Abstract

Citation:Goss EM, Cardenas ME, Myers K, Forbes GA, Fry WE, Restrepo S, et al. (2011) The Plant PathogenPhytophthora andina Emerged via Hybridization of an UnknownPhytophthora Species and the Irish Potato Famine Pathogen,P. infestans. PLoS ONE 6(9): e24543. https://doi.org/10.1371/journal.pone.0024543

Editor:Silvana Allodi, Federal University of Rio de Janeiro, Brazil

Received:May 20, 2011;Accepted:August 12, 2011;Published: September 16, 2011

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Funding:Financial support was provided by the United States Department of Agriculture–Agricultural Research Service (USDA-ARS) CRIS 5358-22000-034-00. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Emerging plant pathogens threaten natural ecosystems, food security, and commercial interests. Major mechanisms underlying plant pathogen emergence include host range expansion and host jumps[1],[2]. Recently, these events have largely been the result of migration or movement of pathogens or hosts into new geographic regions[3],[4],[5]. Another mechanism is hybridization between species or individuals[6]. Known hybrid plant pathogens include the alder pathogenPhytophthora alni[7], the poplar rustMelampsora×columbiana[8], the crucifer pathogenVerticillium longisporum[9], the onion pathogenBotrytis allii[10],[11], andHeterobasidion forest pathogens[12],[13]. Hybridization and introgression are also hypothesized to be behind the continued epidemic of Dutch elm disease in Europe[14]. Hybridization between a recently introduced exotic pathogen and a resident pathogen may allow rapid evolution and adaptation to new hosts or environments[14],[15],[16],[17], because hybridization introduces genetic variation that has already been “tested by selection” in the resident parental species[18]. The continuing global movement of plant pathogens may be creating opportunities for new and virulent hybrid pathogens to arise[15],[19].

Hybrid plant pathogens have traditionally been difficult to detect or confirm and have generally been investigated for their unusual morphology, pathogenicity, or other phenotypic characters and subsequently identified as hybrids[15],[19]. Modern molecular techniques are currently the gold standard for identifying hybrid pathogens, in particular the sequencing of nuclear loci for which genealogies can be constructed and ancestral and derived states inferred. Based on DNA sequences, hybrids have been identified when sampled individuals consistently have genes or alleles with phylogenetically divergent origins. In diploids or polyploids one may observe that alleles at any one locus are from divergent origins. However, in the case of introgression, when hybrid offspring are not sterile and can backcross to one or the other parental species or strains, the hybridization event may be more difficult to detect if limited DNA sequences are available. Modern molecular methods and especially whole genome sequencing will likely identify additional ‘atypical’ plant pathogens as being hybrids or as having introgressed genes from past hybridization events.

The oomycete pathogenPhytophthora infestans is one of the most widely known emerging plant pathogens. It initially emerged in the early 1840s in the United States and Europe and rapidly spread across potato-growing regions, leading to the Irish potato famine. It causes an aggressive disease of potato and tomato, and is still considered a major threat to global food security[20]. In the 1950s, a diverse and sexual population ofP. infestans was found in the Toluca Valley of central Mexico, on commercial potatoes and then wild relatives of potato, leading to the conventional wisdom that this devastating pathogen evolved in association with the diverse tuber-bearingSolanum plant community in the central highlands of Mexico[21],[22]. This scenario is supported by the presence of two closely related species,P. mirabilis andP. ipomoeae, also found in the Toluca Valley[23],[24]. However, the center of origin and primary center of diversity of potatoes is in the Andean highlands of South America, thus a competing hypothesis is that the Andean highlands are the center of origin ofP. infestans. This scenario is supported by a genealogical analysis ofP. infestans using two mitochondrial DNA loci and one nuclear locus that showed old lineages of the pathogen in the Andes and not Mexico[25]. One of the arguments for an Andean origin ofP. infestans has also been that the closest known relative ofP. infestans,P. andina (formerly known asP. infestans sensu lato), is morphologically indistinguishable fromP. infestans and is found only in the Andean highlands[25],[26]. Furthermore, several apparent lineages ofP. infestans-like pathogens, all now classified asP. andina, has led to the suggestion that the Andes are a hotspot ofPhytophthora diversification[25].

Phytophthora andina was originally discovered when a broader range of blightedSolanum species, particularly non-tuber-bearing species, were sampled in Ecuador[26],[27],[28]. These isolates were quickly identified as being genetically distinct fromP. infestans despite their shared morphology. Specifically, they had new RFLP fingerprints (EC-2 and EC-3) and some EC-2 isolates had a distinct mtDNA haplotype, designated Ic[26],[28]. There are currently three distinct clonal lineages withinP. andina, defined by RFLP fingerprint (also readily distinguished by AFLP), mitochondrial DNA haplotype, and mating type[26],[29]. Initially these lineages were referred to asP. infestans sensu lato, but recently they were all reclassified asP. andina Adler & Flier, sp. nov.[29]. Due to the genetic differences among theP. andina lineages, this species description is controversial[30]. Host use byP. infestans andP. andina in Ecuador overlap minimally, withP. infestans found infectingS. tuberosum (potato),S. lycopersicum (tomato), and close relatives (Solanum sections Petota, Lycopersicon, and Juglandifolium), andP. andina primarily infectingS. betaceum (section Pachyphylla),S. muricatum (section Basarthrum),S. quitoense (section Lasiocarpa),S. hispidum (section Torva), and species in the section Anarrhichomenum[29],[31]. Both species have been isolated fromS. muricatum,S. quitoense, andS. ochranthum[26],[29]. Genetic variation withinP. andina may be correlated with host use, suggesting the possibility of host specialization byP. andina lineages in the field[26],[29],[31].

P. infestans andP. andina share identical or nearly identical ITS sequences[29],[32], which is the traditional molecular marker used in species definition in oomycetes and fungi.P. mirabilis andP. ipomoeae also have identical or nearly identical ITS sequences toP. infestans[23]. These four closely related species, plusP. phaseoli, make upPhytophthora clade 1c[33],[34],[35]. Direct sequencing of nuclear genes inP. andina produced identical sequences in allP. andina isolates examined[29],[32], but also revealed high levels of heterozygosity with several of these sites differentiatingP. infestans fromP. mirabilis sequences[29],[32],[35]. Based on the observed heterozygous sites, it was hypothesized thatP. andina may be a hybrid betweenP. infestans andP. mirabilis[32] or betweenP. infestans and another unspecified parent[29],[35], but the question was not investigated further. Resolution of the ancestry ofP. andina, particularly whether it is of hybrid origin, is necessary for accurate interpretation of its population structure, evolution, and genetics. Here, we investigate the evolutionary history ofP. andina and determine whetherP. andina is in fact a hybrid ofP. infestans and another species by cloning four nuclear loci to obtain haplotypes to infer the phylogenetic relationships of these alleles in relation toP. infestans and related species. Because of the considerable methodological and analytical challenges posed by both the large (∼240 Mb) and highly repetitive (∼74%)P. infestans genome[36] and the phasing of haplotypes in short-read, high throughput sequencing approaches, our work relied on traditional PCR cloning of coding sequences.

Results

EveryP. andina isolate was heterozygous at each of the four loci sequenced, as evidenced by double peaks in chromatograms from direct sequencing of PCR products. The total number of heterozygous sites summed across the four sequenced loci was significantly higher inP. andina compared toP. infestans,P. ipomoeae, andP. mirabilis (Figure 1;P<0.0001 for each comparison withP. andina by Tukey HSD). On average,P. andina isolates had greater than seven times more heterozygous sites than the other three species (Figure 1). Heterozygosity for indels was also observed in both regions ofypt1,btub and PITG11126, such that chromatograms showed overlapping PCR products of different lengths. Heterozygosity was observed inP. infestans,P. mirabilis, and in one isolate ofP. ipomoeae, but with many fewer heterozygous sites per locus. When maximum likelihood gene trees were constructed using genotypes,P. andina could not be distinguished fromP. infestans (Figure S1).

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Figure 1. Total number of heterozygous sites across four nuclear loci sequenced in each isolate by species.

Lines represent mean values for each species and circles represent values of individual isolates (circles are overlapping). Lowercase letters above graph indicate significance, such that significantly different means (P<0.05) by Tukey's HSD are shown by different letters. The number of heterozygous sites observed inP. andina isolates was at least two to three times higher than isolates from the other species andP-values of comparisons withP. andina were less than 0.0001.

https://doi.org/10.1371/journal.pone.0024543.g001

Sequencing of cloned PCR products in every case revealed two distinct haplotypes forP. andina isolates (Table 1). Forbtub,trp1, and PITG11126, one haplotype was identical to the most commonP. infestans haplotype, found in isolates from the Andes, the United States, Mexico, and the United Kingdom (Table S1). Forypt1,P. andina isolates had one of twoP. infestans haplotypes (H9 or H10) differing by 2 bp, with the exception of EC_3678, which had aP. infestans-like haplotype (H8) that differed from H9 at one nucleotide site (Table S2A). The second haplotype in each isolate was more or less distantly related toP. infestans depending on the locus (Figure 2). There were two versions of the non-P. infestans haplotypes fortrp1 and PITG11126, which differed by 1 and 5 bp, respectively (Table S2). Much of the observed variation withinP. andina segregates between the threeP. andina lineages (Table 2).

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Figure 2. Maximum likelihood trees of haplotypes for each locus sequenced.

Loci areA.ypt1,B.trp1,C.btub, andD. PITG11126, sequenced inP. andina (Pa) and four other closely related species (Pi:P. infestans;Pm:P. mirabilis;Po:P. ipomoeae; andPp:P. phaseoli). The haplotype designation is shown for each branch tip, corresponding toTables 1,2, andS1,S2,S3,S4,S5,S6,S7,S8.P. andina haplotypes are bolded. Trees have been rooted withP. phaseoli. Bootstrap support values obtained by maximum likelihood are shown above branches and Bayesian posterior probabilities are shown below branches. Values are not shown for branches that had less then 80% support/probability by both methods.

https://doi.org/10.1371/journal.pone.0024543.g002

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Table 1.P. andina isolates and haplotypes of each locus sequenced.

https://doi.org/10.1371/journal.pone.0024543.t001

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Table 2. Summary of sequence variation amongP. andina lineages.

https://doi.org/10.1371/journal.pone.0024543.t002

The non-P. infestans P. andina haplotypes (hereafterPa-unknown) forypt1,btub, andtrp1, were related toP. mirabilis, but clearly distinct. Different possible phylogenetic relationships amongPa-unknown,P. infestans,P. mirabilis, andP. ipomoeae were statistically tested using the approximately unbiased (AU) test and Shimodaira–Hasegawa (SH) tests (Table S9). All trees were essentially star phylogenies fortrp1, thus the AU test failed and no trees were rejected by the SH test. Forypt1, trees that did not contain a derived {Pa-unknown,P. mirabilis} clade had lowP values by the AU and SH tests (0.05<P<0.1), but no trees were rejected at theP = 0.05 level. Forbtub, trees with the complex {Pa-unknown,P. mirabilis} clade had highP values. But one tree with monophyleticP. mirabilis as sister species toPa-unknown was also not rejected, as well as two trees in whichP. infestans andP. ipomoeae formed a derived clade (P>0.1 by the AU test, 0.05<P<0.1 by the SH test). Species relationships were qualitatively different for the PITG11126 locus. ThePa-unknown haplotype was more closely related toP. infestans thanP. mirabilis (Fig. 2D). Unlike the other loci, sites in PITG11126 that differed betweenP. infestans andP. phaseoli,P. mirabilis, andP. ipomoeae were in theP. infestans state inP. andina (Table S2D). The AU test rejected all trees that did not include a derived {Pa-unknown,P. infestans} clade or a derived {Pa-unknown,P. ipomoeae} clade.

Discussion

We tested the hypothesis thatPhytophthora andina is a hybrid pathogen and found that it is a hybrid betweenP. infestans and an unknown species that is closely related but distinct fromP. mirabilis andP. ipomoeae. Cloning and sequencing of four nuclear loci clearly shows thatP. andina isolates have one allele derived from aP. infestans parent and a second divergent allele from the unknown species. TheP. infestans haplotypes found inP. andina appear to be common worldwide, found in North and South America, Europe, and Asia. BecauseP. andina has a different host range thanP. infestans and the threeP. andina lineages may have different host ranges themselves[26],[29],[31], it is probable that hybridization led to host range expansion or shifts.

Host shifts are likely to require several rapid genetic changes, butP. andina may have been in a unique position to undergo the necessary changes and rapidly adapt to new hosts. First, hybridization may facilitate adaptation to a new environment by rapidly introducing genetic variation, and not random variation but rather a complement of alleles that have been subjected to selection in the parental species[18]. Second,P. infestans has a genome structure which may have contributed to its ability to rapidly evolve virulence to resistant potato varieties in the near absence of sexual reproduction[36]. In particular,P. infestans has a very large genome with expanded repeat-rich gene-sparse regions where pathogenicity effectors, genes involved in virulence and host range, are primarily located. Comparisons to the genome sequences of two distantly relatedPhytophthora species show considerable expansion of effector genes inP. infestans and suggest that the repeat-rich gene-sparse regions are highly dynamic, exhibiting gene duplications and gene loss by tandem duplication, non-allelic homologous recombination, and pseudogenization.P. ipomoeae,P. mirabilis, andP. phaseoli have similar genome structures toP. infestans and comparisons among these species show greater gene copy number variation and presence/absence polymorphisms in the repeat-rich regions compared to the gene-dense repeat-poor regions where core orthologs are found[37]. The repeat-rich regions are also enriched in genes induced during infection.P. infestans is also known to exhibit aneuploidy, particularly in the clonal lineages that dominate much of its current geographic distribution[38],[39],[40]. Thus,P. andina had a potential mechanism for rapid change in its genic and allelic composition following hybridization. Different evolutionary paths taken by hybrid progeny could also explain the genetic variation observed withinP. andina.

We cloned parental haplotypes at different frequencies fromP. andina isolates for several loci, and while it is possible thatP. andina is tetraploid or aneuploid and that the haplotypes are actually present inP. andina at different frequencies, it is more likely that there is a bias in the efficiency of the primers between haplotypes. The primers were designed fromP. infestans and may contain mismatches with the sequences of the otherPhytophthora clade 1c species. Cloned recombinant haplotypes are likely to be chimeras from PCR error, as PCR conditions were not optimized to reduce these sorts of errors[9],[41]. Illumina sequence reads fromP. andina graciously shared with us [S. Kamoun personal communication,[37]] were examined, but these data could not be analyzed because depth of coverage was not sufficient to call heterozygous sites with high confidence[42] and the short reads were problematic for determining haplotype phase. More extensive deep sequencing may elucidate the genome composition ofP. andina, particularly using sequencing technologies that generate longer read lengths. Genome-wide analysis would also allow for examination of alternate hypotheses for the observed pattern of phylogenetically distinct alleles at each locus, including mechanisms such as gene duplication or horizontal gene transfer.

The hybridP. alni, a lethal pathogen of alder, is another example of hybridization between closely relatedPhytophthora species resulting in a host range expansion or shift[7],[43],[44]. Additional examples include hybridPhytophthora pathogens causing disease on loquat trees in Peru and Taiwan[45],[46] and in ornamental nurseries where exotic pathogens are brought together under artificial conditions[47],[48],[49]. Host range expansions byPhytophthora hybrids have been documented for both these naturally occurring hybrids and for hybrids created in the laboratory[6].P. infestans andP. mirabilis are outcrossers, occur in sympatry on different hosts in the Toluca Valley of central Mexico, and are thought to have evolved by sympatric speciation via host shifts[21], thus the potential for interspecific mating between these species has been investigated. Population genetic analysis suggested some gene flow betweenP. infestans andP. mirabilis populations[21],[50]. However, initial crosses betweenP. infestans andP. mirabilis produced hybrid offspring that were largely unable to infect either host group and had poor viability[51]. Nevertheless, a recent cross between aP. infestans isolate (virulent on potato and tomato) and aP. mirabilis isolate (virulent onMirabilis jalapa), both from central Mexico, produced F1 and F2 progeny that were pathogenic on tomato and one F2 isolate had an expanded host range, able to infect all parental hosts[52]. Interestingly, the ability to infect tomato segregated as a dominant single locus trait in this cross. Sexual crosses have also been attempted betweenP. infestans andP. andina[53]. A limited number of viable progeny were obtained, but further crosses with these progeny were not successful. Here we examined only four nuclear loci, yet we observed both parental haplotypes at each locus for each isolate, which suggests that theseP. andina lineages were not the result of backcrosses.

Reproductive barriers between closely related species are usually stronger when the species occur in sympatry than when the species have evolved in allopatry[54]. There is not yet strong evidence for this pattern specifically for fungal or oomycete pathogens, in part because the native distributions of many of these pathogens are not well known. Essentially, it is not clear where pathogens evolved and therefore whether sister species evolved in sympatry or allopatry. On the other hand, host shift speciation may also occur without intrinsic reproductive barriers when pathogens must sexually reproduce on their host[1]. It has nevertheless been hypothesized thatPhytophthora hybrids are offspring of two exotic species or of an exotic and resident species[6],[48]. If this pattern does hold true forPhytophthora, it would suggest that at least one of theP. andina parent species is introduced and did not co-evolve with the AndeanSolanum host community[52].

Synthetic hybridization experiments have been used to recreate hybrids, to a certain extent, observed in the wild in order to validate the hybrid origin of species (e.g.[18],[55],[56]). ForP. andina, one of the hybrid parents remains unknown and so these experiments remain to be conducted, pending the collection and identification of the unknown parent. However, locating this species could be challenging. Disease epidemics caused byP. mirabilis andP. ipomoeae are infrequent and incidence of infection is low (N. J. Grünwald, personal observation). This would probably also be true of other relatives ofP. infestans that infect wild and patchy host populations. The unknown species suggests that there is undiscovered diversity inPhytophthora clade 1c that may be found in the Andes, although the evolutionary origin of this species in relationship toP. infestans and its Mexican sister species remains unclear.

Phytophthora diseases are currently being managed with fungicides or, preferably, resistant plant varieties when available. Global movement and interspecific hybridization of plant pathogens multiply the considerable challenges already faced by crop breeding programs and growers trying to manage disease. The global movement of plant pathogens may increase the risk of formation of novel hybridPhytophthora pathogens if hybridization is more likely between previously allopatric species brought together by migration events. Understanding the ecological and genetic processes that result in hybrid pathogens with novel host ranges or virulence, as appears to be the case forP. andina, should suggest conditions under which special vigilance and increased monitoring for emerging pathogens is warranted.

Materials and Methods

Nuclear gene sequencing

The following genes known to contain variation within and amongPhytophthora species were chosen for sequencing: the Ras geneypt1[25],[59];trp1,btub,[33],[35],[60], and an additional gene that also exhibited variation within and among 1c species in preliminary sequencing (PITG11126,[61]). Primers forypt1 were from Gomez-Alpizar et al.[25]. These amplify a fragment of the 5′ untranslated region of the gene (intron1, IR) and a larger downstream fragment including both exons and introns (RAS). These two fragments were concatenated for analysis. Primers for the other genes were designed from theP. infestans genome sequence[36] (Table 3). All isolates were directly sequenced from the PCR product. For each locus, two to sixP. andina isolates were selected for cloning of the PCR product to obtain haplotypes (Table 3). Forypt1, 6 isolates were additionally cloned across the entire region to obtain haplotypes across both amplified fragments. SeveralP. infestans andP. mirabilis isolates with heterozygous sites were also cloned to obtain haplotypes. When a chromatogram indicated that the isolate was heterozygous for an indel at a sequenced locus, the preliminary sequence was determined using the sequence obtained from each primer up to the indel (i.e. sequence was inferred from one strand). Then, isolates representing each inferred genotype were cloned to obtain haplotypes and confirm the genotype inferred from direct sequencing. Specific cloning and sequencing methods and protocols differed among the laboratories (Fry, Grünwald, Restrepo) where they were performed and are available upon request (see also[60],[62]).

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Table 3. Loci sequenced andP. andina isolates cloned.

https://doi.org/10.1371/journal.pone.0024543.t003

The number of heterozygous sites was summed across the four sequenced loci for each isolate for which sequence was obtained for all loci. This total per isolate was used to examine differences in the number of heterozygous sites among species using analysis of variance, implemented in R 2.11.1 for Mac OS. Post-hoc multiple comparisons were conducted using Tukey's HSD.

Supporting Information

Acknowledgments

We thank Valerie Fieland, Karan Fairchild, Kim Henslee, and Caroline Press for excellent technical support. Mention of trade names or commercial products in this manuscript are solely for the purpose of providing specific information and do not imply recommendation or endorsement.

Author Contributions

Conceived and designed the experiments: EMG GAF WEF SR NJG. Performed the experiments: EMG MEC KM. Analyzed the data: EMG MEC KM NJG. Contributed reagents/materials/analysis tools: GAF NJG SR WEF. Wrote the paper: EMG NJG.

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