to have anti-inflammatory, neuroprotective, and cytostatic and anti-tumour activity. The non-alkaloid ergochromes play a role in toxicity. The SAs are unique lattice-works of carbons oxygens and hydrogens (Tbl 1) that have a few different variations. Notable amongst them are SA-A which has been shown to have to have antitumor properties [Zhai 2011]. This same compound, with a slight change in structure alone (called an enantiomere) you get SA-D, which is a major constituent in ruining corn crops [Masters 2012].
One of the more important aspects the scientist noticed was concentration of EAs in sclerotia, they were mostly testing for presence and absence, but note that "average content of EAs in C. purpurea is 0.26mg/g, rarely reaching 1–10mg/g of sclerotial dry weight. This is comparable to the concentration of SA's in sclerotia, which typically reaches 5–20mg/g." However, they were surprised to find that the distribution of theses entheogens (amongst others) across species and their environmental impact of these compounds was not very well investigated, "despite their high biological activity against mammalian cells."
Understanding Phylogenies & Fungal Targets Revisited
Many people have been exposed to the phylogenies like the' tree of life' before, but not many understand how to break it down into understandable sections. First, lets start with the basic structures: forks and nodes. Forks are points on the tree where one line splits into two, these are located either to the centered, left or below the rest of the 'tree' representing the 'trunk' or most recent common ancestor lineage. Nodes are the endpoints of each 'branch' of the 'tree' and can be found at the terminating ends of the phylogeny, typically the outer, right or above the rest of the 'branches'. Once we have these basic structures understood, we can zoom out and talk about distinct lineages, or clades, on the 'tree of life'. Clades are a shared lineages: fungi is a clade, primates are a clade, and the genus homo that humanity belongs, is a clade. A genus, like Claviceps, would need to have all of the species classified within it to share the same common ancestor (Fig 4).
Always of interest: genetics, barcoding, and organism ID is crucial to better understanding the organism and the role it plays within its ecosystem. Genomes for organisms can be widely variable, and we have previously looked into some genetics of other entheogenic fungi for reference. For fungi the genes we use for barcoding are centered around the genes that build ribosomes (ITS1, ITS2, & LSU) as well as a the genes that build RNA polymerase (RPB1 & RPB2). For this research, the scientists selected five (5) gene sequences for building their phylogenetic analysis around. They chose the nuclear ribosomal DNA (ITS-LSU) containing internally transcribed spacers (ITS1 and ITS2), 5.8S and the 5′ end of the 28S region, part of the RNA polymerase II gene (RPB2), a fragment of the a protein complex required for DNA replication (MCM7) gene, a region of the translation elongation factor 1α gene (TEF-1α), and part of beta-tubulin (TUB2).
Among the many benefits of data-mining, with the wealth of information emerging from genomic sequencing projects, data can be mined to uncover candidate genes as barcoding targets. Using a bioinformatics approach, Aguileta et al.  mined genomic sequences among fungi to identify clusters of [species-similar] single-copy genes." one found was the MCM7 gene. [Tretter 2013] While previously mentioned, TEF-1α is responsible for the enzymatic delivery of transfer RNA's to the ribosome. It use as a barconde gene is, overall, consistent with ITS phylogeny, but also provided a "higher discriminatory power" than ITS alone. [Mirhendi 2015] That being said, TUB2, which codes for a protein involved in the generation of microfilaments; has been used successfully for species delineation in other groups of fungi such as Aspergillus, Penicillium, Scedosporium, and Phaeoacremonium. The gene cluster "includes some introns, which are known to be good estimators for distinction of closely related species." [Rezaei-Matehkolaei 2014]
Conclusion and Caveats
To better root their phylogentic tree historically the scientists opted to use two fossil fungi: Palaeoclaviceps parasiticus and Paleoophiocordyceps coccophagus; one to anchor the main Claviceps genus in the Cretaceous era (about 75 million years ago) [Poinar 2017], the other to anchor the out-group Ophiocordyceps genus in the Cretaceous (about 145-66 million years ago) [Sung 2008] (Fig 5). Through their work they were able to discern that of the organisms tested "59 species are considered members of the genus Claviceps." The distribution range of Claviceps species was divided into six regions: Africa, South America, Eurasia, North America, Australia and India (Fig 6). Each region was inteded to be representative of a biogeographic regions: North America is adequate for the Nearctic biogeographic region and South America for the Neotropical region; Mexico spans both biogeographic realms, and three Mexican species (C. gigantea, C. clavispora and C. citrina) are recorded in the Nearctic part of the country. The hosts were categorized into seven groups by family or subfamily: Panicoideae, Chloridoideae, Arundinoideae, Pooideae, Bambusoideae, Ehrhartoideae and Cyperaceae (Fig 6). The group even notes that through their analysis they were able to reconstruct the transition from symbiote to ovarian-strict parasite.
Among the interesting caveats was the additional proof, with mass spectrometry, that "EAs are also absent from numerous Claviceps species"; pointing towards the impacts of ergotism not being widespread like the genus as a whole. There were also a few discrepancies with the three species C. citrina, C. clavispora, and C. gigantea. C. citrina was only recorded once on the host Distichlis spicata, and is considered native in South and North America, the scientists assumed the occurrence of C. citrina to be both North and South American. They were unable to identity the host of C. clavispora nor C. gigantea, where the later is restricted to high, humid locations in central Mexico. The distribution of C. gigantea and C. clavispora was decided to be treated as North American and of C. citrina as both North and South American. These are human choices, and might not reflect the actual distributions in nature and are best left as 'Unresolved'.
Project Proposal Success or Failure?
Molecular and chemical analysis supports the idea of Claviceps as a clade.
However, the analysis of the data was not very robust and provided unsatisfactory statistical support.
Reviewing the data and the conclusions put forth, the hypothesis proposed by the Scientists was supported. The reason(s) for acceptance are quoted below (emphasis and parentheticals mine):
"Our analysis support the current concept of Claviceps, which includes non-systemic parasites exclusively infecting unfertilized ovaria, albeit with unsatisfactory statistical support. "
"Our analyses confirm the presence of four lineages inside of Claviceps and the congenerity of Aciculosporium, Cepsiclava and Neoclaviceps."
"The deep divergence is highlighted by the fact that all four lineages, described here as new sections, can be sharply distinguished based on the combination of the asexual spore type, host range, geography and secondary metabolite production."
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