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 of R. secalis that evolution and the diseases they cause on these crops (Couch et al. was characterized previously (McDermott et al. 1989; McDonald 2005; Stukenbrock et al. 2007). We refer to this very recent origin et al. 1999; Salamati et al. 2000; Linde et al. 2003; Zaffarano of the pathogens associated with the domestication of the host et al. 2006). Detailed descriptions of the isolates are found in as the “domestication hypothesis.” The domestication hypothesis Table 1. Each isolate had a unique multilocus genotype based on
EVOLUTION JUNE 2008 1419 PASCAL L. ZAFFARANO ET AL.
Table 1. Origin of the Rhynchosporium isolates used in the study.
Host Geographic No. of Previous publication or source and origin isolates year of collection (in parentheses) Agropyron caninum Switzerland 1 C.C. Linde (2002) Agropyron repens Switzerland 50 P. L. Zaffarano, M. Zala, C.C. Linde (2004–2005) Barley Australia 7 McDonald et al. (1999) Barley Azerbaijan 2 A. Yahyaoui (2003) Barley Ethiopia 13 A. Yahyaoui (2003), Zaffarano et al. (2006) Barley Eritrea 6 A. Yahyaoui (2003) Barley Finland 8 Salamati et al. (2000) Barley France 5 Zaffarano et al. (2006) Barley Germany 7 Zaffarano et al. (2006) Barley Jordan 4 Zaffarano et al. (2006) Barley Kyrgistan 2 A. Yahyaoui (2003) Barley New Zealand 8 M. Cromey (2004) Barley Norway 16 Salamati et al. (2000) Barley South Africa 3 Linde et al. (2003) Barley Sweden 1 S. Salamati (1996) Barley Switzerland 45 Linde et al. (2003) Barley Syria 5 Linde et al. (2003) Barley Tunisia 3 A. Yahyaoui (2003) Barley Turkey 2 Zaffarano et al. (2006) Barley United Kingdom 3 Zaffarano et al. (2006) Barley USA 6 McDermott et al. (1989) Bromus diandrus Australia 2 McDonald et al. (1999) Dactylis glomerata Italy 7 C.C. Linde (2004) Dactylis glomerata Switzerland 6 P. L. Zaffarano, M. Zala, C.C. Linde (2004–2005) Hordeum leporinum Australia 18 McDonald et al. (1999) Hordeum murinum Switzerland 4 C.C. Linde, P. L. Zaffarano (2004) Hordeum murinum USA 3 C.C. Linde (2003) Hordeum spontaneum Syria 5 M. Abang (2003) Rye Russia 25 L. Lebedeva (2003) Rye Switzerland 27 C.C. Linde (2002), Zaffarano et al. (2006) Triticale Switzerland 6 C.C. Linde (2002) Triticale France 16 A. Bouguennec, L. Jestin (2002) Total 316
RFLP loci, RAPD fingerprints, mating types (Linde et al. 2003; 728F and EF1-986R (Carbone and Kohn 1999). Portions of - Zaffarano et al. 2006), and microsatellites (Linde et al. 2005). tubulin were amplified in three steps. The first primer set used was ATUB-25F (5-GAGAAGCTATTAGCATCAACG-3) and DNA EXTRACTION, AMPLIFICATION, AND ATUB-649R (5 -CTCCTTTCCAACAGTGTAGTGAC-3 ), the SEQUENCING second ATUB-540F (5 -TCCCTAGAACCATCTACTGCG-3 ) Isolation and culturing of fungal isolates were as described in and ATUB-1206R (5 -CTTTGGCGGCAGACAACTG-3 ), and McDonald et al. (1999). DNA was extracted from lyophilized tis- the third ATUB-1103F (5 -ACAGTTGTCTCCTCCATTACCG- sue with either the method described in McDonald et al. (1999) 3 ) and ATUB-1603R (5 -TGGACGAAGGCACGCTTAGAG- or the DNeasy Plant Mini DNA extraction kit (Qiagen GmbH, 3 ). Part of the -tubulin gene was amplified with the Hilden, Germany) according to the manufacturer’s instructions. primers BTUB-21F (5 -ATGCGTGAAATCGTACGTCAC-3 ) Four nuclear DNA loci were analyzed. Sequences of the ITS and BTUB-615R (5 -TGACCGAAAGGACCAGCACG-3 ). PCR region (ITS1, 5.8S rRNA gene, ITS2) were obtained by PCR reactions were carried out in 20 l volumes containing 2 l10× amplification of genomic DNA of isolates using the primers PCR buffer, 0.1 mM of each dNTP, 0.5 M of each primer, ITS4 and ITS5 (White et al. 1990). The translation elonga- 0.5 U of Taq polymerase (New England BioLabs, Allschwil, tion factor 1 alpha (EF-1 ) was amplified with primers EF1- Switzerland), and 5 l of genomic DNA (5–20 ng final DNA
1420 EVOLUTION JUNE 2008 RECENT PATHOGEN ORIGINS
concentration). PCR conditions for ITS included a denaturing bootstrap replicate. All characters were equally weighted and un- step at 96◦C for 2 min, followed by 35 cycles at 96◦C for 1 ordered. min, 55◦C for 1 min, and 72◦C for 1 min. Finally, a 5-min Combining several independent loci can increase the accu- PCR extension was carried out at 72◦C. PCR amplifications for racy and confidence of phylogenetic inference (Pamilo and Nei the other three genes were the same as for ITS except that an- 1988; Takahata 1989; Rosenberg 2002). To test whether the data nealing cycles were carried out at 50◦CforEF-1 ,at56◦Cfor of individual loci might be combined, that is, whether the gene -tubulin and at 65◦Cfor -tubulin. For the R. orthosporum genealogies from the four loci were significantly different from isolates the annealing temperature had to be lowered to 53◦Cto each other, the partition homogeneity test (PHT) (Farris et al. amplify -tubulin and -tubulin. 1995) was used. The PHT used only informative characters and Amplification products were electrophoresed on 1% agarose 100 replicates of simple stepwise-addition MP heuristic searches gels to verify the amplification of a single fragment of the appro- (TBR; maxtrees = 500). However, methods to detect conflicts priate length. Amplified products were purified using a Millipore among data partitions, such as the incongruence length difference multiscreen PCR plate (MANU 030 PCR) following the manufac- (ILD) test (implemented as the PHT in PAUP) could be poor indi- turer’s instructions. The purified PCR products were resuspended cators of dataset combinability (e.g., Cunningham 1997; Dolphin in 20 l of water and sequenced bidirectionally with ABI PRISM et al. 2000; Barker and Lutzoni 2002; Darlu and Lecointre 2002; BigDye Terminator v3.0 and 3.1 ready reaction cycle sequencing Dowton and Austin 2002). Therefore, congruence between gene kit (Applied Biosystems, Foster City, CA). The sequencing reac- phylogenies was also estimated by visual inspection of topologies tion was conducted in a final volume of 10 l containing 0.4 l and statistical support (Mason-Gamer and Kellogg 1996; Wiens ready reaction mix, 1.6 l5× reaction buffer (400 mM Tris-HCL, 1998). pH 9.0, 10 mM MgCl2), 1 M primer, and 5 l of the purified For BML analyses the optimal model selected under the AIC PCR product. The DNA samples were sequenced with an ABI- implemented in Modeltest was specified as the prior for each 3100 automated sequencer. gene. MrBayes allowed different data partitions to be modeled separately for the combined dataset (Ronquist and Huelsenbeck PHYLOGENETIC ANALYSES 2003). One cold and three incrementally heated Markov chains DNA sequences were aligned and edited manually with Se- were run simultaneously starting from random trees for 5,000,000 quencher 4.5 (Gene Codes Corporation, Ann Arbor, MI). Iso- generations for the single loci, and for 10,000,000 generations for lates were assigned to haplotypes, that is, to unique DNA the combined dataset. Trees were sampled every 500th generation sequences at each sequence locus. Haplotypes and their frequen- for the single loci and every 1000th generation for the combined cies were obtained with the program MAP (Aylor et al. 2006) dataset, resulting in 10,000 trees of which 1000 were discarded and SITES version 1.1 (Hey and Wakeley 1997) by recoding as the “burn-in.” At least two independent runs were performed insertions or deletions (indels) and removing infinite site vi- to ensure analyses were not converging on a local optimum. The olations, as implemented in the SNAP Workbench (Price and replicate runs were compared to confirm that the analyses reached Carbone 2005). Redundant sequences were removed from the stationarity at similar likelihood scores by plotting the −lnL per datasets by choosing only one individual for each sequence generation in the program Tracer 1.3 (Rambaut and Drummond haplotype. 2003). After confirming that the replicate runs reached stationarity Two tree-building methods were used, namely maximum par- at similar likelihood scores and that the topologies were similar, simony (MP) and Bayesian maximum likelihood (BML), which the remaining trees of the separate runs were pooled together and were performed in PAUP∗ version 4.0b10 (Swofford 2002) and used for calculating the posterior probabilities in the 50% majority MrBayes 3.0b4 (Ronquist and Huelsenbeck 2003), respectively. rule consensus tree in PAUP. The program Modeltest 3.7 (Posada and Crandall 1998) was used to assess which model of nucleotide substitution best fit the data COALESCENT ANALYSES of each locus for BML under the Akaike information criterion The ancestral history of the host-associated populations of the (AIC). The trees were rooted with sequences from the only other fungus was inferred by coalescent-based gene genealogies. The described Rhynchosporium species, R. orthosporum. For MP anal- analysis was conducted in the SNAP Workbench (Price and Car- ysis, gaps were treated as fifth character states in heuristic searches bone 2005) that contains a series of programs described below that were conducted following 100 replicates of random step- to reconstruct the history of haplotypes. Guidelines in Carbone wise addition and tree bisection-reconnection (TBR) for branch- et el. (2004) were followed for each analysis. Sequences were swapping. Branch support for all parsimony analyses was esti- collapsed into unique haplotypes using SNAP MAP (Aylor et al. mated by performing 1000 bootstrap replicates with a heuris- 2006) and SITES version 1.1 (Hey and Wakeley 1997) by recoding tic search consisting of 100 random-addition replicates for each indels and removing infinite site violations. Prior to the application
EVOLUTION JUNE 2008 1421 PASCAL L. ZAFFARANO ET AL.
of coalescent methods, the absence of selection and recombina- demographic history of lineages in a Bayesian coalescent-based tion had to be verified. Therefore, deviation from neutrality was framework. The Bayesian skyline plot (BSP) (Drummond et al. measured by Fu and Li’s D∗ and Fu and Li’s F∗ (Fu and Li 1993) 2005) was specified as demographic model because it can fit a and Tajima’s D (Tajima 1989) with DnaSP version 4.0 (Rozas wide range of demographic scenarios. The MCMC analyses were et al. 2003). Incompatibility matrices were generated to detect in- first performed with short runs with chain length of 106 to op- compatibility among segregating sites in SNAP Clade (Markwordt timize the scale factors of the priors. The analysis was then run et al. 2004) and SNAP Matrix (Markwordt et al. 2004). for 108 generations sampling every 1000th iteration after an ini- Migration matrices indicating the number and direction of tial burn-in of 10%. The performance of the MCMC process was migrants exchanged between populations were constructed in checked for stationarity and large effective sample sizes in Tracer. the program MIGRATE (Beerli and Felsenstein 1999, 2001). The mean and corresponding credibility intervals of the estimated MIGRATE estimates the product of effective population size and parameters and the BSP were depicted using Tracer. mutation rate, and the amount and direction of gene flow between the host-associated populations. The analysis included 20 short PATHOGENICITY ASSAYS chains with 500 sampled genealogies each and 5 long chains with To determine whether Rhynchosporium isolates from each phy- 5000 sampled genealogies each. Chain heating was adaptive, with logenetic lineage (see Results) could infect hosts of other phy- four different temperatures. The migration matrices were used as logenetic lineages, barley, rye, and H. murinum were inoculated starting backward migration matrices for coalescent analysis with with representative isolates from each lineage originating from the population subdivision in the program GENETREE version 9.0 same geographical region, in this case Switzerland. Pathogenic- (Griffiths and Tavare´ 1994; Bahlo and Griffiths 2000) as incor- ity is here defined as the ability of a fungal isolate to infect a porated in the SNAP workbench. GENETREE reconstructs the host species. In this case pathogenicity was used as a phenotypic ancestral history of haplotypes showing a coalescence tree with marker to assess species boundaries and host specialization. relative time of divergence between host-associated pathogen pop- The isolates for the pathogenicity tests were a representa- ulations. The genealogy with the highest root probability was de- tive sample including different Swiss locations, collection years, termined by performing 500,000 simulations of the coalescent and genotypes. Two trials were undertaken. In the first trial, with five different starting random number seeds. From these runs, only isolates from cultivated grasses were used. The 16 selected the tree with the highest root probability was selected showing isolates originated from fields planted to barley, rye, and triti- the distribution of mutations along the branches of the pathogen cale. The seven barley-infecting isolates 99CH2A2B, 99CH5E4A, populations. 99CH5E6B, 99CH5H10A, 99CH6C3A, 99CH6E3B, and 00A1B, originated from collections of 1999 and 2000 that were de- DIVERGENCE TIME ESTIMATES AND DEMOGRAPHIC scribed previously (Linde et al. 2003; Zaffarano et al. 2006). ANALYSIS Four of the eight rye-infecting isolates (99CH1E7A, 99CH1B8, The Bayesian Markov Chain Monte Carlo (MCMC) method im- 99CH1H10B, and 99CH1D4A) were collected in 1999 as de- plemented in the program BEAST version 1.4.1 (Drummond and scribed earlier (Zaffarano et al. 2006). The remaining isolates Rambaut 2005) was used to estimate time of divergence between (02CH4-14a.1, 02CH4-9a.2, 02CH4-5a.1, and 02CH4-6a.1) were host-associated populations of the pathogen, that is the time to collected in 2002 from a rye field near Maur in the canton of the most recent common ancestor (TMRCA), and past popula- Zurich, whereas the isolate 02CH2-3c.1 was collected from triti- tion dynamics. Kasuga et al. (2002) proposed a range of mutation cale at the experimental station of Changins in the canton of Vaud rates for the Eurotiomycetes, a monophyletic class of Ascomycota. in 2002. We applied these mutation rates representing the lower end, the To produce inoculum, all isolates were grown from sil- mean, and the upper end of the range, that is, 0.9 × 10−9, 8.8 × ica gel storage onto Difco lima bean agar (Becton, Dickinson 10−9, and 16.7 × 10−9 mutations per site and per year. The anal- and Co., Sparks, MD) amended with kanamycin (50 mg/L). ysis was conducted with the multilocus dataset as the program Plates were incubated for 14 days at 18◦C in the dark. Colonies allows partitioning the combined datasets. Different evolutionary were then transferred to fresh lima bean agar plates and in- substitution models can be included for each partition and ap- cubated under the same conditions as above. After 14 days plied simultaneously. The substitution models specified for each spores were harvested by adding 2 mL of sterile water to each gene were the same as obtained under the AIC in Modeltest. Esti- plate and scraping spores off the agar surface with a steril- mates assuming a strict molecular clock were compared to those ized microscope slide. A portion of the spores from each iso- performed using the relaxed molecular clock option with uncor- late was transferred into 1.8 mL CryoTubes (Nunc Cryoline Sys- related, branch-specific rates following lognormal or exponen- tems, Roskilde, Denmark) containing anhydrous silica gel (Fluka tial distribution (Drummond et al. 2006). BEAST also infers the Chemie GmbH, Steinheim, Germany) for long-term storage at
1422 EVOLUTION JUNE 2008 RECENT PATHOGEN ORIGINS
−80◦C. The rest of the spore suspension was spread across mental station of Changins in the canton of Vaude in Switzerland, the surface of 20–50 fresh lima bean agar plates. The plates except SEG-A3.2.1.1 which was collected from A. repens plants were incubated at 18◦C and after 14 days spores were harvested bordering the road on the Sattelegg pass in canton Schwyz in as above and filtered through two layers of cheesecloth. Spore Switzerland. The isolates from H. murinum were collected from concentrations were adjusted to 2 × 105 spores/mL with a hema- weedy plants growing alongside streets in Zurich. Seed was har- cytometer (Thoma cell, 0.1-mm depth, 0.0025 mm2) in a spore vested from these plants for inoculation trials. Two previously used solution of 150 mL per isolate. isolates, one each from barley (00CHA1B) and rye (99CH1E7a), Inoculations were conducted on four barley (Chariot, Julia, were included as control isolates. The barley and rye varieties as Pasadena, and Plaisant) and four rye varieties (Avanti, Born, well as H. murinum were represented by three pots each with five Danko, and Picasso). These varieties ranged from moderately to seedlings per pot. In total 10 inoculations were applied for a total highly susceptible to scald. All host varieties were grown in pots of 150 pots. The negative control, sprayed with water and Tween, with a diameter of 13 cm filled with soil mixture Rasenerde Top was represented by 60 pots, that is, six pots containing all host va- Dressing (containing sand, compost, perlite, white peat, and min- rieties and H. murinum per inoculated isolate. The second trial was eral fertilizer; Ricoter AG, Aarberg, Switzerland). Seeds of each repeated once. Procedures for the isolate culturing, inoculation, host variety were sown separately in pots and thinned to five plants and disease assessment were the same as for the first inoculation per pot. trial, except that the spore concentration was 106 spores/mL for The plants were grown in a single greenhouse chamber with both the first experiment and the repetition. a photoperiod of 14 h-day at 18◦C and a 10 h-night period at 15◦C. Relative humidity was set at 60%. The seedlings were inoculated when they had two to three fully emerged leaves. Two drops of Results Tween 20 (Sigma-Aldrich, Buchs, Switzerland) were added to 150 NUCLEOTIDE SEQUENCES AND PHYLOGENETIC mL of spore suspension. For each isolate, three pots of each variety ANALYSES were inoculated. For inoculation, all 24 pots were placed onto a The ITS region (848 bp), portions of the -tubulin (1609 bp), - rotating table in a semiautomatic inoculation chamber and the tubulin (609 bp), and the EF-1 (365 bp) (sizes including gaps) leaves were sprayed with a fine mist until run-off. An additional were amplified for all isolates. Summaries of the phylogenetic in- set of 96 pots, that is, 6 pots per each inoculated isolate, containing formation for the four loci are shown in Table 2. There were 24, 20, all host varieties, was sprayed with sterilized water amended with 15, and 11 parsimony informative characters, respectively for each two drops of Tween 20 as a negative control. The inoculated pots locus (Table 2). Haplotypes representing one of each set of iden- were kept for 48 h at a relative humidity of 90–100%. tical sequences were used for analysis. The haplotypes defined by After 14 days disease was assessed on the second and third the variable sites of the datasets are reported in the online Supple- leaves following the scale described in Ali and Boyd (1973). The mentary Table S1. Haplotypes were shared only between either ratings were 0 = no visible lesion or symptoms, 1 = small lesions populations from rye and triticale, or between populations from at the tip or on the margin and base of the leaf blades, 2 = nar- barley, H. leporinum, H. murinum, and H. spontaneum (Hordeum row band of lesions extending down the length of the leaf blade, spp.) and B. diandrus for all loci. The single isolate from A. can- 3 = broad well-developed lesions covering large areas across leaf inum always grouped with the haplotypes formed by the A. repens blade, 4 = leaves wilted, no evidence of discrete lesions. Reac- isolates. Haplotype H1 occurred most frequently for each respec- tion 0 was considered highly resistant, reaction 1 was resistant, tive locus, and was represented in most of the sampled geograph- reaction 2 was intermediate, and reactions 3 and 4 susceptible ical populations originating from barley, as well as in Hordeum and highly susceptible, respectively. This inoculation trial was re- spp. and B. diandrus. Conversely, many haplotypes were unique peated one month later using a higher inoculum concentration of to one population or host species (see online Supplementary 106 spores/mL. Table S1). In the second trial, Rhynchosporium isolates from uncul- The MP and BML analyses were used to infer genealogies tivated grasses were included for pathogenicity testing. Six of the haplotypes from the four single-locus alignments (Table 2, Rhynchosporium isolates from A. repens (Danikon-1.1.1,¨ WPK- Fig. 1, and see online Supplementary Figs. S1–S4). BML analysis 5b.1A3.1, K2B-2C1.1, Brutten-3.1.2,¨ RAC-2-A9.1, and SEG of the four loci followed an optimal evolutionary model selected A3.2.1.1) and two from H. murinum (WPK-2.1 and WPK-A8.4) under the AIC in Modeltest. These models were different for each that were inoculated onto two of the previously used barley (Char- gene (Table 2). All repeated runs of the BML analyses converged iot and Pasadena) and rye varieties (Danko and Picasso), as well as on the same topology. The topological patterns were consistent on H. murinum. The isolates from A. repens were collected at the across the MP and BML estimations of the phylogeny for all loci borders of cereal fields in the canton of Zurich and at the experi- except for minor differences as described below.
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Table 2. Phylogenetic information of the genomic regions used in this study for the individual and combined datasets.
DNA sequence region
-tubulin -tubulin EF-1 ITS Combinedb Number of isolates 316 316 316 316 316 Number of sites 1609 609 365 848 3066 Number of haplotypes 18 25 15 25 46 Nucleotide characters, excluding indels 1592 592 348 843 3027 Coded characters (indels) 17 17 17 5 39 Constant charactersa 1571 575 332 812 2958 Parsimony informative charactersa 20 15 11 24 59 Variable, parsimony uninformative 1 2 5 7 10 charactersa Percent informative characters (%)a 1.26 2.53 3.16 2.85 1.95 MP tree length 233 135 106 105 488 No. of equally parsimonious trees 30 >10,000 8 354 720 CIc 0.996 0.926 0.979 0.876 0.926 RIc 0.997 0.963 0.987 0.935 0.974 Modeld F81+I HKY+I F81 Tnef+I Base frequencies A 0.2635 0.2861 0.2492 Equal frequencies C 0.2186 0.1929 0.3161 G 0.2901 0.2702 0.1662 T 0.2278 0.2508 0.2685 Substitution model All rates equal Ti/Tve ratio = 0.1688 All rates equal A→C = 1.000 A→G = 3.184 A→T = 1.000 C→G = 1.000 C→T = 8.649 G→T = 1.000 Among-site rate variation If 0.9315 0.9447 0 0.8583 Gg Equal rates for Equal rates for Equal rates for Equal rates for all sites all sites all sites all sites aRefers to ingroup taxa only. bCombined -tubulin, -tubulin, ITS. cRefers to the strict consensus tree. dBest-fit evolutionary models and parameters for each dataset selected by the Akaike information criterion (AIC) in Modeltest (Posada and Crandall 1998). eRatio between transitions and transversions. fProportion of invariable sites. gVariable sites gamma distribution parameter.
-tubulin bpp) but less supported by the MP analysis (62% bootstrap sup- Both MP and BML methods showed three lineages of haplotypes port). Lineage C consisted of two clades with 85–99% bootstrap based on host association, although branch support and resolution support and 1.0 bpp, respectively. was poor (see online Supplementary Fig. S1). Lineage A consisted of those haplotypes representing the isolates from barley, other -tubulin Hordeum spp. and B. diandrus, lineage B the haplotypes from The tree topology was identical for the MP and the BML trees. A. caninum and A. repens (Agropyron spp.), and lineage C those As for -tubulin, three lineages of haplotypes were identified (see from rye and triticale consisting of two monophyletic clades. Lin- online Supplementary Fig. S2). Again, lineage A contained all eage B was well supported (98% bootstrap support, 1.0 Bayesian haplotypes infecting barley, Hordeum spp., and B. diandrus.As posterior probability, bpp), but formed a clade with lineage A. for -tubulin, the bootstrap support for this monophyletic clade This larger clade was well supported in the BML analysis (0.94 was not high using the MP method (67%) but was high using
1424 EVOLUTION JUNE 2008 RECENT PATHOGEN ORIGINS
Figure 1. Phylogeny inferred by maximum parsimony from the combined -tubulin, -tubulin, and ITS DNA sequence loci. The 50% majority-rule consensus tree is shown. Bayesian maximum-likelihood analysis recovered the same topology. The numbers above branches are bootstrap frequencies of 1000 replicates and those below are Bayesian posterior probabilities. Branch lengths are proportional to the number of steps (character changes) along the branch. Labels on the phylogeny are: H1–H45 haplotypes obtained from 316 DNA sequences of 3066 bp representing Rhynchosporium isolates from different hosts (Agropyron spp., barley, Bromus diandrus, Dactylis glomerata, Hordeum spp., rye and triticale). Haplotypes H2–H4 representing Rhynchosporium orthosporum were used as outgroup.
EVOLUTION JUNE 2008 1425 PASCAL L. ZAFFARANO ET AL.
the BML method (0.97 bpp). The monophyletic lineage B in- The phylogeny of the combined -tubulin, -tubulin, and ITS cluded haplotypes infecting Agropyron spp. (69% bootstrap datasets resulted in three well-supported distinct monophyletic support, 1.0 bpp). Lineage C contained the haplotypes infect- lineages (Fig. 1) using both BML and MP analyses. Lineage A ing rye and triticale. This lineage could be distinguished from consisted of haplotypes present on barley, Hordeum spp., and lineages A and B, but resolution was poor and did not form a B. diandrus. In contrast to the phylogenies of the single loci, this monophyly. lineage was well supported by the MP method (83% bootstrap support) and by the BML analysis (1.0 bpp). Lineage B contained EF-1 all Agropyron spp. infecting haplotypes. Lineage C contained all Both MP and BML showed a tree topology consisting of host- rye- and triticale-infecting haplotypes and was highly supported associated lineages A and B (see online Supplementary Fig. S3). (100% bootstrap support) in MP and BML trees (1.0 bpp). The Resolution and branch support for the lineages were low (see phylogenetic analyses thus support that R. secalis should be split online Supplementary Fig. S3). Lineage A contained isolates in- into three Rhynchosporium species, corresponding to isolates from fecting barley, Hordeum spp., B. diandrus, rye, and triticale. The lineages A–C. support for this monophyletic lineage A was weak (bootstrap sup- port 63% and 0.62 bpp). COALESCENT ANALYSES ITS Incompatible sites due to recombination or homoplasy were iden- The phylogeny of the ITS region (see online Supplementary Fig. tified in the incompatibility matrix for all four loci and removed S4) showed the same host-associated lineages as - and -tubulin from the datasets as follows: one site was removed for -tubulin, for both the MP analysis and the BML analysis. However, the six sites for -tubulin, two sites for EF-1 , and seven sites for ITS. ∗ ∗ rye- and triticale-infecting haplotypes(lineage C) formed a mono- Both Fu and Li’s D and F tests, as well as Tajima’s D were not > phyletic clade that was well supported (99% bootstrap support; significantly different from 0 (P 0.10; see online Supplementary 1.00 bpp). Similar tree topologies were obtained with MP and Table S2). Therefore, the hypothesis of selective neutrality could BML. The BML analysis gave higher support, and in contrast to not be rejected for any of the four loci. The datasets from which the MP method, lineage C was placed into a larger clade with the incompatible segregating sites were removed were then used lineage A. Furthermore, in the BML analysis not all haplotypes for coalescent analysis. Independent estimates of gene flow for the from lineage B formed a unique monophyly. four single loci gave similar results (data not shown). Migration matrices calculated by MIGRATE indicated low gene flow be- Combined dataset tween the different Rhynchosporium host-associated populations PHTs were performed in pairwise comparisons and globally (i.e., Nm < 1) except between the populations on rye and triti- to determine which datasets could be combined for phyloge- cale (Nm = 1.46-7.85) and between populations on barley and netic analyses. The PHT of the full dataset, combining all four Hordeum spp. (Nm = 0.45-4.60). loci, indicated that the datasets could not to be combined (P < Simulations in GENETREE provided coalescent-based ge- 0.01). Although EF-1 had the highest percentage of informa- nealogies for each locus showing the ancestral distribution of mu- tive characters (Table 2), these differences were mostly confined tations and coalescence events (Figures not shown). These results to haplotypes associated with A. repens. When EF-1 was re- were congruent with the phylogenetic trees inferred by MP and moved from the dataset, the P-value increased to 0.18. The host- BML. All four loci showed three distinct lineages as inferred by associated haplotype distinctions were the same for all four in- the phylogenetic analysis. When included as an outgroup, the R. dividual loci (see online Supplementary Table S1). Therefore, orthosporum population branched at the deepest point of the ge- we believe that the short sequence length of the EF-1 gene nealogies for separate as well as combined sequence loci (Figures led to a low phylogenetic resolution detected by the PHT rather not shown). All three lineages coalesced to a single common an- than representing a different evolutionary history compared to cestor. To provide an enhanced resolution of the ancestral history other genes. The P-value testing congruency between ITS and of these fungal populations, -tubulin, -tubulin, and ITS were -tubulin was lower (0.35) than between ITS and -tubulin combined in another coalescent analysis. In this analysis, 26 in- (P = 0.87). Compatibility between the -tubulin and -tubulin compatible sites had to be removed from the combined dataset datasets was low (P < 0.05), although the three sequence loci resulting in 27 haplotypes. The two distinct lineages A (on barley, could still be combined according to the PHT. The -tubulin, B. diandrus and Hordeum spp.), and B (on Agropyron spp.) were -tubulin, and ITS datasets were combined despite the minor dif- derived from the same common ancestor (Fig. 2), with coales- ferences in tree topologies because the PHT was not significant cence at a relative coalescent time of approximately 0.7. Most of and the associations between haplotypes and different hosts were the mutations separating the three distinct lineages emerged re- consistent. cently, that is, between the relative coalescent times of 0 to 0.2.
1426 EVOLUTION JUNE 2008 RECENT PATHOGEN ORIGINS
Figure 2. Coalescent-based gene genealogy of the combined -tubulin, -tubulin, and ITS DNA sequence loci showing the distribution ∗ of mutations in Rhynchosporium populations on different hosts (barley; Hordeum spp. = H. leporinum, H. murinum, H. spontaneum; = includes two isolates from Bromus diandrus; Agropyron spp. = Agropyron repens and Agropyron caninum; triticale, rye) that correspond to the three phylogenetic lineages as shown in Figure 1. The numbers below the tree branches describe the different haplotypes and the number of occurrences in total and on the different hosts. The temporal scale of divergence is given on the right. The scale is in coalescent units of effective population size. The direction of the appearance of mutations and bifurcations is from the top (past) to the bottom (present).
EVOLUTION JUNE 2008 1427 PASCAL L. ZAFFARANO ET AL.
DIVERGENCE TIME ESTIMATES AND DEMOGRAPHIC netic lineages. Due to difficulties in propagating A. repens plants, ANALYSIS this host could not be included in the trial as a positive control for The Bayesian MCMC sampling procedures implemented in isolates from A. repens. Disease severity on infected plants ranged BEAST allowed estimating the posterior distribution of the TM- from 1 to 4 based on the disease assessment scale of Ali and Boyd RCA and effective population size through time. The analysis (1973) (Table 4). included credibility intervals [highest posterior density (HPD)] representing both phylogenetic and coalescent uncertainty. The posterior mean estimates of the TMRCAs and corresponding Discussion 95% HPDs are shown in Table 3. Estimates assuming a strict In a previous population study on R. secalis using RFLP markers, molecular clock were similar to those based on a relaxed clock high population subdivision and a low number of shared alleles with exponential distribution. Under the relaxed clock model with among barley- and rye-infecting populations were interpreted as log-normal distribution the majority of the divergence time esti- evidence for genetic isolation between the host populations de- mates were higher. No fossil ages were available for R. secalis spite the geographical proximity of the two hosts in Switzerland to be specified as node priors to estimate the mutation rates for (Zaffarano et al. 2006). In this study additional isolates infect- the three loci used. To compensate, three mutation rates span- ing rye and other uncultivated grasses were included to further ning the entire range of mutation rates suggested for other fungi investigate genetic isolation between host-associated populations (Kasuga et al. 2002) were chosen in the analysis. The posterior of the pathogen. Our analyses assigned R. secalis isolates to three mean estimate for the TMRCAs and corresponding HPDs that distinct lineages: Lineage A infecting cultivated barley, Hordeum were obtained using these three mutation rates were combined spp. and B. diandrus; Lineage B infecting rye and triticale, and; and averaged in the program Tracer. Thus, we here refer to the av- Lineage C infecting Agropyron spp. We propose that these lin- eraged posterior mean estimates of the TMRCA and 95% HPDs. eages should be defined as three species of Rhynchosporium in Depending on the clock model implemented, the posterior mean addition to R. orthosporum infecting D. glomerata. estimates of the TMRCA of all isolates, including R. orthosporum, Several lines of evidence support the proposed species split: were between 14,443 to 35,199 years B.P. (95% HPD, 1246 to (1) population genetic data (Zaffarano et al. 2006) demonstrated 101,891) (Table 3) suggesting an older split from the ancestor the absence of gene flow between populations of Rhynchosporium of the lineages A–C. The posterior mean estimates of the TM- from rye and Hordeum spp., (2) sequences from four independent RCA of all three lineages A–C were between 1281 to 3627 years loci indicated that three evolutionary lineages could be assigned B.P. (95% HPD, 113 to 12,431). The diversification within the to unique haplotypes according to their hosts, showing ecological three lineages began more recently and started during a similar specialization, (3) the phylogeny of the combined loci showed a time frame for all three lineages with posterior means of the TM- highly supported monophyletic grouping of these haplotypes, (4) RCA between 459 and 1139 years B.P. (95% HPD, 21 to 3,853) coalescence analysis revealed independent evolution of the three (Table 3 and Fig. 3). lineages, and (5) host-association for the three lineages was con- The historical demographic reconstructions (BSP) shown in firmed with pathogenicity tests. Because R. secalis was first de- Figure 4 depict similar demographic histories for all three muta- scribed on rye (Oudemans 1897), this name should be retained for tion rates and clock options used (data not shown). The population Rhynchosporium isolates infecting rye and triticale. Rhynchospo- size of the three lineages started to decline almost simultaneously rium isolates infecting cultivated barley and other Hordeum spp. with the split of the three lineages ∼ 1200 to 3600 years ago (Fig. and B. diandrus, belong to a different species that should be given 4) until they reached a maximum decline between ∼ 250 and a new name. Similarly, isolates infecting Agropyron spp. should 500 years ago, followed by a rapid expansion that recovered the also be described as a new species of Rhynchosporium. prebottleneck population sizes (Fig. 4). The genetic isolation and subsequent speciation among these lineages is likely due to host specialization, which is common PATHOGENICITY ASSAYS in pathogenic fungi (Leppik 1965; Parlevliet 1986; Wyand and A total of 24 isolates from different hosts were inoculated onto Brown 2003). Domestication of plant hosts can be a factor driv- barley and rye cultivars, and a subset of these isolates was inocu- ing host specialization and subsequent fungal speciation (Kohn lated onto H. murinum. Isolate 02CH2-3c.1 from triticale was able 2005). As the evolutionary history of a pathogen is often tightly to infect the four rye cultivars and isolates WPK-2.1 and WPK- linked to that of its host (Fisher et al. 2001; Falush et al. 2003), re- A8.4 from H. murinum were able to infect two barley cultivars in constructing the population history of one component of the host- addition to H. murinum plants. All other isolates infected only the pathogen association may be useful to reconstruct the evolutionary host from which they were originally isolated. Thus there was no history of the other component if they have coevolved (Wirth et al. cross pathogenicity on hosts of isolates from different phyloge- 2005; Blaser 2006; Nieberding and Olivieri 2006). We therefore
1428 EVOLUTION JUNE 2008 RECENT PATHOGEN ORIGINS
Table 3. Bayesian estimates of the time (in thousands of years) to the most recent common ancestor (TMRCA) of Rhynchosporium populations on different hosts corresponding to three phylogenetic lineages A–C. Values in parentheses are 95% highest posterior densities intervals. The divergence time estimates were calculated under the assumption of three mutation rates adopting different molecular clock models as implemented in BEAST version 1.4.1 (Drummond and Rambaut 2005). The mean values of the three analyses were estimated with the program TRACER 1.3 (Rambaut and Drummond 2003).
Mutation rates per site per year
TMRCA Clock model Combined 0.9×10−9 8.8×10−9 16.7×10−9 Clock All sequences including Rhynchosporium 35.199 90.837 9.509 5.010 orthosporum (3.628–101.891) (71.093–112.121) (7.407–11.773) (4.965–3.876) Lineage A on barley/Hordeum spp./ 0.510 1.322 0.137 0.072 Bromus diandrus (0.036–1.638) (0.775–1.952) (0.080–0.201) (0.042–0.106) Lineage B on Agropyron spp. 0.459 1.194 0.120 0.063 (0.021–1.636) (0.491–2.023) (0.052–0.206) (0.026–0.107) Lineage C on rye/triticale 0.463 1.194 0.127 0.067 (0.027–1.560) (0.566–1.922) (0.061–0.203) (0.032–0.106) Lineages A+B on barley/Hordeum 1.291 3.347 0.345 0.181 spp./Bromus diandrus and on (0.095–4.028) (2.127–4.632) (0.218–0.475) (0.116–0.252) Agropyron spp. All three lineages A+B+C 1.399 3.625 0.375 0.198 (0.120–4.247) (2.487–4.791) (0.260–0.498) (0.136–0.262) Relaxed exponential All sequences including 14.443 37.800 3.577 1.953 Rhynchosporium orthosporum (1.246–43.732) (25.419–48.987) (2.450–4.751) (1.376–2.570) Lineage A on barley/Hordeum spp./ 0.811 2.111 0.214 0.108 Bromus diandrus (0.063–2.499) (1.398–2.878) (0.142–0.294) (0.072–0.148) Lineage B on Agropyron spp. 0.685 1.772 0.184 0.098 (0.050–2.179) (1.082–2.558) (0.111–0.268) (0.058–0.145) Lineage C on rye/triticale 0.568 1.474 0.152 0.077 (0.043–1.761) (0.955–2.030) (0.098–0.208) (0.050–0.104) Lineages A+B on barley/Hordeum 1.211 3.142 0.321 0.169 spp./Bromus diandrus and on (0.104–3.669) (2.192–4.144) (0.226–0.426) (0.119–0.225) Agropyron spp. All three lineages A+B+C 1.281 3.319 0.343 0.180 (0.113–3.846) (2.371–4.360) (0.241–0.454) (0.126–0.241) Relaxed lognormal All sequences including Rhynchosporium 26.853 69.562 7.281 3.715 orthosporum (1.685–89.040) (37.060–120.359) (3.844–12.519) (2.006–6.355) Lineage A on barley/Hordeum spp./ 1.139 2.958 0.299 0.158 Bromus diandrus (0.069–3.853) 1.411–4.855) (0.142–0.488) (0.079–0.258) Lineage B on Agropyron spp. 1.015 2.633 0.269 0.141 (0.046–3.576) (1.054–4.733) (0.103–0.493) (0.057–0.257) Lineage C on rye/triticale 0.614 1.594 0.161 0.088 (0.029–2.151) (0.633–2.905) (0.064–0.300) (0.035–0.161) Lineages A+B on barley/Hordeum 3.359 8.725 0.887 0.465 spp./Bromus diandrus and on (0.101–12.019) (2.797–15.468) (0.257–1.551) (0.146–0.819) Agropyron spp. All three lineages A+B+C 3.627 9.410 0.967 0.502 (0.187–12.431) (4.073–15.942) (0.419–1.637) (0.220–0.842) evaluated what role the history of barley and rye cultivation played evolved with them during domestication at the place of domestica- in promoting genetic isolation of Rhynchosporium populations on tion (the domestication hypothesis). It was shown that M. oryzae, different hosts. Plant pathogens are usually assumed to have origi- an important pathogen on rice, originated from a host shift from nated on the direct ancestors of their modern hosts and to have co- Setaria millet (Couch and Kohn 2002; Couch et al. 2005). This
EVOLUTION JUNE 2008 1429 PASCAL L. ZAFFARANO ET AL.
Figure 3. Bayesian posterior probability densities (bppds) of the TMRCA of Rhynchosporium on different hosts corresponding to three − phylogenetic lineages A–C as shown in Figure 1. These bppds assumed a strict molecular clock model and a mutation rate of 0.9 × 10 9 per site per year. Higher mutation rates and relaxed molecular clock models gave similar values. host shift was possibly associated with rice domestication and was the process of host domestication, not longer than 10,000 years brought about by the loss of an avirulence gene that allowed the ago. fungus to adapt to rice and diverge from other ancestral lineages Divergence time estimates were calculated for the three Rhyn- (Couch et al. 2005). In another study (Stukenbrock et al. 2007), chosporium lineages A–C in this study. The means of the com- the wheat pathogen M. graminicola was shown to be derived from bined estimates calculated in BEAST indicated that the split an ancestral population of Mycosphaerella species present on wild of the three lineages occurred recently, between ∼ 1200 and grasses in the Middle East. Coalescent analysis indicated that the ∼ 3600 years B.P. with a 95% HPD ranging from ∼ 100 pathogen started to infect wheat around 10,000 years ago, during to ∼ 12,000 years B.P. depending on the implemented clock the same time frame when the progenitors of wheat began to be do- models. These dates suggest that the emergence of the scald mesticated in the Middle East. Gradual adaptation of the fungus to pathogen was associated with the onset of agriculture. The up- domesticated wheat and a gradual decrease in gene flow between per ends of the HPD intervals indicate that the adaptation of the Mycosphaerella population infecting wheat and the population the pathogens to their hosts coincides with the beginning of the on wild grasses led to today’s host-specialized M. graminicola, domestication of barley around 10,000 years B.P. (Badr et al. which has spread to cultivated wheat populations around the 2000; Salamini et al. 2002) thus supporting the domestication world. In both cases the pathogen species fit the domestica- hypothesis. Similar to the Mycosphaerella pathogen on wheat, tion hypothesis, rapidly adapting to their modern host during the Rhynchosporium pathogen could have shifted and adapted
1430 EVOLUTION JUNE 2008 RECENT PATHOGEN ORIGINS
from an ancestral population on wild grasses onto domesticated barley and rye during the beginning of agriculture in the Fertile Crescent. As H. spontaneum is the progenitor of cultivated barley (Zo- hary and Hopf 1988; Badr et al. 2000) and still widely distributed in the Fertile Crescent, the Middle Eastern populations from H. spontaneum included in this study were expected to represent the closest relatives of the ancestral population of the pathogen on barley. Moreover, as domesticated rye is a younger crop than barley (Zohary and Hopf 1988; Bushuk 2001), the fungal populations on rye were expected to be derived from those on H. spontaneum or cultivated barley. Surprisingly, the coales- cent analysis indicated that the mutations found in isolates in- fecting H. spontaneum were all relatively recent. Furthermore, Rhynchosporium associated with rye did not originate from Hordeum spp. or cultivated barley populations, but instead the lin- eages evolved in parallel. The rooted coalescent genealogy showed that all three lineages A–C split from a common ancestor and none of the lineages can be regarded as ancestral to the other. Thus the barley- and rye-infecting populations most likely did not origi- nate from each other, or from H. spontaneum, but instead origi- nated from an unidentified common ancestor. Rhynchosporium orthosporum diverged from the other Rhynchosporium lin- eages prior to the domestication of cereals. Hence the split of R. orthosporum from the ancestor of lineage A–C is ancient and not associated with the advent of agriculture. In contrast, the means of the divergence time estimates and the lower ends of the HPD intervals suggest an even more re- cent emergence of the scald pathogen. These values are consistent with the divergence time estimates based on the nucleotide se- quences of the NIP1 avirulence gene present in R. secalis. The sampled isolates from barley fields were shown to coalesce back to a common ancestor that occurred between 2500 and 5000 years B.P. (Brunner et al. 2007). We hypothesize that the ancestor of the pathogen came into contact with the crops after the hosts had been domesticated and probably after the hosts were introduced Figure 4. Bayesian Skyline Plots (BSP) (Drummond et al. 2005) into Europe, hence the recent divergence time. We support this show fluctuations of effective population sizes through time. The hypothesis with the findings of our previous studies (Zaffarano x-axis is in units of thousands of years before present, and the y- et al. 2006; Brunner et al. 2007). Genetic diversity is expected to axis is equal to Ne (the product of the effective population size and the generation length). The thick solid line is the median esti- be greatest in ancient populations compared to more recent, de- mate, and the gray areas show the 95% highest posterior density rived populations because of their long-standing accumulation of (HPD) limits. The thin-dashed line shows the lower 95% HPD limit polymorphism. Allele richness measured at RFLP loci (Zaffarano of the time to the most recent common ancestor (TMRCA) and the et al. 2006), microsatellite loci (C. C. Linde and B. A. McDonald, thick-dashed line the mean of the TMRCA of lineages A–C corre- unpubl. ms), and additional sequence loci (P. L. Zaffarano, B. A. sponding to Figure 1. The presented BSPs were based on analyses McDonald, and C. C. Linde, unpubl. ms) was shown to be highest that assumed a strict molecular clock model and a mutation rate − in the Rhynchosporium populations from northern Europe and low of 0.9 × 10 9 per site per year. Higher mutation rates and relaxed in regions of ancient barley cultivation including the Middle East molecular clock models gave similar demographic histories. The profiles indicate a decline of population size since the TMRCA of and Ethiopia (Zaffarano et al. 2006). These findings are consistent all three lineages and rapid recoveries from the bottleneck ∼ 250 with a center of origin located away from the Fertile Crescent and years B.P. most probably in northern Europe as suggested in Zaffarano et al.
EVOLUTION JUNE 2008 1431 PASCAL L. ZAFFARANO ET AL.
Table 4. Reaction of Rhynchosporium secalis isolates from different hosts on barley and rye varieties, and on Hordeum murinum.
Source of Isolate Disease reactiona isolate Barley cultivars Rye cultivars H. murinum Control plants Chariot Julia Pasadena Plaisant Avanti Born Danko Picasso seedlings Barley 99CH2A2b 3–41–43–43–40 00 0 0 0 99CH5E4a 2–42–43–42–40 00 0 − 0 99CH5E6b 4 2–43–44 0000 − 0 99CH5H10a 3–43–44 3–40 00 0 − 0 99CH6C3a 2–41–33–44 0000 − 0 99CH6E3b 3–41–43–42–30 00 0 − 0 00CHA1b 2–41–41–41–40 00 0 − 0 Rye 99CH1E7a 0 0 0 0 3–43–42–41–40 0 99CH1B8 0 0 0 0 2–42–43–42–4 − 0 99CH1H10b 0 0 0 0 1–43–42–43–4 − 0 99CH1D4a 0 0 0 0 2–43–41–43–4 − 0 02CH4-5a.1 0 0 0 0 1–41–43–42–4 − 0 02CH4-6a.1 0 0 0 0 2–42–32 1–4 − 0 02CH4-9a.2 0 0 0 0 3–43–43–41–4 − 0 02CH4-14a.1 0 0 0 0 2–42–43–42–4 − 0 Triticale 02CH2-3c.1 0 0 0 0 3–42–41–41–4 − 0 A. repens Brutten-3.1.2¨ 0 − 0 −−−00 0 0 Danikon-1.1.1¨ 0 − 0 −−−00 0 0 K2B-2C1.1 0 − 0 −−−00 0 0 RAC-2-A9.1 0 − 0 −−−00 0 0 SEG-A3.2.1.1 0 − 0 −−−00 0 0 WPK-5b.1A3.1 0 − 0 −−−00 0 0 H. murinum WPK-2.1 0–3 − 3 −−−00 2–30 WPK-A8.4 2–4 − 3–4 −−−00 3–40 aDisease assessment was according to the scale described in Ali and Boyd (1973). Host-isolate combinations marked with ”-”were not tested.
(2006). Archeological remains of barley grains suggest that bar- causing late blight on potatoes and the fungus Colletotrichum lin- ley was first domesticated around 10,000 years B.P. (Badr et al. demuthianum causing anthracnose in common bean both have 2000; Salamini et al. 2002) in the Fertile Crescent. As a result of their highest levels of genetic diversity in Mesoamerica whereas Neolithic migrations, agriculture was brought to Europe ∼ 7500 their hosts originated in the Andes (Fry et al. 1992; Sicard et al. B.P. (Salamini et al. 2002; Haak et al. 2005). The emergence of 1997; Grunwald¨ and Flier 2005). However, coalescent analy- the pathogen lineages coincides with the introduction of barley ses showed that ancestral mutations in P. infestans originated and rye cultivation into northern Europe which has been dated to from the Andes, suggesting a South American origin of the ∼5000 to 3000 B.P. (Price 1996; Bushuk 2001). Given that the pathogen (Gomez-Alpizar´ et al. 2007). For C. lindemuthianum, highest diversity for all genetic markers is in northern Europe pop- incongruence between the geographical origin of the pathogen ulations and that there is a decreasing gradient of genetic diversity and its hosts is not completely confirmed because the com- running from northern Europe to the Middle East (Brunner et al. mon bean experienced independent domestication events in both 2007), we consider it most likely that Rhynchosporium emerged Mesoamerica and the Andes, and both areas developed into cen- recently as a barley and rye disease via a host shift that occurred ters of diversity (Debouck 1986; Kami et al. 1995; Pickersgill in northern Europe ∼ 1200 to 3600 years ago. Under this scenario 2007). It is important to note that general conclusions regard- we falsify the domestication hypothesis, although the spread of ing geographical origins of pathogens based solely on compar- agriculture played a significant role in bringing a host into contact isons of genetic diversity are problematic. In this study, how- with a new pathogen. ever, a combination of genetic diversity studies and divergence For other plant pathogens it was suggested that their center time estimates for Rhynchosporium populations provide support of origin does not necessarily coincide with the center of origin for a recent origin of the pathogen outside the host’s center of of their hosts. For example, the oomycete Phytophthora infestans origin.
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An alternative scenario considers the possible loss of genetic graphic history as on the other domesticated hosts. Agropyron spp. diversity in Rhynchosporium through a series of selective sweeps are commonly found in cereal fields and pastures. Their close as- associated with adaptation to its domesticated hosts. The BSPs sociation with cereal crops might have allowed this host species indicate a decline in population size for all three lineages after to increase in population size and spread globally as a weed while they started diverging from each other. This could reflect a bottle- the barley and rye populations increased. The coalescent genealo- neck that was experienced after the host shift from the ancestral gies showed that lineages A and C infecting barley and Agropyron population onto the domesticated hosts through a combination of spp., respectively, share a common ancestor from which they di- selection for host specialization and genetic drift. Under this alter- verged soon after their ancestor split from lineage B infecting rye. native scenario, the pathogen populations currently present on the It is probable that lineage C mirrored the demographic develop- sampled hosts would have lost most of their ancient diversity, lead- ment of lineage A and was spread globally through barley cultiva- ing to an inability to reconstruct the true lines of descent among tion and trade. Interestingly, the geographic area from which iso- the host-specialized lineages through the last 10,000 years. In this lates infecting Agropyron spp. were obtained is small (only from case a divergence time coinciding with the beginning of barley Switzerland) compared to that of rye-infecting isolates (Russia, domestication would favor an origin of the pathogen in the Fertile Switzerland, France) or barley-infecting isolates (many fields on Crescent (i.e., supporting the domestication hypothesis). We con- five continents). However, lineage C contained more than half of sider this alternative scenario to be less likely because it suggests the haplotype diversity present in lineage A and more haplotype that selective sweeps occurred independently in all three lineages diversity than present in lineage B. It is possible that the genotypic and affected all the genes used in this study and in Brunner et al. diversity of the scald pathogen on Agropyron spp. is higher than (2007). Moreover, although the sexual stage of the fungus has not on cultivated hosts because of a higher genetic diversity in mainly been observed in the field or induced in vitro, there is strong in- outbreeding Agropyron hosts, compared to genetically uniform direct evidence for regular sexual reproduction in the field (Linde cultivated crops. et al. 2003; Zaffarano et al. 2006). A recombining genome would Agriculture played a crucial role in the evolution of the scald prevent single haplotypes from becoming fixed in field popula- pathogen by first bringing the new hosts and the pathogen together tions of the pathogen. The selective sweep scenario is therefore and then by driving the pathogen adaptation and transmission on a considered less likely. global scale. The expansion of agriculture and resulting increase The diversification within lineages started between ∼ 460 and in host population size allowed these new pathogens to diverge 1100 years B.P. with the 95% HPDs ranging from ∼ 20 to 3800 rapidly into new species with large population sizes. This study years B.P. The accumulation of polymorphism in the pathogen highlights the importance of agriculture in the emergence of fungal populations likely increased with the expansion of the host popu- pathogens of crops and illustrates how the introduction of a crop lations in Eurasia and later in other continents. The demographic to new areas can select for new pathogens that emerge through growth reconstructions shown by the BSPs point to a recent re- host shifts. A short time frame was sufficient to allow the scald covery of the pathogen populations from the bottleneck that was pathogens to adapt to new hosts and to evolve into new species. experienced after the host shift onto domesticated hosts. The rapid Although this study has focused on Rhynchosporium pathogens increase in pathogen population sizes occurred during the last ∼ of cereals, it is likely that many other plant pathogens with life 250 years, coinciding with the global expansion and industrializa- histories similar to Rhynchosporium have emerged in the same tion of agriculture and the Green Revolution. Recent population way. It also is likely that more pathogens will emerge via host shifts expansion and diversification is also supported by the finding that in the future as old crops are introduced into newly developed the majority of the mutations present in all three lineages were lo- agricultural areas around the world. cated at the tips of the coalescent-based genealogies. As a result of Further evidence that agriculture is driving the adaptation of intensified cereal production, changed cultivation practices, and fungal pathogens to their hosts can be found in the rye/triticale- increased global seed trade, populations of the fungus reached a infecting populations. The genealogies suggest that rye- and magnitude that allowed scald to become one of the most impor- triticale-infecting isolates belong to the same gene pool and tant diseases on barley. The scald disease was first described at thus share a common origin. The crop triticale (× Triticosecale the end of the 19th century (Frank 1897; Oudemans 1897; Hein- Wittmack) is a man-made crop derived from hybridizing wheat sen 1901). It is not known whether the scald disease was already (Triticum aestivum L.) with rye (S. cereale L.). Triticale is of very present before that time. However, it is possible that the disease recent origin and was commercialized at the end of the 1960s was perceived only about 100 years ago after the pathogen popu- (Ammar et al. 2004; Oettler 2005). Although the disease was first lations increased and began to cause significant yield losses. recorded on rye in the late 19th century (Oudemans 1897), it was Although Agropyron spp. are weeds and not cultivated, Rhyn- only noticed on triticale in the 1990s, 30 years after the introduc- chosporium on these host species experienced a similar demo- tion of triticale as a crop (Welty and Metzger 1996). This recent
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host shift from rye to triticale may have occurred due to the loss of Cavalli-Sforza, L. L, P. Menozzi, and A. Piazza. 1994. The history and geog- scald resistance in the genome supplied by the wheat parent or be- raphy of human genes. Princeton Univ. Press, Princeton, NJ. cause new pathogenic forms of Rhynchosporium recently emerged Couch, B. C., and L. M. Kohn. 2002. A multilocus gene genealogy concor- dant with host preference indicates segregation of a new species, Mag- from the rye-infecting population (Welty and Metzger 1996). In naporthe oryzae, from M. grisea. Mycologia 94:683–693. the latter case, 30 years were sufficient to allow the rye-infecting Couch, B. C., I. Fudal, M-H. Lebrun, D. Tharreau, B. Valent, P. van Kim, J-L. population to adapt to triticale. By breeding a new host species Notteghem,´ and L. M. Kohn. 2005. 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Supplementary Material The following supplementary material is available for this article:
Figure S1. Phylogeny inferred by maximum parsimony from the -tubulin DNA sequence locus. The 50% majority-rule consensus tree is shown. Bayesian maximum likelihood analysis recovered the same topology. The numbers above branches are bootstrap frequencies of 1000 replicates and those below are Bayesian posterior probabilities. Branch lengths are proportional to the number of steps (character changes) along the branch. Labels on the phylogeny are: H1-H18 haplotypes obtained from 316 DNA sequences of 1609 bp representing Rhynchosporium isolates from different hosts (Agropyron spp., barley, Bromus diandrus, Dactylis glomerata, Hordeum spp., rye and triticale). Haplotypes H2-H4 representing Rhynchosporium orthosporum were used as outgroup. Figure S2. Phylogeny inferred by maximum parsimony from the -tubulin DNA sequence locus. The 50% majority-rule consensus tree is shown. Bayesian maximum likelihood analysis recovered the same topology. The numbers above branches are bootstrap frequencies of 1000 replicates and those below are Bayesian posterior probabilities. Branch lengths are proportional to the number of steps (character changes) along the branch. Labels on the phylogeny are: H1-H25 haplotypes obtained from 316 DNA sequences of 609 bp representing Rhynchosporium isolates from different hosts (Agropyron spp., barley, Bromus diandrus, Dactylis glomerata, Hordeum spp., rye and triticale). Haplotypes H2-H4 representing Rhynchosporium orthosporum were used as outgroup. Figure S3. Phylogeny inferred by maximum parsimony from the EF-1 DNA sequence locus. The 50% majority-rule consensus tree is shown. Bayesian maximum likelihood analysis recovered a similar topology. The numbers above branches are bootstrap frequencies of 1000 replicates and those below are Bayesian posterior probabilities. Branch lengths are proportional to the number of steps (character changes) along the branch. Labels on the phylogeny are: H1-H15 haplotypes obtained from 316 DNA sequences of 365 bp representing Rhynchosporium isolates from different hosts (Agropyron spp., barley, Bromus diandrus, Dactylis glomerata, Hordeum spp., rye and triticale). Haplotypes H2-H4 representing Rhynchosporium orthosporum were used as outgroup. Figure S4. Phylogeny inferred by maximum parsimony from the ITS DNA sequence locus. The 50% majority-rule consensus tree is shown. Bayesian maximum likelihood analysis recovered a similar topology. The numbers above branches are bootstrap frequencies of 1000 replicates and those below are Bayesian posterior probabilities. Branch lengths are proportional to the number of steps (character changes) along the branch. Labels on the phylogeny are: H1-H25 haplotypes obtained from 316 DNA sequences of 848 bp representing Rhynchosporium isolates from different hosts (Agropyron spp., barley, Bromus diandrus, Dactylis glomerata, Hordeum spp., rye and triticale). Haplotypes H2-H4 representing Rhynchosporium orthosporum were used as outgroup. Table S1. Distribution of the different haplotypes of Rhynchosporium species on different hosts corresponding to three phylo- genetic lineages A–C and Rhynchosporium orthosporum based on four DNA sequence loci. Table S2. Results from neutrality tests [Tajima’s D (Tajima 1989) and Fu and Li’s D∗ and Fu and Li’s F∗ (Fu and Li 1993)].
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