The evolutionary origins of three new Neotyphodium endophyte species from grasses indigenous to the Southern Hemisphere

Members of the genus Neotyphodium are asexual, seedborne, protective fungal endophytes of cool season grasses that have likely evolved either directly from sexual Epichloe species, or by the interspecific hybridization of distinct lineages of Epichloe and Neotyphodium. We investigated the evolutionary origins of Neotyphodium endophytes from several grasses that are indigenous to the Southern Hemisphere using a multiple-gene phylogenetic approach. Intron regions of the genes encoding P-tubulin (tub2), translation elongation factor 1-alpha (tef1) and actin (act1) were amplified by polymerase chain reaction and sequenced. Phylogenetic analyses of these sequences, aligned with homologous sequences from Epichloe spp., revealed the evolutionary origins of the Southern Hemisphere endophytes, where one lineage of apparently non-hybrid origin, and three lineages of unique interspecific hybrid origin were identified. On the basis of morphology; host range and evolutionary history, we propose three new species of Neotyphodium. Neotyphodium aotearoae was isolated from Echinopogon ovatus populations from New Zealand and Australia, and comprised a unique, apparently non-hybrid lineage within the Epichloe species phylogeny. In contrast, an interspecific hybrid lineage was identified from two Australian Ec. ovatus populations, whose ancestry apparently involved lineages closely related to extant E. festucae and all E. typhina genotype similar to that of isolates from Poa pratensis. Endophytes infecting South African Melica racemosa and M. decumbens (dronkgras) appeared to be hybrids of E. festucae and X aotearoae or close relatives. The names N. australiense and N. melicicola are proposed for these two hybrid lineages, respectively. The origin of N tembladerae, an established endophyte species from South American Poa and Festuca spp., was also investigated. Neotyphodium tembladerae appeared to be of hybrid origin, involving E. festucae and an E. typhina genotype similar to that of isolates from Poa nemoralis. The results of this study highlight the widespread occurrence of interspecific hybrid Neotyphodium lineages on a global scale, and the extent of endophyte gene-flow between the Northern and Southern Hemispheres.