A new species of Periglandula symbiotic with the morning glory Ipomoea tricolor

Many morning glories in the family Convolvulaceae contain ergot alkaloids, which are important agricultural and pharmaceutical compounds produced exclusively by fungi. In the few morning glories that have been investigated in detail, the ergot alkaloids are associated with the presence of a symbiotic fungus from the family Clavicipitaceae. The genus Periglandula was erected in the family Clavicipitaceae in 2011 for two species of morning glory symbionts. Biochemical and limited sequence data indicate that Ipomoea tricolor, a commonly cultivated morning glory from Mexico, contains a Periglandula species but the fungus is uncharacterized, and no signs of it have ever been detected in the plant. Our goal was to isolate and characterize this fungus. We isolated the fungus in pure culture, characterized it morphologically, and prepared sufficient DNA from it to sequence its genome. Phylogenetic analyses based on multiple genes indicate that the symbiont of I. tricolor is distinct from, but related to, the two described species of Periglandula previously observed in other species of morning glories.

Methods
The genome of the fungal symbiont of Ipomoea tricolor was sequenced (GenBank accession JBIMFC000000000) from DNA obtained from an axenic culture. Sequence reads were obtained by Illumina NextSeq technology at the West Virginia University and Marshall University Genomics Core facilities and assembled with SPAdes version 3.15.4. Homologs of the following loci were extracted from the genome of the symbiont of I. tricolor for phylogenetic analyses based on identity with homologs from the Periglandula ipomoeae genome: γ-actin (actG), β-tubulin (tubB), mitochondrial ATPase 6 (atp6), largest subunit of RNA polymerase II (rpbA), dimethylallyltryptophan synthase (dmaW), and the ribosomal internal transcribed spacer region (ITS). Sequences were aligned by MUSCLE as found in MEGA X (Kumar et al. 2018) and trimmed by eye. Maximum likelihood analyses with 1000 bootstrap replications were performed on ITS, dmaW, and concatenated actG, tubB, atp6, and rpbA loci. Those loci were selected for further analysis because they were available from both described Periglandula species––P. ipomoeae, whose genome was sequenced, and P. turbinae whose genome had not been sequenced and for which only limited sequence data were available. For the concatenated actG, tubB, atp6, and rpbA data set, we included members of each of the genera in the Clavicipitaceae that had sequenced genomes in which each of the four selected loci aligned well. Since multiple species in the genera Epichloë and Metarhizium met these criteria, three-well-characterized members of each of those genera were included. For ITS and dmaW trees, sequences from a greater number of undescribed Periglandula species were available in GenBank and thus were included in those analyses. Additional members of the Hypocreales were included as outgroups. Maximum likelihood analyses were conducted in MEGA X (Kumar et al. 2018) with general time reversible model for protein-coding sequences and the Tamura-Nei model for the internal transcribed spacer. Analyses were bootstrapped 1000 times, and the tree with the highest log likelihood is presented for each analysis.

Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molec Biol Evol. 35(6):1547–1549.