Anthracnose of lentil caused by the fungal pathogen Colletotrichum lentis is an economically important disease in Western Canada. The pathogen population is divided into two races (0 and 1) and two sexual incompatibility groups (IG-1 and IG-2). Resistance to anthracnose race 1 is found in cultivated Lens cultivars whereas for the more aggressive race 0, higher levels of resistance have been reported only from wild Lens species. Furthermore, C. lentis seems to only possess one (MAT1-2) of the two mating type idiomorphs commonly present in heterothallic ascomycete fungi with the typical bipolar mating system. The purpose of this study was to verify the phylogenetic relationship between race 0 and 1 isolates of C. lentis and to sequence and characterize the MAT1-2 of C. lentis. A morphological, multi-locus phylogenetic and host-range study was conducted with isolates of C. lentis, C. truncatum (from various host species and the epitype), C. destructivum, C. dematium, C. higginsianum, C. linicola and C. lindemuthianum. Sequence data from six conserved loci displayed 100% identity for C. lentis isolates of both races that formed a single cluster separate from other Colletotrichum species including C. destructivum, the epitype of C. truncatum and isolates from other hosts identified as C. truncatum. Conidia of C. lentis were slightly falcate with obtuse apices compared to cylindrical conidia with rounded ends of C. destructivum, and longer lunate to falcate conidia of the epitype C. truncatum. Host range tests undertaken on Lens culinaris, Pisum sativum, Cicer arietinum, Vicia faba, Glycine max, Phaseolus vulgaris, Phaseolus lunatus, Trifolium pratense, Medicago sativa, Medicago truncatula, Brassica chinensis and Arabidopsis thaliana under controlled environmental conditions revealed that the host ranges of C. linicola and C. higginsianum overlapped with that of lentil isolates. In contrast, the epitype specimen of C. truncatum was pathogenic on Pisum sativum, Phaseolus vulgaris, T. pratense and Medicago sativa, but not on L. culinaris. All Colletotrichum spp. infected Medicago truncatula and all but the lentil isolates caused disease on G. max. The mating type gene MAT1-2 of C. lentis contained two introns and three exons and an open reading frame of 726 bp coding for a putative protein of 241 amino acids including the high mobility group (HMG) domain characteristic of the MAT1-2 in fungi. The MAT1-2 nucleotide sequences of C. lentis isolates were identical irrespective of IG. An isolate from each of the two IGs, CT-21 (IG-2), CT-30 (IG-1) and a co-culture of CT-21 and CT-30 was used to study the expression levels of MAT1-2 at seven different in vitro time points (0h, 6h, 12h, 18h, 24h, 36, 48h after inoculation in glucose yeast media) and investigate for possible alternative splicing events. MAT1-2 expression for CT-21, CT-30 and the co-culture was observed at all seven time points indicating that it is constitutively expressed, and no differences in the transcript size were seen, ruling out the possibility of a splicing event.

The aim of this thesis was to study the evolution of reproductive systems and reproductive traits in the fungal genus Neurospora. More specifically, I have investigated the evolutionary forces shaping the genes involved in sexual reproduction, focusing on mating-type (mat) and pheromone receptor (pre) genes. Neurospora contains species exhibiting three different mating systems, i.e., heterothallism (self-incompatibility), homothallism (self-compatibility) and pseudohomothallism (partial self-incompatibility). First, a robust phylogeny of Neurospora was established. The phylogenetic analyses revealed multiple independent transitions in reproductive life style during the evolutionary history of the genus. We argued for a heterothallic ancestor of the genus, although our subsequent ancestral reconstruction analyses favored a homothallic ancestor. To be able to settle the ancestral mating system, we zoomed in on the structural architecture of the mat-locus in four homothallic species of Neurospora, thought to have arisen from independent transitions. Our results led us to suggest two different genetic mechanisms (translocation and unequal crossover) to explain the transitions in mating system from heterothallism to homothallism. We pointed out that the mating-system transitions in Neurospora are unidirectional, and suggested that transposable elements might be driving the transitions. In conclusion, we suggest a heterothallic ancestor for Neurospora, and that at least six transitions to homothallism and two transitions to pseudohomothallism have occurred in its evolutionary history. Further, we used the phylogeny of Neurospora as a framework to test if the evolution of pre-genes (pre-1 and pre-2) in hetero- and homothallic Neurospora is dependent on mating systems and/or even the homothallic clades themselves (i.e., mating-system and/or switch-dependent). The molecular evolution results suggest that pre-1 and pre-2 are overall functional in both homothallic and heterothallic Neurospora. The molecular evolution of pre-1 seems to be independent of mating-system or homothallic clade, and we detected signs for positive selection in the C-terminal tail. For pre-2 we found no support for mating-system dependent evolution, but indications for switch-dependent evolution. In this study we also included expression analyses of both pre- as well as mat-genes, with the prospect to assess functionality and regulation. During this thesis work, we also performed a phylogenetic study were we found that reproductive genes might be more permeable to introgression than other genes, which is in contrast to theoretical expectations. In the last study, we confirmed the co-existence of two alternative splice variants of the pheromone receptor gene pre-1 in Neurospora crassa, and performed expression profiles studies using quantitative RT-PCR. I hope this thesis work will further strengthen Neurospora as a model for research in evolutionary genetics.