The evolutionary origins of Epichloe endophytes from annual ryegrasses

The annual Lolium (ryegrass) species were surveyed for seedborne, asexual Epichloe: endophytes (i.e., Neotyphodium spp.). Endophytes that were extremely slow growing in culture were identified from all seven annual ryegrass spp. Sequences of beta -tubulin gene (tu62) introns and the internal transcribed spacers of the nuclear ribosomal RNA (rDNA-ITS) were determined, as were profiles based on five microsatellite loci. Two taxonomic groups of annual ryegrass endophytes were distinguishable by morphology, culturability and genotypes. One group, for which the name Neotyphodium occultans is proposed, comprised only isolates that could not be subcultured on potato dextrose agar medium. These isolates contained multiple tub2 genes and multiple alleles at microsatellite loci, suggestive of an interspecific hybrid origin. The results of phylogenetic analysis implied that the ancestors of N, occultans included Epichloe; baconii and E. bromicola, or close relatives. The second taxonomic group of annual ryegrass endophytes, exemplified by isolate Lc4, was found only in L. canariense. This taxon had a single tub2 sequence and microsatellite genotype that was nearly identical to those of extant Epichloe typhina isolates that infect Poa nemoralis. Therefore, it is proposed that Lc4 is an asexual derivative of an E. typhina genotype closely related to extant strains from P. nemoralis. We propose the name Neotyphodium typhinum var. canariense for this taxon. These two new endophyte taxa from the annual ryegrasses are further examples of asexual Neotyphodium species that have arisen from sexual Epichloe; spp. either directly by loss of the sexual state, or by interspecific hybridization events.