LOL is a novel secondary metabolite gene cluster associated with the production of loline alkaloids (saturated 1-aminopyrrolizidine alkaloids with an oxygen bridge) exclusively in closely related grass-endophyte species in the genera Epichloë and Neotyphodium. In this study I characterize the LOL cluster in E. festucae, including the presentation of sequence corresponding to 10 individual lol genes as well as defining the boundaries of the cluster and evaluation of the genomic DNA region flanking LOL in E. festucae. In addition to characterizing the LOL cluster in E. festucae, I present LOL sequence from two additional species, Neotyphodium coenophialum and Neotyphodium sp. PauTG-1. Together with two recently published LOL clusters from N. uncinatum, these data allow for a powerful phylogenetic comparison of five clusters from four closely related species. There is a high degree of microsynteny (conserved gene order and orientation) among the five LOL clusters, allowing us to predict potential transcriptional co-regulatory binding motifs in lol promoter regions. The relatedness of LOL clusters is especially interesting in light of the history of interspecific hybridizations that generated the asexual, Neotyphodium lineages. In fact, three of the clusters appear to have been introduced to different Neotyphodium species by the same ancestral Epichloë species, for which present day isolates are no longer able to produce lolines. To address the evolutionary origins of the cluster we have investigated the phylogenetic relationships of particular lol ORFs to their paralogous primary metabolism genes (and gene families) from endophytes, other fungi and even other kingdoms. I present extensive evidence that at least two individual lol genes have evolved from primary metabolism genes within the fungal ancestors of endophytes, rather than being introduced via horizontal gene transfer. I also present complementation studies in Neurospora crassa exploring the functional divergence of one lol gene from its primary metabolism paralog. While it is clear that these insecticidal compounds should convey a selective advantage to the fungus and its host, thus explaining preservation of the trait, this analysis provides an exploration into the evolutionary origin and maintenance of the genes that comprise the LOL and the cluster itself.

LOL is a novel secondary metabolite gene cluster associated with the production of loline alkaloids (saturated 1-aminopyrrolizidine alkaloids with an oxygen bridge) exclusively in closely related grass-endophyte species in the genera Epichloë and Neotyphodium. In this study I characterize the LOL cluster in E. festucae, including the presentation of sequence corresponding to 10 individual lol genes as well as defining the boundaries of the cluster and evaluation of the genomic DNA region flanking LOL in E. festucae. In addition to characterizing the LOL cluster in E. festucae, I present LOL sequence from two additional species, Neotyphodium coenophialum and Neotyphodium sp. PauTG-1. Together with two recently published LOL clusters from N. uncinatum, these data allow for a powerful phylogenetic comparison of five clusters from four closely related species. There is a high degree of microsynteny (conserved gene order and orientation) among the five LOL clusters, allowing us to predict potential transcriptional co-regulatory binding motifs in lol promoter regions. The relatedness of LOL clusters is especially interesting in light of the history of interspecific hybridizations that generated the asexual, Neotyphodium lineages. In fact, three of the clusters appear to have been introduced to different Neotyphodium species by the same ancestral Epichloë species, for which present day isolates are no longer able to produce lolines. To address the evolutionary origins of the cluster we have investigated the phylogenetic relationships of particular lol ORFs to their paralogous primary metabolism genes (and gene families) from endophytes, other fungi and even other kingdoms. I present extensive evidence that at least two individual lol genes have evolved from primary metabolism genes within the fungal ancestors of endophytes, rather than being introduced via horizontal gene transfer. I also present complementation studies in Neurospora crassa exploring the functional divergence of one lol gene from its primary metabolism paralog. While it is clear that these insecticidal compounds should convey a selective advantage to the fungus and its host, thus explaining preservation of the trait, this analysis provides an exploration into the evolutionary origin and maintenance of the genes that comprise the LOL and the cluster itself.