Author response: The rise and fall of the Phytophthora infestans lineage that triggered the Irish potato famine
2013-05-08

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Material and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Phytophthora infestans, the cause of potato late blight, is infamous for having triggered the Irish Great Famine in the 1840s. Until the late 1970s, P. infestans diversity outside of its Mexican center of origin was low, and one scenario held that a single strain, US-1, had dominated the global population for 150 years; this was later challenged based on DNA analysis of historical herbarium specimens. We have compared the genomes of 11 herbarium and 15 modern strains. We conclude that the 19th century epidemic was caused by a unique genotype, HERB-1, that persisted for over 50 years. HERB-1 is distinct from all examined modern strains, but it is a close relative of US-1, which replaced it outside of Mexico in the 20th century. We propose that HERB-1 and US-1 emerged from a metapopulation that was established in the early 1800s outside of the species' center of diversity. https://doi.org/10.7554/eLife.00731.001 eLife digest Few crop failures have been as devastating as those caused by potato late blight in the 1840s. This disease is caused by a filamentous microbe called Phytophthora infestans, which spread from North America to Europe in 1845, leading to the Great Famine in Ireland and to severe crop losses in the rest of Europe. Phytophthora is thought to have originated in the Toluca valley of Mexico, where many different strains evolve alongside wild potato relatives, but the exact strain that caused the Great Famine, and how it is related to modern strains of the pathogen, has remained a mystery. Yoshida et al. have used a technique call 'shotgun' sequencing to map the genomes of 11 historical strains of P. infestans and 15 modern strains. The historical strains were extracted from the leaves of potato and tomato plants that were collected in North America and Europe, including Ireland and Great Britain, from 1845 onwards and stored in herbaria for future research. By comparing the genomes of the historical and modern samples, Yoshida et al. found that the historical strains all belonged to a single lineage that shows very little genetic diversity. Previously it has been proposed that this lineage was the same as US-1, which was the dominant strain of potato blight in the world until the end of the 1970s, or that it was more closely related to modern strains than to US-1. Yoshida et al. now rule out both of these possibilities and show that the lineage that caused the great famine, which they call HERB-1, is clearly distinct from US-1, although they are closely related, and they conclude that both HERB-1 and US-1 might have dispersed from a common ancestor that existed outside of Mexico in the early 1800s. Why US-1 later replaced HERB-1 as the dominant strain in the world is an important question for future studies. https://doi.org/10.7554/eLife.00731.002 Introduction Potato late blight's impact on humankind is rivaled by few other plant diseases. The Spanish introduced Europeans to the South American staple crop potato shortly after their conquest of the New World, but for three centuries Europe stayed free of P. infestans, the causal agent of late blight. In 1845, the oomycete P. infestans finally reached Europe, spreading rapidly from Belgium to other countries of mainland Europe and then to Great Britain and Ireland. The impact of the epidemic reached catastrophic levels in Ireland, where the population was more dependent on potato for their subsistence than in other parts of Europe (Bourke, 1964; Reader, 2009). The subsequent Great Famine killed around 1 million people, and an additional million were forced to leave the island (Turner, 2005). Even today, the Irish population remains less than three quarters of what it was at the beginning of the 1840s. These dramatic consequences of the P. infestans epidemic were due to the absence of chemical and genetic methods to combat it; such means became available only several decades later. Ever since triggering the Irish famine, P. infestans has continued to wreak havoc on potato fields throughout the world. Late blight remains the most destructive disease of the third largest food crop, resulting in annual losses of potatoes that would be sufficient to feed anywhere from 80 to many hundreds of millions of people (Fisher et al., 2012). Phytophthora infestans is an extraordinarily virulent and adaptable pathogen (Fry, 2008; Haas et al., 2009). In agricultural systems, sexual reproduction may trigger explosive population shifts that are driven by the emergence and migration of asexual lineages (Fry et al., 1992, 2009; Cooke et al., 2012). The species is thought to originate from Toluca Valley, Mexico, where it infects wild relatives of potato, frequently undergoes sexual reproduction and co-occurs with the two closely related species P. mirabilis and P. ipomoeae (Tooley et al., 1985; Goodwin et al., 1994; Flier et al., 2003; Grünwald and Flier, 2005). In its center of origin, P. infestans is characterized by high levels of genetic and phenotypic diversity (Grünwald and Flier, 2005). The genomes of a few P. infestans strains have been described (Haas et al., 2009; Raffaele et al., 2010a; Cooke et al., 2012). Compared to other species in the genus, the 240 Mb T30-4 reference genome of P. infestans is large, with three quarters of the genome consisting of repetitive DNA. A large number of genes codes for effector proteins, many of which are delivered inside plant cells to promote host colonization, for instance by suppressing plant immunity. RXLR proteins, the main class of host-translocated effectors, are encoded by about 550 genes in the P. infestans T30-4 genome. RXLR effectors that can be recognized by plant immune receptors, known as Resistance (R) proteins, are said to have 'avirulence' activity. Upon introduction of a cognate R gene into the host population, such avirulence effectors become a liability for the pathogen, and natural selection favors the spread of pseudogenized or mutated alleles (Vleeshouwers et al., 2011). The detailed descriptions and drawings of Heinrich Anton de Bary and the reports of several other pioneers of plant pathology leave no doubt that the 19th century blight epidemic was triggered by P. infestans (de Bary, 1876; Bourke, 1964). What remains controversial is the relationship of the 19th century strains to modern isolates. The quest for understanding the origin of the strain that resulted in the Irish famine began with extant samples. Prior to the late 1970s, global P. infestans populations outside of South America and Mexico, the centers of diversity of the host and the pathogen, were dominated by a single clonal lineage that had the mitochondrial (mtDNA) haplotype Ib and was called US-1 (Goodwin et al., 1994). It was therefore proposed that the US-1 lineage was a direct descendant of the strain that first caused widespread late blight in North America from 1843 on, and then triggered the Irish famine beginning in 1845 (Bourke, 1964; Goodwin et al., 1994). This hypothesis was subsequently directly addressed by PCR analysis of infected 19th century potato leaves stored in herbaria. The conclusion from these studies was that the historic strains belonged to a mtDNA haplotype, Ia, that was distinct from that of the US-1 lineage (Ristaino et al., 2001; May and Ristaino, 2004). Because Ia was at the time not only the predominant haplotype in the Toluca Valley in Mexico (Gavino and Fry, 2002; Flier et al., 2003), but had also been found in South America (Perez et al., 2001), May and Ristaino (2004) speculated that the 19th century and US-1 lineages represented two independent epidemics of divergent lineages that had both originated in South America and spread from there to North America and Europe. A caveat was that these far-reaching conclusions were based on only three mtDNA SNPs (Ristaino et al., 2001; May and Ristaino, 2004). Since these first herbarium analyses, the retrieval and sequencing of DNA from museum specimens, fossil remains and archaeological samples—collectively known as ancient DNA (aDNA) (Pääbo et al., 2004)—have seen impressive advances thanks to the advent of high-throughput sequencing technologies. The combined analysis of modern and ancient genomes of human pathogens has begun to solve important questions about their history and evolution (Bos et al., 2011, 2012). Here we show that aDNA methods hold similar promise for plant pathology and that they can improve our understanding of historically important plant pathogen epidemics. To determine how the historic P. infestans strain(s) relate to extant isolates, we shotgun-sequenced 11 herbarium samples of infected potato and tomato leaves collected from continental Europe, Great Britain, Ireland, and North America in the period from 1845 to 1896, and extracted information on P. infestans mitochondrial and nuclear genomes. To understand the subsequent evolution of the pathogen, we compared the historic P. infestans genomes to those of 15 modern 20th century strains that span the genetic diversity of the species, and to the two sister species P. ipomoeae and P. mirabilis. Our analyses revealed that the 19th century epidemic was caused by a single genotype, HERB-1, that persisted for at least 50 years. While it is distinct from all examined modern strains, HERB-1 is closely related to the 20th century US-1 genotype, suggesting that these two pandemic genotypes may have emerged from a secondary metapopulation rather than from the species' Mexican center of diversity. Results Preservation of ancient DNA and genome statistics Nineteenth-century samples of potato and tomato leaves with P. infestans lesions were obtained from the herbaria of the Botanische Staatssammlung München and the Kew Royal Botanical Gardens (Table 1 and Figure 1). DNA was extracted under clean room conditions and two genomic libraries were prepared from each sample for Illumina sequencing. The preparations were expected to comprise P. infestans DNA, host DNA from potato or tomato as well as DNA from microbes that had colonized either the living material at the time of its collection, or the dried material during its storage in the herbaria. Table 1 Provenance of P. infestans samples https://doi.org/10.7554/eLife.00731.003 IDCountry of originCollection yearHost speciesReference*Herbarium samplesKM177500England1845Solanum tuberosum1KM177513Ireland1846Solanum tuberosum1KM177502England1846Solanum tuberosum1KM177497England1846Solanum tuberosum1KM177514Ireland1847Solanum tuberosum1KM177548England1847Solanum tuberosum1KM177507England1856Petunia hybrida1M-0182898GermanyBefore 1863Solanum tuberosum2KM177509England1865Solanum tuberosum1M-0182900Germany1873Solanum lycopersicum2M-0182907Germany1875Solanum tuberosum1KM177517Wales1875Solanum tuberosum1M-0182897USA1876Solanum lycopersicum2M-0182906Germany1877Solanum tuberosum2M-0182896Germany1877Solanum tuberosum2M-0182904Austria1879Solanum tuberosum2M-0182903Canada1896Solanum tuberosum2KM177512EnglandNASolanum tuberosum1Modern samples06_3928AEngland2006Solanum tuberosum3DDR7602Germany1976Solanum tuberosum4P1362Mexico1979Solanum tuberosum5P6096Peru1984Solanum tuberosum5P7722 (P. mirabilis)USA1992Solanum lycopersicum5P9464USA1996Solanum tuberosum5P12204Scotland1996Solanum tuberosum5P13527Ecuador2002Solanum andreanum5P10127USA2002Solanum lycopersicum5P13626Ecuador2003Solanum tuberosum5P10650Mexico2004Solanum tuberosum5LBUS5South Africa2005Petunia hybrida6P11633Hungary2005Solanum lycopersicum5NL07434Netherlands2007Solanum tuberosum3P17777USA2009Solanum lycopersicum5P17721USA2009Solanum tuberosum5 * 1, Kew Royal Botanical Gardens; 2, Botanische Staatssammlung München; 3, Cooke et al. (2012); 4, Kamoun et al. (1999); 5, World Oomycete Genetic Resource Collection at UC Riverside, CA; 6, Dr Adele McLeod, Univ. of Stellenbosch, South Africa. Figure 1 Download asset Open asset Countries of origin of samples used in whole-genome, mtDNA genome or both analyses. Red indicates number of historic and blue of modern samples. More information on the samples is given in Tables 1 and 2. https://doi.org/10.7554/eLife.00731.004 The first set of libraries was used for verification of aDNA-like characteristics, and the second set was used for production. In this second set we used a repair protocol that removes aDNA-associated lesions, mainly characterized by cytosine deamination to uracil (U), which is subsequently converted to and read as thymine (T) (Hofreiter et al., 2001; Briggs et al., 2007, 2010; Brotherton et al., 2007). The combination of uracil-DNA-glycosylase (UDG) and endonuclease VIII, which removes uracil residues and repairs abasic sites, reduces the overall per-base error rate to as low as one 20th of unrepaired aDNA (Briggs et al., 2010). Ancient DNA fragments are typically shorter than 100 bp (Pääbo, 1989), and paired-end reads of 100 bases each will therefore substantially overlap. Forward and reverse reads from the unrepaired libraries (Table 2) were merged, requiring at least 11 base overlap. Merging of short-insert libraries considerably decreases the error-rate and also generates sequences that reflect the original molecule length (Kircher, 2012). The median length of merged reads was in the range of approximately 50–85 bp (Figure 2A,B). Table 2 Sequencing strategy https://doi.org/10.7554/eLife.00731.005 IDInstrument and read typeSequencing centerCoverageM-0182896HiSeq 2000 (2 × 101 bp)MPIHighM-0182897HiSeq 2000 (2 × 101 bp)MPILow*M-0182898HiSeq 2000 (2 × 101 bp)MPILowM-0182900HiSeq 2000 (2 × 101 bp)MPILow†M-0182903HiSeq 2000 (2 × 101 bp)MPILowM-0182904HiSeq 2000 (2 × 101 bp)MPILow*M-0182906HiSeq 2000 (2 × 101 bp)MPILow†M-0182907HiSeq 2000 (2 × 101 bp)MPILowKM177497MiSeq (2 × 150 bp)MPILowKM177500MiSeq (2 × 150 bp)MPILow*KM177502AMiSeq (2 × 150 bp)MPILow*KM177507MiSeq (2 × 150 bp)MPILow*KM177509MiSeq (2 × 150 bp) and HiSeq 2000 (2 × 101 bp)MPILowKM177512MiSeq (2 × 150 bp) and HiSeq 2000 (2 × 101 bp)MPILowKM177513MiSeq (2 × 150 bp) and HiSeq 2000 (2 × 101 bp)MPILowKM177514MiSeq (2 × 150 bp) and HiSeq 2000 (2 × 101 bp)MPILowKM177517MiSeq (2 × 150 bp) and HiSeq 2000 (2 × 101 bp)MPILowKM177548MiSeq (2 × 150 bp) and HiSeq 2000 (2 × 101 bp)MPILow06_3928AGAIIX (2 × 76 bp)TSLHighDDR7602GAIIX (2 × 76 bp)TSLHighLBUS5GAIIX (2 × 76 bp)TSLHighNL07434GAIIX (2 × 76 bp)TSLHighP10127HiSeq 2000 (2 × 101 bp)MPILowP10650HiSeq 2000 (2 × 101 bp)MPILowP12204HiSeq 2000 (2 × 101 bp)MPILowP13527GAIIX (2 × 76 bp)TSLHighP1362HiSeq 2000 (2 × 101 bp)MPILowP13626GAIIX (2 × 76 bp)TSLHighP11633HiSeq 2000 (2 × 101 bp)MPILowP17721HiSeq 2000 (2 × 101 bp)MPILowP17777GAIIX (2 × 76 bp)TSLHighP6096HiSeq 2000 (2 × 101 bp)MPILowP7722HiSeq 2000 (2 × 101 bp)MPILowP9464HiSeq 2000 (2 × 101 bp)MPILow*PIC99114GAIIX (2 × 76 bp)TSLHighPIC99167GAIIX (2 × 76 bp)TSLHigh * Samples not included in any analysis due to extremely low coverage. † Samples used only in mtDNA analysis. Figure 2 Download asset Open asset Ancient DNA-like characteristic of historic samples. (A) Lengths of merged reads from historic sample M-0182898. (B) Mean lengths of merged reads from historic samples. (C) Nucleotide mis-incorporation in reads from the historic sample M-0182898. (D) Deamination at first 5′ end base in historic samples. (E) Percentage of merged reads that mapped to the P. infestans reference genome. https://doi.org/10.7554/eLife.00731.006 Merged sequences were mapped to the P. infestans T30-4 reference genome (Haas et al., 2009). Deamination of C to U in aDNA is highest at the first base (Briggs et al., 2007), and C-to-T was the predominant substitution at the 5′-end of molecules (Figure 2C,D). Based on mapping against the reference genome, we estimated the fraction of P. infestans DNA in the samples to be between 1% and 20% (Figure 2E). Most of the remaining reads could be mapped to the reference genomes for potato and tomato (Potato Genome Sequencing Consortium, 2011; The Tomato Genome Consortium, 2012). In addition to 11 historic samples from Ireland, Great Britain, continental Europe and North America (Figure 1 and Table 1), we shotgun sequenced 14 modern strains from Europe, the Americas and Africa (Figure 1 and Table 1). These had been selected based on preliminary mtDNA information to present a cross section of P. infestans diversity. Finally, we sequenced two strains of P. mirabilis, P7722 and PIC99114, and a single strain of P. ipomoeae, PIC99167. Both species are closely related to P. infestans and served as outgroups (Kroon et al., 2004; Raffaele et al., 2010a). We considered genomes with mean-fold coverage of at least 20 as high coverage; one historic, seven modern and both outgroup genomes fulfilled this condition (Figure 3A). We identified single nucleotide polymorphisms (SNPs) in each sample independently by comparison with the P. infestans reference T30-4 genome (Figure 3B). Thresholds for calling homozygous and heterozygous SNPs were determined from simulated data from high- and low-coverage genomes (Figure 3—figure supplement 1). We accepted SNPs from low-coverage genomes if the variants had also been called in a high-coverage genome. Inverse cumulative coverage plots indicated how many high- or low-coverage samples were needed to cover different fractions of SNPs (Figure 3C,D). A total of 4.5 million non-redundant SNPs were called. Eighty percent of all homozygous SNPs were found in at least eight samples, and only 20% of all SNPs were found in fewer than 10 strains. Thus, the great majority of polymorphic sites were shared by several strains and thus informative for phylogenetic analyses. Figure 3 with 1 supplement see all Download asset Open asset Coverage and SNP statistics. (A) Mean nuclear genome coverage from historic (red) and modern (blue) samples. (B) Homo- and heterozygous SNPs in each sample. (C) Inverse cumulative coverage for all homozygous SNPs across all samples. (D) Same as (C) for homo- and heterozygous SNPs. https://doi.org/10.7554/eLife.00731.007 A unique type I mtDNA haplotype in 19th century P. infestans strains We reconstructed the mtDNA genomes from historic and modern strains using an iterative mapping assembler (Green et al., 2008) and estimated a phylogenetic tree from complete mtDNA genomes, with one of the P. mirabilis mtDNA genomes as outgroup. Previous studies have recognized four P. infestans mtDNA haplotype groups (Ia, Ib, IIa and IIb), based on a small number of restriction fragment length polymorphisms (RFLPs) (Carter et al., 1990). Surprisingly, a comparison of the complete mtDNA genomes revealed that the historic samples did not fit into any of these groups, and instead formed an independent clade, called HERB-1 from here on. Among the HERB-1 mtDNA genomes, there were very few differences, with a mean pair-wise difference of only 0.2 bp, compared to 3.9 bp for the modern haplotype I strains, and 9.0 bp for modern haplotype II strains. The origin of HERB-1 relative to haplotypes Ia and Ib could not be unequivocally resolved, and a polytomy was inferred for these three groups or support for branches were low (Figure 4 and Figure 4—figure supplement 1). Figure 4 with 2 supplements see all Download asset Open asset Maximum-parsimony phylogenetic tree of complete mtDNA genomes. Sites with less than 90% information were not considered, leaving 24,560 sites in the final dataset. Numbers at branches indicate bootstrap support (100 replicates), and scale indicates The clonal lineage US-1, with the mtDNA haplotype Ib, was the predominant throughout the world until about (Goodwin et al., 1994). The two US-1 in our and with the Ib reference genome and samples and (Figure 4 and Figure 4—figure supplement 1), these two samples had not been as US-1 isolates. the US-1 is closely related to the herbarium strains, US-1 is not a of HERB-1 and US-1 are sister groups that a common are three that the US-1 clade, and two that the HERB-1 In with the by Ristaino et al. all historic samples had a at the for haplotype Ib (Figure 4—figure supplement which from the US-1 which instead a C at this In to the (Ristaino et al., 2001), we found no other around this between HERB-1 and modern strains and the HERB-1 strains were in the 19th their genomes fewer compared to modern strains, which have continued to evolve for over a years. This can be to directly substitution and using the as in a analysis. time into in phylogenetic that both modern and ancient pathogen strains (Bos et al., 2011). By the nucleotide to the outgroup all HERB-1 strains were found to show fewer mtDNA than modern strains with haplotype Ia or Ib of the strain and the number of mtDNA were Figure Figure Download asset Open asset between nucleotide of mtDNA genomes of Ib to the outgroup and sample in the between sample and the number of mtDNA a of complete modern and the HERB-1 mtDNA genomes was used as for a analysis using in the et al., 2012). The hypothesis for the modern strains could not be at a a and a tree were used for the analysis. Collection for all herbaria samples and the for all modern strains were used as that substitution time could be (Table 1). with million were of the estimated and and were in the throughout the merged using the and 2007). this we estimated the mtDNA substitution rate to be × and × This rate resulted in a mean time for P. infestans and P. mirabilis of and for P. infestans type I and type II mtDNA haplotypes of The origin of the 19th century herbarium was estimated to around (Figure and Table Figure Download asset Open asset of mtDNA genomes. tree from inferred support is to each the for the indicate historical in the Figure and Table 3 for detailed at the four main in P. Table 3 time to most common ancestor for different in the mtDNA tree To understand the between historical and modern strains in more we also compared their nuclear genomes. We phylogenetic with high-coverage genomes using (Figure (Figure and (Figure supplement We included in the analysis heterozygous SNPs by an from each of In all the HERB-1 formed a that as a sister to US-1 and a the of SNPs at heterozygous will bootstrap a map (Figure that the two US-1 strains are both most closely related to HERB-1 and the most distinct modern strains. analyses that included the low-coverage genomes (Figure supplement were with a close relationship between the HERB-1 and US-1 Figure with 1 supplement see all Download asset Open asset of high-coverage nuclear genomes using both homozygous and heterozygous SNPs. (A) Maximum-parsimony only sites with at least leaving sites in the final dataset. Numbers at branches indicate bootstrap support (100 replicates), and scale indicates genetic (B) (C) map of genetic scale indicates SNP US-1 strains and have the genomes sequences to The two US-1 in are compared to all other modern strains by a in modern strains The independent of the pandemic HERB-1 and US-1 lineages with a very common ancestor are with both lineages having originated from the same To the global of HERB-1 by US-1 in the 20th century was due to we for selected genes using 2007). We did not any for genes or sites that had selection in any of the only a with selected genes shared across could be that were more important than or that overall diversity and of gene variants by to the of US-1. Genetic diversity can be by which has been in of P. infestans et al., et al., and which has for asexual To in our specimens, we the of read at SNPs for high-coverage genomes. In a species, the mean of read at heterozygous have a single at there be two and for genomes, and three and for genomes. We compared the of read with of and genomes. Based on the and of the we that the historic genome was the modern genomes, only and were the including the two US-1 strains and as well as and were strain, to be (Figure and Figure supplement 1). This conclusion was by strains having for at many more sites than (Figure Figure with 1 supplement see all Download asset Open asset analysis. (A) of expected read of reads at SNPs for and genomes. (B) read at SNPs in gene for the historic sample two modern samples, and simulated and genomes. The simulated genome is to have 20% of 1 and of 3 in The and of the are similar to the simulated (C) with more than one in the To the we SNPs as or based on information from the outgroup species P. mirabilis and P. We then compared the rate of at alleles in and In with the had more than as many homozygous