ORIGINAL ARTICLE doi:10.1111/j.1558-5646.2008.00390.x RAPID SPECIATION FOLLOWING RECENT HOST SHIFTS IN THE PLANT PATHOGENIC FUNGUS RHYNCHOSPORIUM Pascal L. Zaffarano,1,2,3 Bruce A. McDonald,1 and Celeste C. Linde4 1Plant Pathology, Institute of Integrative Biology, ETH-Zurich, LFW, CH-8092 Zurich,¨ Switzerland 2E-mail: [email protected] 4School of Botany and Zoology, Building 116, Daley Rd, Australian National University, Canberra ACT 0200, Australia Received July 6, 2007 Accepted March 11, 2008 Agriculture played a significant role in increasing the number of pathogen species and in expanding their geographic range during the last 10,000 years. We tested the hypothesis that a fungal pathogen of cereals and grasses emerged at the time of domestication of cereals in the Fertile Crescent and subsequently speciated after adaptation to its hosts. Rhynchosporium secalis, originally described from rye, causes an important disease on barley called scald, although it also infects other species of Hordeum and Agropyron. Phylogenetic analyses based on four DNA sequence loci identified three host-associated lineages that were confirmed by cross-pathogenicity tests. Bayesian analyses of divergence time suggested that the three lineages emerged between ∼1200 to 3600 years before present (B.P.) with a 95% highest posterior density ranging from 100 to 12,000 years B.P. depending on the implemented clock models. The coalescent inference of demographic history revealed a very recent population expansion for all three pathogens. We propose that Rhynchosporium on barley, rye, and Agropyron host species represent three cryptic pathogen species that underwent independent evolution and ecological divergence by host-specialization. We postulate that the recent emergence of these pathogens followed host shifts. The subsequent population expansions followed the expansion of the cultivated host populations and accompanying expansion of the weedy Agropyron spp. found in fields of cultivated cereals. Hence, agriculture played a major role in the emergence of the scald diseases, the adaptation of the pathogens to new hosts and their worldwide dissemination. KEY WORDS: Barley, coevolution, crop domestication, host shift, plant pathogens, TMRCA. Agriculture began with the domestication of the plants and ani- by the corresponding increase in human population density that mals that enabled the rapid human population expansion of the allowed (1) the maintenance of stable pathogen populations, (2) last 10,000 years (Cavalli-Sforza et al. 1994; Diamond 1997). the increase of interspecies transmission from domesticated ani- As agriculture spread, populations of pathogens on humans and mals, and (3) the expansion of human populations into novel en- their domesticated animals and plants expanded. Agriculture may vironments and resulting exposure to novel pathogens (Diamond also have contributed to the number of pathogen species and their 2002; Armelagos and Harper 2005). A similar process affected the current geographic ranges through the anthropogenic modifica- pathogens colonizing agricultural crops. The high host densities tion of the environment (Schrag and Wiener 1995; Kolar and and genetic uniformity of host populations coupled with cultiva- Lodge 2001; Diamond 2002; Anderson et al. 2004; Armelagos tion practices and trade created more uniform environments that and Harper 2005). The expansion of human pathogens follow- maintained stable pathogen populations and were conducive for ing the shift of human societies to agriculture has been explained disease development and transmission. The movement of domes- ticated plants and their respective pathogens into new areas could 3Corresponding author. simultaneously introduce “domesticated” pathogens into new C 2008 The Author(s) . Journal compilation C 2008 The Society for the Study of Evolution. 1418 Evolution 62-6: 1418–1436 RECENT PATHOGEN ORIGINS areas in which they could colonize “wild” hosts and expose the was not supported for fungal pathogens of the genera Ustilago and domesticated crops to new pathogens that could shift from wild Sporisorium causing smut on Poaceae such as maize, sorghum, to domesticated hosts. Anthropogenic influences on the environ- and sugarcane. Divergence time estimates for these species were ment such as intensification of crop production and global trade millions of years, indicating that speciation occurred long be- or other factors such as climate change are thought to promote fore their hosts were domesticated less than 10,000 years ago the emergence of new plant diseases (Anderson et al. 2004; Slip- (Munkacsi et al. 2007). pers et al. 2005; Woolhouse et al. 2005; Money 2007). However, Rhynchosporium secalis (Oudem.) J.J. Davis. causes an im- the history and the processes that led wild pathogens to become portant disease called scald on barley, rye, and other grasses. domesticated and vice versa have been poorly studied. Analyses of nucleotide sequences of NIP1, a gene encoding a Many human pathogens may have originated since the rise toxin involved in pathogenicity, indicated that R. secalis started of agriculture as a result of host shifts from domestic animals to colonize barley ∼5000–7500 years after the domestication of (Pearce-Duvet 2006; Wolfe et al. 2007). Although this hypothesis barley (Brunner et al. 2007) that occurred ∼10,000 B.P. (Badr has been widely proposed for many human diseases, unequivo- et al. 2000). This led to the hypothesis that domestication and cal evidence based on phylogenetics and estimates of divergence agricultural practices affected the emergence and global spread of times are rarely presented (Pearce-Duvet 2006 for review). For the this barley pathogen. In this study we investigate how agriculture tuberculosis bacterium Mycobacterium tuberculosis, tapeworms shaped the demography of the pathogen through time and deter- of the genus Taenia, and the protozoan Plasmodium falciparum mine whether the emergence of the pathogen fits the domestication causing falciparal malaria, there is evidence that their progenitors hypothesis. The evolutionary history of R. secalis was investigated predate the rise of agriculture and may have already been hu- using nucleotide sequences of several housekeeping genes not in- man pathogens before animal domestication (Pearce-Duvet 2006 volved in pathogenicity, in contrast to NIP1. We included 316 for review). In a similar way, the progenitors of modern plant isolates from barley, rye, and uncultivated grasses from different pathogens may have been present already on the progenitors of continents. Population genetic analysis, multiple gene genealo- crop plants and diverged with them after domestication (Munkacsi gies, coalescent-based approaches, and analyses of pathogenicity et al. 2007). Alternatively, domestication might have strongly in- were combined to determine if cryptic species exist on cultivated fluenced host shifts leading to the emergence of new diseases on and wild hosts of the pathogen. We then determined whether the crops (Couch et al. 2005). Agricultural practices might have sub- fungal populations on uncultivated grasses were ancestral to pop- sequently favored host specialization, reproductive isolation, and ulations on cereal hosts or originated from the same or different speciation of plant pathogens on new hosts (Hansen 1987; Kohn ancestors. Divergence time between and within host-associated 2005). The role that agriculture has played in the emergence of populations was estimated and the demographic history of the a plant disease can be evaluated by dating the divergence of the pathogen reconstructed. We present evidence that agriculture has causal agent. For the most important group of plant pathogens, the driven the evolution of R. secalis since the Neolithic through a fungi, few studies have attempted to date divergence from their host shift from wild grasses to cultivated barley, falsifying the progenitors (Couch et al. 2005; Munkacsi et al. 2007; Stukenbrock domestication hypothesis for this plant pathogen. et al. 2007), mainly due to the lack of fossil records and large er- rors associated with molecular clocks. In contrast, the evolutionary Material and Methods history and time of domestication has been well studied for most FUNGAL ISOLATES of the important staple crops infected by these fungal pathogens. Isolates of R. secalis from nine different hosts including culti- The origins of the fungal pathogens Mycosphaerella gramini- vated barley (Hordeum vulgare), rye (Secale cereale) and triticale cola causing septoria leaf blotch on wheat and Magnaporthe (× Triticosecale Wittmack), as well as six wild grasses; Agropy- oryzae causing rice blast coincided with the domestication of their ron caninum, Agropyron repens, Bromus diandrus, Hordeum lep- current hosts (Couch et al. 2005; Stukenbrock et al. 2007), start- orinum, Hordeum murinum and Hordeum spontaneum, were in- ing ∼10,000 years before present (B.P.) for wheat in the Fertile cluded in this study. Isolates of Rhynchosporium orthosporum (the Crescent (Flannery 1973; Lev-Yadun et al. 2000; Salamini et al. only other described Rhynchosporium species) infecting Dactylis 2002) and ∼7000 years B.P. for rice in East Asia (Flannery 1973; glomerata were included as outgroup. The R. secalis isolates orig- Crawford and Shen 1998; Higman and Lu 1998). Domestication, inated from 21 countries representing five continents. Many of agricultural practices, and trade strongly influenced the pathogen’s these isolates were representatives of a collection