The evolutionary origins of Epichloe endophytes from annual ryegrasses

The annual Lolium (ryegrass) species were surveyed for seedborne, asexual E pichloe: endophytes (i.e., Neotyphodium spp.). Endophytes that were extreme ly slow growing in culture were identified from all seven annual ryegrass s pp. Sequences of beta -tubulin gene (tu62) introns and the internal transcr ibed spacers of the nuclear ribosomal RNA (rDNA-ITS) were determined, as we re profiles based on five microsatellite loci. Two taxonomic groups of annu al ryegrass endophytes were distinguishable by morphology, culturability an d genotypes. One group, for which the name Neotyphodium occultans is propos ed, comprised only isolates that could not be subcultured on potato dextros e agar medium. These isolates contained multiple tub2 genes and multiple al leles at microsatellite loci, suggestive of an interspecific hybrid origin. The results of phylogenetic analysis implied that the ancestors of N, occu ltans included Epichloe; baconii and E. bromicola, or close relatives. The second taxonomic group of annual ryegrass endophytes, exemplified by isolat e Lc4, was found only in L. canariense. This taxon had a single tub2 sequen ce and microsatellite genotype that was nearly identical to those of extant Epichloe typhina isolates that infect Poa nemoralis. Therefore, it is prop osed that Lc4 is an asexual derivative of an E. typhina genotype closely re lated 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.