Many species of fungal plant pathogens coexist as multiple lineages on the same host, but the factors underlying the origin and maintenance of population structure remain largely unknown. The rice blast fungus Pyricularia oryzae is a widespread model plant pathogen displaying population subdivision. However, most studies of natural variation in P. oryzae have been limited in genomic or geographic resolution, and host adaptation is the only factor that has been investigated extensively as a contributor to population subdivision. In an effort to complement previous studies, we analyzed genetic and phenotypic diversity in isolates of the rice blast fungus covering a broad geographical range. Using single-nucleotide polymorphism genotyping data for 886 isolates sampled from 152 sites in 51 countries, we showed that population subdivision of P. oryzae in one recombining and three clonal lineages with broad distributions persisted with deeper sampling. We also extended previous findings by showing further population subdivision of the recombining lineage into one international and three Asian clusters, and by providing evidence that the three clonal lineages of P. oryzae were found in areas with different prevailing environmental conditions, indicating niche separation. Pathogenicity tests and bioinformatic analyses using an extended set of isolates and rice varieties indicated that partial specialization to rice subgroups contributed to niche separation between lineages, and differences in repertoires of putative virulence effectors were consistent with differences in host range. Experimental crosses revealed that female sterility and early post-mating genetic incompatibilities acted as strong additional barriers to gene flow between clonal lineages. Our results demonstrate that the spread of a fungal pathogen across heterogeneous habitats and divergent populations of a crop species can lead to niche separation and reproductive isolation between distinct, widely distributed, lineages.

Blast disease is a notorious fungal disease leading to dramatic yield losses on major food crops such as rice and wheat. The causal agent, Pyricularia oryzae, encompasses different lineages, each having a different host range. Host shifts are suspected to have occurred in this species from Setaria sp. to rice and from Lolium sp. to wheat. The emergence of blast disease on maize in Iran was observed for the first time in the North of the country in 2012. We later identified blast disease in two additional regions of Iran, Gilan in 2013, and Golestan in 2016. Epidemics on the weed barnyard grass (Echinochloa spp.) were also observed in the same maize fields. Here, we showed that P. oryzae is the causal agent of this disease on both hosts. Pathogenicity assays in the greenhouse revealed that strains from maize can infect barnyard grass and conversely. However, genotyping with SSR markers and comparative genomics showed that strains causing field epidemics on maize and on barnyard grass are different, although they belong to the same previously undescribed clade of P. oryzae. Phylogenetic analyses including these strains and a maize strain collected in Gabon in 1985, revealed two independent host-range expansion events from barnyard grass to maize. Comparative genomics between maize and barnyard grass strains revealed the presence/absence of five candidate genes associated with host specificity on maize, with the deletion of a small genomic region possibly responsible for adaptation to maize.

12Natural variation in plant pathogens has an impact on food security and ecosystem health. The rice 13 blast fungus Pyricularia oryzae, which limits rice production in all rice-growing areas, is structured into 14 multiple lineages. Diversification and the maintenance of multiple rice blast lineages have been 15 proposed to be due to separation in different areas and differential adaptation to rice subspecies. 16However, the precise world distribution of rice blast populations, and the factors controlling their 17 presence and maintenance in the same geographic areas, remain largely unknown. We used 18 genotyping data for 886 isolates from more than 185 locations in 51 countries to show that P. oryzae is 19 structured into one recombining and three clonal lineages, each with broad geographic distributions. 20No evidence was found for admixture in clonal lineages, and crossing experiments revealed that female 21 sterility and early postmating genetic incompatibilities acted as strong barriers to gene flow between 22 these lineages. An analysis of climatic and geographic data indicated that the four lineages of P. oryzae 23 were found in areas differing in terms of the prevailing environmental conditions and types of rice 24 2 grown. Pathogenicity tests with representatives of the five main rice subspecies revealed differences 25 in host range between pathogenic lineages, highlighting a contribution of specialization to niche 26 separation between lineages, despite co-existence on the same host species. Our results demonstrate 27 that the spread of a pathogen across heterogeneous habitats and divergent populations of a crop 28 species can lead to niche separation and incipient speciation in the pathogen. 29 Short title: Incipient speciation in the rice blast pathogen 30 31