Sympatry and interference of divergent Microbotryum pathogen species

The impact of infectious diseases in natural ecosystems is strongly influenced by the degree of pathogen specialization and by the local assemblies of potential host species. This study investigated anther‐smut disease, caused by fungi in the genus Microbotryum, among natural populations of plants in the Caryophyllaceae. A broad geographic survey focused on sites of the disease on multiple host species in sympatry. Analysis of molecular identities for the pathogens revealed that sympatric disease was most often due to co‐occurrence of distinct, host‐specific anther‐smut fungi, rather than localized cross‐species disease transmission. Flowers from sympatric populations showed that the Microbotryum spores were frequently moved between host species. Experimental inoculations to simulate cross‐species exposure to the pathogens in these plant communities showed that the anther‐smut pathogen was less able to cause disease on its regular host when following exposure of the plants to incompatible pathogens from another host species. These results indicate that multi‐host/multi‐pathogen communities are common in this system and they involve a previously hidden mechanism of interference between Microbotryum fungi, which likely affects both pathogen and host distributions.     

Congruent population genetic structures and divergence histories in anther‐smut fungi and their host plants Silene italica and the Silene nutans species complex

Study of the congruence of population genetic structure between hosts and pathogens gives important insights into their shared phylogeographical and coevolutionary histories. We studied the population genetic structure of castrating anther‐smut fungi (genus Microbotryum) and of their host plants, the Silene nutans species complex, and the morphologically and genetically closely related Silene italica, which can be found in sympatry. Phylogeographical population genetic structure related to persistence in separate glacial refugia has been recently revealed in the S. nutans plant species complex across Western Europe, identifying several distinct lineages. We genotyped 171 associated plant–pathogen pairs of anther‐smut fungi and their host plant individuals using microsatellite markers and plant chloroplastic single nucleotide polymorphisms. We found clear differentiation between fungal populations parasitizing S. nutans and S. italica plants. The population genetic structure of fungal strains parasitizing the S. nutans plant species complex mirrored the host plant genetic structure, suggesting that the pathogen was isolated in glacial refugia together with its host and/or that it has specialized on the plant genetic lineages. Using random forest approximate Bayesian computation (ABC‐RF), we found that the divergence history of the fungal lineages on S. nutans was congruent with that previously inferred for the host plant and probably occurred with ancient but no recent gene flow. Genome sequences confirmed the genetic structure and the absence of recent gene flow between fungal genetic lineages. Our analyses of individual host–pathogen pairs contribute to a better understanding of co‐evolutionary histories between hosts and pathogens in natural ecosystems, in which such studies remain scarce.     

A genome-wide identification of genes potentially associated with host specificity of <Emphasis Type="Italic">Brucella</Emphasis> species

Brucella species are facultative intracellular pathogenic α-Proteobacteria that can cause brucellosis in humans and domestic animals. The clinical and veterinary importance of the bacteria has led to well established studies on the molecular mechanisms of Brucella infection of host organisms. However, to date, no genome-wide study has scanned for genes related to the host specificity of Brucella spp. The majority of bacterial genes related to specific environmental adaptations such as host specificity are well-known to have evolved under positive selection pressure. We thus detected signals of positive selection for individual orthologous genes among Brucella genomes and identified genes related to host specificity. We first determined orthologous sets from seven completely sequenced Brucella genomes using the Reciprocal Best Hits (RBH). A maximum likelihood analysis based on the branch-site test was accomplished to examine the presence of positive selection signals, which was subsequently confirmed by phylogenetic analysis. Consequently, 12 out of 2,033 orthologous genes were positively selected by specific Brucella lineages, each of which belongs to a particular animal host. Extensive literature reviews revealed that half of these computationally identified genes are indeed involved in Brucella host specificity. We expect that this genome-wide approach based on positive selection may be reliably used to screen for genes related to environmental adaptation of a particular species and that it will provide a set of appropriate candidate genes.     

Hidden diversity in the non‐caryophyllaceous plant‐parasitic members of Microbotryum (Pucciniomycotina: Microbotryales)

Abstract

Members of the fungal genus Microbotryum are well‐known parasites on eudicotyledonous plant hosts. However, recent studies focused exclusively on Microbotryum species being parasites in the anthers of Caryophyllaceae in which strong host‐specificity was confirmed by molecular analyses. Consequently, species numbers have risen considerably as multi‐host parasites were split up in so‐called cryptic species. We subjected three non‐caryophyllaceous Microbotryum groups to molecular phylogenetic analyses to see whether we would confirm multi‐host morphospecies or if host‐specific cryptic species in these selected groups could be revealed as well (i.e. a group of non‐caryophyllaceous anther smuts, parasites on different Fallopia species, and parasites on Polygonum bistorta and Polygonum vi‐viparum). We applied a multiple analysis strategy to correct for varying alignment effects on a two‐locus dataset (ITS and LSU rDNA). The results obtained by the different approaches are uniform; high host‐specificity exists in the non‐ caryophyllaceous anther smuts, but overlapping host ranges occur in the parasites of Fallopia species. Results for the parasites of Polygonum are similar, with Microbotryum bistortarum being separated into three lineages and M. marginale forming a lineage on P. bistorta which apparently is conspecific with M. bistortarum p.p. Our study shows that phylogenetic patterns within Microbotryum are much more complicated than deduced from morphological observations alone. Even though Microbotryum species are highly host‐specific, it is impossible to identify species based solely on host taxa affiliation. Species status is reinstated for the anther smut on Salvia pratensis.     

Host association influences variation at salivary protein genes in the bat ectoparasite Cimex adjunctus

Parasite–host relationships create strong selection pressures that can lead to adaptation and increasing specialization of parasites to their hosts. Even in relatively loose host–parasite relationships, such as between generalist ectoparasites and their hosts, we may observe some degree of specialization of parasite populations to one of the multiple potential hosts. Salivary proteins are used by blood‐feeding ectoparasites to prevent hemostasis in the host and maximize energy intake. We investigated the influence of association with specific host species on allele frequencies of salivary protein genes in Cimex adjunctus, a generalist blood‐feeding ectoparasite of bats in North America. We analysed two salivary protein genes: an apyrase, which hydrolyses ATP at the feeding site and thus inhibits platelet aggregation, and a nitrophorin, which brings nitrous oxide to the feeding site, inhibiting platelet aggregation and vasoconstriction. We observed more variation at both salivary protein genes among parasite populations associated with different host species than among populations from different spatial locations associated with the same host species. The variation in salivary protein genes among populations on different host species was also greater than expected under a neutral scenario of genetic drift and gene flow. Finally, host species was an important predictor of allelic divergence in genotypes of individual C. adjunctus at both salivary protein genes. Our results suggest differing selection pressures on these two salivary protein genes in C. adjunctus depending on the host species.     

Host resistance and pathogen infectivity in host populations with varying connectivity

Theory predicts that hosts and pathogens will evolve higher resistance and aggressiveness in systems where populations are spatially connected than in situations in which populations are isolated and dispersal is more local. In a large cross‐inoculation experiment we surveyed patterns of host resistance and pathogen infectivity in anther‐smut diseased Viscaria alpina populations from three contrasting areas where populations range from continuous, through patchy but spatially connected to highly isolated demes. In agreement with theory, isolated populations of V. alpina were more susceptible on average than either patchily distributed or continuous populations. While increased dispersal in connected systems increases disease spread, it may also increase host gene flow and the potential for greater host resistance to evolve. In the Viscaria–Microbotryum system, pathogen infectivity mirrored patterns of host resistance with strains from the isolated populations being the least infective and strains from the more resistant continuous populations being the most infective on average, suggesting that high resistance selects for high infectivity. To our knowledge this study is the first to characterize the impacts of varying spatial connectivity on patterns of host resistance and pathogen infectivity in a natural system.     

A genome scan of diversifying selection in Ophiocordyceps zombie‐ant fungi suggests a role for enterotoxins in co‐evolution and host specificity

Identification of the genes underlying adaptation sheds light on the biological functions targeted by natural selection. Searches for footprints of positive selection, in the form of rapid amino acid substitutions, and the identification of species‐specific genes have proved to be powerful approaches to identifying the genes involved in host specialization in plant‐pathogenic fungi. We used an evolutionary comparative genomic approach to identify genes underlying host adaptation in the ant‐infecting genus Ophiocordyceps, which manipulates ant behaviour. A comparison of the predicted genes in the genomes of species from three species complexes—O. unilateralis, O. australis and O. subramanianii—revealed an enrichment in pathogenesis‐associated functions, including heat‐labile enterotoxins, among species‐specific genes. Furthermore, these genes were overrepresented among those displaying significant footprints of positive selection. Other categories of genes suspected to be important for virulence and pathogenicity in entomopathogenic fungi (e.g., chitinases, lipases, proteases, core secondary metabolism genes) were much less represented, although a few candidate genes were found to evolve under positive selection. An analysis including orthologs from other entomopathogenic fungi in a broader context showed that positive selection on enterotoxins was specific to the ant‐infecting genus Ophiocordyceps. Together with previous studies reporting the overexpression of an enterotoxin during behavioural manipulation in diseased ants, our findings suggest that heat‐labile enterotoxins are important effectors in host adaptation and co‐evolution in the Ophiocordyceps entomopathogenic fungi.     

Specificity and seasonal prevalence of anther smut disease Microbotryum on sympatric Himalayan Silene species

Host sympatry provides opportunities for cross‐species disease transmission and compounded disease effects on host population and community structure. Using the Silene–Microbotryum interaction (the castrating anther smut disease), eleven Himalayan Silene species were assessed in regions of high host diversity to ascertain levels of pathogen specificity. We also investigated disease prevalence, seasonal dynamics of infection and flowering patterns in five co‐blooming Silene species. We identified several new Microbotryum lineages with varying degrees of specialization that is likely influenced by degrees of host divergence and ecological similarities (i.e. shared pollinator guilds). Affected species had 15%–40% of plants infected by anther smut. Flowering was seasonally overlapping among host species (except for the species pair S. asclepiadea and S. atrocastanea), but diseased flowering onset was earlier than healthy plants, leading to dramatic seasonal shifts in observed disease prevalence. Overlapping distributions and flowering provides opportunities for floral pathogen movement between host species, but host specialization may be constrained by the plant phylogenetic relatedness, adaptation to micro‐habitats and difference in pollinator/vector guilds.     

The emergence of the multi-species NIP1 effector in Rhynchosporium was accompanied by high rates of gene duplications and losses

Plant pathogens secrete effector proteins to manipulate the host and facilitate infection. Cognate hosts trigger strong defence responses upon detection of these effectors. Consequently, pathogens and hosts undergo rapid coevolutionary arms races driven by adaptive evolution of effectors and receptors. Because of their high rate of turnover, most effectors are thought to be species-specific and the evolutionary trajectories are poorly understood. Here, we investigate the necrosis-inducing protein 1 (NIP1) effector in the multihost pathogen genus Rhynchosporium. We retraced the evolutionary history of the NIP1 locus using whole-genome assemblies of 146 strains covering four closely related species. NIP1 orthologues were present in all species but the locus consistently segregated presence–absence polymorphisms suggesting long-term balancing selection. We also identified previously unknown paralogues of NIP1 that were shared among multiple species and showed substantial copy-number variation within R. commune. The NIP1A paralogue was under significant positive selection suggesting that NIP1A is the dominant effector variant coevolving with host immune receptors. Consistent with this prediction, we found that copy number variation at NIP1A had a stronger effect on virulence than NIP1B. Our analyses unravelled the origins and diversification mechanisms of a pathogen effector family shedding light on how pathogens gain adaptive genetic variation.     

Variation in resistance to multiple pathogen species: anther smuts of Silene uniflora

The occurrence of multiple pathogen species on a shared host species is unexpected when they exploit the same micro‐niche within the host individual. One explanation for such observations is the presence of pathogen‐specific resistances segregating within the host population into sites that are differentially occupied by the competing pathogens. This study used experimental inoculations to test whether specific resistances may contribute to the maintenance of two species of anther‐smut fungi, Microbotryum silenes‐inflatae and Microbotryum lagerheimii, in natural populations of Silene uniflora in England and Wales. Overall, resistance to the two pathogens was strongly positively correlated among host populations and to a lesser degree among host families within populations. A few instances of specific resistance were also observed and confirmed by replicated inoculations. The results suggest that selection for resistance to one pathogen may protect the host from the emergence via host shifts of related pathogen species, and conversely that co‐occurrence of two species of pathogens may be dependent on the presence of host genotypes susceptible to both.     

Genomewide signatures of selection in Epichloë reveal candidate genes for host specialization

Host specialization is a key process in ecological divergence and speciation of plant‐associated fungi. The underlying determinants of host specialization are generally poorly understood, especially in endophytes, which constitute one of the most abundant components of the plant microbiome. We addressed the genetic basis of host specialization in two sympatric subspecies of grass‐endophytic fungi from the Epichloë typhina complex: subsp. typhina and clarkii. The life cycle of these fungi entails unrestricted dispersal of gametes and sexual reproduction before infection of a new host, implying that the host imposes a selective barrier on viability of the progeny. We aimed to detect genes under divergent selection between subspecies, experiencing restricted gene flow due to adaptation to different hosts. Using pooled whole‐genome sequencing data, we combined F_ST and D_XY population statistics in genome scans and detected 57 outlier genes showing strong differentiation between the two subspecies. Genomewide analyses of nucleotide diversity (π), Tajima's D and dN/dS ratios indicated that these genes have evolved under positive selection. Genes encoding secreted proteins were enriched among the genes showing evidence of positive selection, suggesting that molecular plant–fungus interactions are strong drivers of endophyte divergence. We focused on five genes encoding secreted proteins, which were further sequenced in 28 additional isolates collected across Europe to assess genetic variation in a larger sample size. Signature of positive selection in these isolates and putative identification of pathogenic function supports our findings that these genes represent strong candidates for host specialization determinants in Epichloë endophytes. Our results highlight the role of secreted proteins as key determinants of host specialization.     

Niche‐based host extinction increases prevalence of an environmentally acquired pathogen

Understanding the ecology of environmentally acquired and multi‐host pathogens affecting humans and wildlife has been elusive in part because fluctuations in the abundance of host and pathogen species may feed back onto pathogen transmission. Complexity of pathogen‐host dynamics emerges from processes driving local extinction of the pathogen, its hosts and non‐hosts. While the extinction of species may entail losses in pathogen–host interactions and decrease the proportion of hosts infected by a pathogen (prevalence), some studies suggest the opposite pattern. Niche‐based extinction, based on the species tolerance to environmental conditions, may increase prevalence of infection because the pathogen and its hosts persist, while other species go extinct. Hence, understanding prevalence of infection requires disentangling random‐ and niche‐based extinction processes occurring simultaneously. To contribute to this exercise, we analysed the prevalence of an environmentally acquired, multi‐host pathogen, Mycobacterium ulcerans (MU), in a unique dataset of 16 communities of freshwater animals, surveyed during 12 months in Akonolinga, Cameroon in equatorial Africa. Two different ecosystems were identified: rivers (lotic) and swamps and flooded areas (lentic). Increased prevalence of MU infection was correlated with niche‐based extinction of aquatic host invertebrates and vertebrates in the lentic ecosystems, whereas decreased prevalence was associated with random disassembly of the lotic ecosystems. This finding suggests that random and niche‐based extinction of host taxa are key to assessing the effect of local extinction of species on the ecology of environmentally acquired and multi‐host pathogens.     

Evolution of Drosophila resistance against different pathogens and infection routes entails no detectable maintenance costs

Pathogens exert a strong selective pressure on hosts, entailing host adaptation to infection. This adaptation often affects negatively other fitness‐related traits. Such trade‐offs may underlie the maintenance of genetic diversity for pathogen resistance. Trade‐offs can be tested with experimental evolution of host populations adapting to parasites, using two approaches: (1) measuring changes in immunocompetence in relaxed‐selection lines and (2) comparing life‐history traits of evolved and control lines in pathogen‐free environments. Here, we used both approaches to examine trade‐offs in Drosophila melanogaster populations evolving for over 30 generations under infection with Drosophila C Virus or the bacterium Pseudomonas entomophila, the latter through different routes. We find that resistance is maintained after up to 30 generations of relaxed selection. Moreover, no differences in several classical life‐history traits between control and evolved populations were found in pathogen‐free environments, even under stresses such as desiccation, nutrient limitation, and high densities. Hence, we did not detect any maintenance costs associated with resistance to pathogens. We hypothesize that extremely high selection pressures commonly used lead to the disproportionate expression of costs relative to their actual occurrence in natural systems. Still, the maintenance of genetic variation for pathogen resistance calls for an explanation.     

Disease hotspots or hot species? Infection dynamics in multi‐host metacommunities controlled by species identity,not source location

Pathogen persistence in host communities is influenced by processes operating at the individual host to landscape‐level scale, but isolating the relative contributions of these processes is challenging. We developed theory to partition the influence of host species, habitat patches and landscape connectivity on pathogen persistence within metacommunities of hosts and pathogens. We used this framework to quantify the contributions of host species composition and habitat patch identity on the persistence of an amphibian pathogen across the landscape. By sampling over 11 000 hosts of six amphibian species, we found that a single host species could maintain the pathogen in 91% of observed metacommunities. Moreover, this dominant maintenance species contributed, on average, twice as much to landscape‐level pathogen persistence compared to the most influential source patch in a metacommunity. Our analysis demonstrates substantial inequality in how species and patches contribute to pathogen persistence, with important implications for targeted disease management.     

Parallel evolution of gene classes,but not genes: Evidence from Hawai'ian honeycreeper populations exposed to avian malaria

Adaptation in nature is ubiquitous, yet characterizing its genomic basis is difficult because population demographics cause correlations with nonadaptive loci. Introduction events provide opportunities to observe adaptation over known spatial and temporal scales, facilitating the identification of genes involved in adaptation. The pathogen causing avian malaria, Plasmodium relictum, was introduced to Hawai'i in the 1930s and elicited extinctions and precipitous population declines in native honeycreepers. After a sharp initial population decline, the Hawai'i ‘amakihi (Chlorodrepanis virens) has evolved tolerance to the parasite at low elevations where P. relictum exists, and can sustain infection without major fitness consequences. High‐elevation, unexposed populations of ‘amakihi display little to no tolerance. To explore the genomic basis of adaptation to P. relictum in low‐elevation ‘amakihi, we genotyped 125 ‘amakihi from the island of Hawai'i via hybridization capture to 40,000 oligonucleotide baits containing SNPs and used the reference ‘amakihi genome to identify genes potentially under selection from malaria. We tested for outlier loci between low‐ and high‐elevation population pairs and identified loci with signatures of selection within low‐elevation populations. In some cases, genes commonly involved in the immune response (e.g., major histocompatibility complex) were associated with malaria presence in the population. We also detected several novel candidate loci that may be implicated in surviving malaria infection (e.g., beta‐defensin, glycoproteins and interleukin‐related genes). Our results suggest that rapid adaptation to pathogens may occur through changes in different immune genes, but in the same classes of genes, across populations.     

Pathogen effects on vegetative and floral odours mediate vector attraction and host exposure in a complex pathosystem

Pathogens can alter host phenotypes in ways that influence interactions between hosts and other organisms, including insect disease vectors. Such effects have implications for pathogen transmission, as well as host exposure to secondary pathogens, but are not well studied in natural systems, particularly for plant pathogens. Here, we report that the beetle‐transmitted bacterial pathogen Erwinia tracheiphila – which causes a fatal wilt disease – alters the foliar and floral volatile emissions of its host (wild gourd, Cucurbita pepo ssp. texana) in ways that enhance both vector recruitment to infected plants and subsequent dispersal to healthy plants. Moreover, infection by Zucchini yellow mosaic virus (ZYMV), which also occurs at our study sites, reduces floral volatile emissions in a manner that discourages beetle recruitment and therefore likely reduces the exposure of virus‐infected plants to the lethal bacterial pathogen – a finding consistent with our previous observation of dramatically reduced wilt disease incidence in ZYMV‐infected plants.     

Identifying genomic hotspots of differentiation and candidate genes involved in the adaptive divergence of pea aphid host races

Identifying the genomic bases of adaptation to novel environments is a long‐term objective in evolutionary biology. Because genetic differentiation is expected to increase between locally adapted populations at the genes targeted by selection, scanning the genome for elevated levels of differentiation is a first step towards deciphering the genomic architecture underlying adaptive divergence. The pea aphid Acyrthosiphon pisum is a model of choice to address this question, as it forms a large complex of plant‐specialized races and cryptic species, resulting from recent adaptive radiation. Here, we characterized genomewide polymorphisms in three pea aphid races specialized on alfalfa, clover and pea crops, respectively, which we sequenced in pools (poolseq). Using a model‐based approach that explicitly accounts for selection, we identified 392 genomic hotspots of differentiation spanning 47.3 Mb and 2,484 genes (respectively, 9.12% of the genome size and 8.10% of its genes). Most of these highly differentiated regions were located on the autosomes, and overall differentiation was weaker on the X chromosome. Within these hotspots, high levels of absolute divergence between races suggest that these regions experienced less gene flow than the rest of the genome, most likely by contributing to reproductive isolation. Moreover, population‐specific analyses showed evidence of selection in every host race, depending on the hotspot considered. These hotspots were significantly enriched for candidate gene categories that control host–plant selection and use. These genes encode 48 salivary proteins, 14 gustatory receptors, 10 odorant receptors, five P450 cytochromes and one chemosensory protein, which represent promising candidates for the genetic basis of host–plant specialization and ecological isolation in the pea aphid complex. Altogether, our findings open new research directions towards functional studies, for validating the role of these genes on adaptive phenotypes.     

Phylogenetic congruence between subtropical trees and their associated fungi

Recent studies have detected phylogenetic signals in pathogen–host networks for both soil‐borne and leaf‐infecting fungi, suggesting that pathogenic fungi may track or coevolve with their preferred hosts. However, a phylogenetically concordant relationship between multiple hosts and multiple fungi in has rarely been investigated. Using next‐generation high‐throughput DNA sequencing techniques, we analyzed fungal taxa associated with diseased leaves, rotten seeds, and infected seedlings of subtropical trees. We compared the topologies of the phylogenetic trees of the soil and foliar fungi based on the internal transcribed spacer (ITS) region with the phylogeny of host tree species based on matK, rbcL, atpB, and 5.8S genes. We identified 37 foliar and 103 soil pathogenic fungi belonging to the Ascomycota and Basidiomycota phyla and detected significantly nonrandom host–fungus combinations, which clustered on both the fungus phylogeny and the host phylogeny. The explicit evidence of congruent phylogenies between tree hosts and their potential fungal pathogens suggests either diffuse coevolution among the plant–fungal interaction networks or that the distribution of fungal species tracked spatially associated hosts with phylogenetically conserved traits and habitat preferences. Phylogenetic conservatism in plant–fungal interactions within a local community promotes host and parasite specificity, which is integral to the important role of fungi in promoting species coexistence and maintaining biodiversity of forest communities.