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.     

Co‐occurrence among three divergent plant‐castrating fungi in the same Silene host species

The competitive exclusion principle postulates that different species can only coexist in sympatry if they occupy distinct ecological niches. The goal of this study was to understand the geographical distribution of three species of Microbotryum anther‐smut fungi that are distantly related but infect the same host plants, the sister species Silene vulgaris and S. uniflora, in Western Europe. We used microsatellite markers to investigate pathogen distribution in relation to host specialization and ecological factors. Microbotryum violaceo‐irregulare was only found on S. vulgaris at high elevations in the Alps. Microbotryum lagerheimii could be subdivided into two genetically differentiated clusters, one on S. uniflora in the UK and the second on S. vulgaris in the Alps and Pyrenees. The most abundant pathogen species, M. silenes‐inflatae, could be subdivided into four genetic clusters, co‐occurring in the Alps, the UK and the Pyrenees, and was found on both S. vulgaris and S. uniflora. All three fungal species had high levels of homozygosity, in agreement with the selfing mating system generally observed in anther‐smut fungi. The three pathogen species and genetic clusters had large range overlaps, but occurred at sites with different elevations, temperatures and precipitation levels. The three Microbotryum species thus do not appear to be maintained by host specialization or geographic allopatry, but instead may occupy different ecological niches in terms of environmental conditions.     

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.     

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.     

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.     

Distribution and population structure of the anther smut Microbotryum silenes‐acaulis parasitizing an arctic–alpine plant

Cold‐adapted organisms with current arctic–alpine distributions have persisted during the last glaciation in multiple ice‐free refugia, leaving footprints in their population structure that contrast with temperate plants and animals. However, pathogens that live within hosts having arctic–alpine distributions have been little studied. Here, we therefore investigated the geographical range and population structure of a fungus parasitizing an arctic–alpine plant. A total of 1437 herbarium specimens of the plant Silene acaulis were examined, and the anther smut pathogen Microbotryum silenes‐acaulis was present throughout the host's geographical range. There was significantly greater incidence of anther smut disease in more northern latitudes and where the host locations were less dense, indicating a major influence of environmental factors and/or host demographic structure on the pathogen distribution. Genetic analyses with seven microsatellite markers on recent collections of 195 M. silenes‐acaulis individuals revealed three main genetic clusters, in North America, northern Europe and southern Europe, likely corresponding to differentiation in distinct refugia during the last glaciation. The lower genetic diversity in northern Europe indicates postglacial recolonization northwards from southern refugia. This study combining herbarium surveys and population genetics thus uniquely reveals the effects of climate and environmental factors on a plant pathogen species with an arctic–alpine distribution.     

Host‐specific differentiation in the anther smut fungus Microbotryum violaceum as revealed by microsatellites

The anther smut fungus Microbotryum violaceum ( = Ustilago violacea) is a parasite of many species of the Caryophyllaceae. Its host specificity has been debated since early this century, when cross‐inoculation experiments indicated the existence of host‐specific lineages. Recently, on the basis of spore ultrastructure, all presumed host races were lumped within M. violaceum. To measure gene flow among natural populations of anther smuts from different host species, we used microsatellite variation at 5 loci among samples from 8 Silene, 2 Saponaria, 2 Dianthus and 1 Gypsophila species. Most of the 326 M. violaceum samples investigated originated from the Swiss Alps and close surroundings. Microsatellite variation revealed almost perfect isolation among anther smut fungi from different host species. In addition, differentiation was supported by the nonrandom distribution of null alleles among samples from different host species and host genera. Null alleles were most abundant in anther smut samples from non‐Silene hosts. The resolution of genetic differentiation among anther smuts from different host species was highest in those from Silene species. Genetic relationships among samples as indicated by Neighbour‐Joining analysis based on genetic distances are discussed with respect to host phylogeny and host ecology. One sample was identified as Ustilago gausseni because it had verrucose instead of reticulate spores and was collected from Silene italica. Neighbour‐Joining analysis revealed that this sample was similar to the M. violaceum samples from other Silene host species. Therefore, our data question spore morphology as a reliable character for anther smut systematics.     

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.     

The role of infectious disease in the evolution of females: Evidence from anther‐smut disease on a gynodioecious alpine carnation*

In flowering plants, the evolution of females is widely hypothesized to be the first step in the evolutionary pathway to separate male and female sexes, or dioecy. Natural enemies have the potential to drive this evolution if they preferentially attack hermaphrodites over females. We studied sex‐based differences in exposure to anther‐smut (Microbotryum), a sterilizing pollinator‐transmitted disease, in Dianthus pavonius, a gynodioecious perennial herb. We found that within a heavily diseased population, females consistently had lower levels of Microbotryum spore deposition relative to hermaphrodites and that this difference was driven by rapid floral closing in females following successful pollination. We further show that this protective closing behavior is frequency dependent; females close faster when they are rare. These results indicate that anther‐smut disease is an important source of selection for females, especially since we found in a common garden experiment no evidence that females have any inherent fecundity advantages over hermaphrodites. Finally, we show that among populations, those where anther‐smut is present have a significantly higher frequency of females than those where the disease is absent. Taken together our results indicate that anther‐smut disease is likely an important biotic factor driving the evolution and maintenance of females in this gynodioecious species.     

Life‐history strategy defends against disease and may select against physiological resistance

Host ecological traits may limit exposure to infectious disease, thereby generating the wide variation in disease incidence observed between host populations or species. The exclusion of disease by ecological traits may then allow selection to act against physiological defenses when they are costly to maintain in the absence of disease. This study investigates ecological resistance in the Silene-Microbotryum pathosystem. An estimated 80% of perennial Silene species host the anther-smut disease while no annuals harbor the disease in nature. Artificial inoculations of annual and perennial Silene plants, obtained from both natural and horticultural populations, demonstrate that the absence of disease in annuals is not explained by elevated physiological resistance. The annual habit is thus a powerful form of ecological defense against anther smut. Moreover, the higher susceptibility of annual species to anther smut relative to perennials supports the hypothesis of a loss of costly physiological resistance under ecological protection. The observation in annuals that physiological susceptibility is correlated with lower rates of flowering (i.e., lower fitness) suggests that variation in physiological resistance is costly in the absence of disease, even in a naїve Silene species. The absence of disease in natural populations of annuals combined with their high physiological susceptibility attest to the strength of host ecology in shaping the distribution of disease and to the dynamic nature of disease resistance.     

Host‐specific thermal profiles affect fitness of a widespread pathogen

Host behavior can interact with environmental context to influence outcomes of pathogen exposure and the impact of disease on species and populations. Determining whether the thermal behaviors of individual species influence susceptibility to disease can help enhance our ability to explain and predict how and when disease outbreaks are likely to occur. The widespread disease chytridiomycosis (caused by the fungal pathogen Batrachochytrium dendrobatidis, Bd) often has species‐specific impacts on amphibian communities; some host species are asymptomatic, whereas others experience mass mortalities and population extirpation. We determined whether the average natural thermal regimes experienced by sympatric frog species in nature, in and of themselves, can account for differences in vulnerability to disease. We did this by growing Bd under temperatures mimicking those experienced by frogs in the wild. At low and high elevations, the rainforest frogs Litoria nannotis, L. rheocola, and L. serrata maintained mean thermal regimes within the optimal range for pathogen growth (15–25°C). Thermal regimes for L. serrata, which has recovered from Bd‐related declines, resulted in slower pathogen growth than the cooler and less variable thermal regimes for the other two species, which have experienced more long‐lasting declines. For L. rheocola and L. serrata, pathogen growth was faster in thermal regimes corresponding to high elevations than in those corresponding to low elevations, where temperatures were warmer. For L. nannotis, which prefers moist and thermally stable microenvironments, pathogen growth was fastest for low‐elevation thermal regimes. All of the thermal regimes we tested resulted in pathogen growth rates equivalent to, or significantly faster than, rates expected from constant‐temperature experiments. The effects of host body temperature on Bd can explain many of the broad ecological patterns of population declines in our focal species, via direct effects on pathogen fitness. Understanding the functional response of pathogens to conditions experienced by the host is important for determining the ecological drivers of disease outbreaks.     

Glacial refugia in pathogens: European genetic structure of anther smut pathogens on Silene latifolia and Silene dioica

Climate warming is predicted to increase the frequency of invasions by pathogens and to cause the large-scale redistribution of native host species, with dramatic consequences on the health of domesticated and wild populations of plants and animals. The study of historic range shifts in response to climate change, such as during interglacial cycles, can help in the prediction of the routes and dynamics of infectious diseases during the impending ecosystem changes. Here we studied the population structure in Europe of two Microbotryum species causing anther smut disease on the plants Silene latifolia and Silene dioica. Clustering analyses revealed the existence of genetically distinct groups for the pathogen on S. latifolia, providing a clear-cut example of European phylogeography reflecting recolonization from southern refugia after glaciation. The pathogen genetic structure was congruent with the genetic structure of its host species S. latifolia, suggesting dependence of the migration pathway of the anther smut fungus on its host. The fungus, however, appeared to have persisted in more numerous and smaller refugia than its host and to have experienced fewer events of large-scale dispersal. The anther smut pathogen on S. dioica also showed a strong phylogeographic structure that might be related to more northern glacial refugia. Differences in host ecology probably played a role in these differences in the pathogen population structure. Very high selfing rates were inferred in both fungal species, explaining the low levels of admixture between the genetic clusters. The systems studied here indicate that migration patterns caused by climate change can be expected to include pathogen invasions that follow the redistribution of their host species at continental scales, but also that the recolonization by pathogens is not simply a mirror of their hosts, even for obligate biotrophs, and that the ecology of hosts and pathogen mating systems likely affects recolonization patterns.     

Identifying the abiotic and biotic drivers behind the elevational distribution shift of a parasitic plant

• Climate change will alter the biotic and abiotic environment and dissipate ecological barriers, reorganising maps of current distribution of parasites and their hosts. In this study, we analyse the population dynamics of the parasitic plant Viscum album subsp. austriacum and explore key biotic (host availability and seed dispersal) as well as abiotic (temperature) factors influencing elevational distribution.
• The study was conducted along an elevational gradient of a Mediterranean mountain, covering the distribution belts of three potential pine hosts: Pinus halepensis (1300–1500 m), P. nigra (1300–1900 m) and P. sylvestris var. nevadensis (1600–2000 m). Along this gradient, we measured multiple variables of mistletoe population (prevalence, abundance and demographic profile) and different factors that might define the current mistletoe distribution (host suitability and availability, temperature and seed dispersal services).
• We found a decline in mistletoe prevalence and abundance with increasing elevation, detecting larger values of both variables at lower elevations of the most suitable host (Pinus nigra). Pinus sylvestris var. nevadensis was a suboptimal but suitable host for the parasite at high elevations. Mistletoe found suitable temperatures and seed dispersal services all along the gradient, being able to recruit at any site.
• With warming temperatures, the presence of suitable vectors for parasite dispersion, and the presence of a sub‐optimal host (P. sylvestris var. nevadensis) at the mountain top, mistletoe currently has a window of opportunity to expand its present geographic distribution to the summits.

     

Anther‐smut fungi from more contaminated sites in Chernobyl show lower infection ability and lower viability following experimental irradiation

The long‐term contamination that followed the nuclear disaster at Chernobyl provides a case study for the effects of chronic ionizing radiation on living organisms and on their ability to tolerate or evolve resistance to such radiation. Previously, we studied the fertility and viability of early developmental stages of a castrating plant pathogen, the anther‐smut fungus Microbotryum lychnidis‐dioicae, isolated from field sites varying over 700‐fold in degree of radioactive contamination. Neither the budding rate of haploid spores following meiosis nor the karyotype structure varied with increasing radiation levels at sampling sites. Here, we assessed the ability of the same M. lychnidis‐dioicae strains to perform their whole life cycle, up to the production of symptoms in the plants, that is, the development of anthers full of fungal spores; we also assessed their viability under experimental radiation. Fungal strains from more contaminated sites had no lower spore numbers in anthers or viability, but infected host plants less well, indicating lower overall fitness due to radioactivity exposure. These findings improve our understanding of the previous field data, in which the anther‐smut disease prevalence on Silene latifolia plants caused by M. lychnidis‐dioicae was lower at more contaminated sites. Although the fungus showed relatively high resistance to experimental radiation, we found no evidence that increased resistance to radiation has evolved in populations from contaminated sites. Fungal strains from more contaminated sites even tolerated or repaired damage from a brief acute exposure to γ radiation less well than those from non‐ or less contaminated sites. Our results more generally concur with previous studies in showing that the fitness of living organisms is affected by radiation after nuclear disasters, but that they do not rapidly evolve higher tolerance.     

PATHOGEN LIFE‐HISTORY TRADE‐OFFS REVEALED IN ALLOPATRY

Trade‐offs in life‐history traits is a central tenet in evolutionary biology, yet their ubiquity and relevance to realized fitness in natural populations remains questioned. Trade‐offs in pathogens are of particular interest because they may constrain the evolution and epidemiology of diseases. Here, we studied life‐history traits determining transmission in the obligate fungal pathogen, Podosphaera plantaginis, infecting Plantago lanceolata. We find that although traits are positively associated on sympatric host genotypes, on allopatric host genotypes relationships between infectivity and subsequent transmission traits change shape, becoming even negative. The epidemiological prediction of this change in life‐history relationships in allopatry is lower disease prevalence in newly established pathogen populations. An analysis of the natural pathogen metapopulation confirms that disease prevalence is lower in newly established pathogen populations and they are more prone to go extinct during winter than older pathogen populations. Hence, life‐history trade‐offs mediated by pathogen local adaptation may influence epidemiological dynamics at both population and metapopulation levels.     

The spatial distribution of plant populations, disease dynamics and evolution of resistance

Empirical studies of the interaction between the anther smut fungus Microbotryum violaceum and its host plant Lychnis alpina were combined with modelling approaches to investigate how variation in the spatial distribution of host populations influences disease dynamics and variation in resistance. Patterns of disease incidence and prevalence were surveyed in three contrasting systems of natural L . alpina populations where there is substantial variation in spatial structure, ranging from large continuous populations through to small isolated patches. Disease incidence (fraction of populations where disease was present) was highest in the continuous situation, and lowest in the most isolated populations. The reverse was true for prevalence (fraction of individuals diseased). To better understand the long-term ecological and evolutionary consequences of differences in among population spatial structure, we developed a two-dimensional spatially explicit simulation model in which host-population spacing was modelled by varying the percentage of sites suitable for the host. The general patterns of disease incidence and prevalence generated in the simulations corresponded well with the patterns observed in natural populations of L. alpina and M. violaceum ; i.e. the fraction of sites with disease increased while the average disease prevalence in diseased populations decreased when host populations became more connected. One likely explanation for the differences in disease incidence and prevalence seen in natural populations is that the evolution of host resistance varies as a function of the degree of fragmentation. This is supported by simulation results that were qualitatively similar to the survey data when resistance was allowed to vary, but not when hosts were assumed to be uniformly susceptible. In the former, the frequency of resistance increased markedly as host populations became more connected.     

Loss of pathogens in threatened plant species

Global declines in biodiversity create an urgent need to address the impact of infectious disease in the small and fragmented populations that characterize threatened species. However, the paucity of empirical data provides little ability to predict whether disease generally accelerates threatened species towards extinction or becomes less important as populations decline. This study tests whether plant species threatened with extinction exhibit lower disease frequencies and lower overall parasite species richness while also experimentally testing for the effect of physiological disease resistance. Herbarium surveys of the genus Silene revealed that anther‐smut disease was significantly less frequent in threatened species than non‐threatened species, and this effect was not constrained by the host phylogeny or by physiological resistance. Moreover, analysis across a much broader range of plants (using US Federal designations) revealed that species with endangered status had significantly lower species richness of fungal pathogens than closely‐related, non‐endangered species. These results support the role of host ecology, rather than physiological resistance or phylogeny, in determining overall lower incidences and diversity of diseases in plant species threatened by extinction. Low disease incidence accompanied by susceptibility in threatened species may result from selection against costly resistance genes in the absence of disease.     

Differences in population structure of the anther smut fungus Microbotryum violaceum on two closely related host species, Silene latifolia and S. dioica

We investigated the genetic population structure of the sexually transmitted plant pathogen, the fungus Microbotryum violaceum, on the two closely related host species Silene latifolia and S. dioica using microsatellite markers. We found strong deviations from Hardy-Weinberg expectations, with significant heterozygote deficiency in almost all populations. Fungal strains from the two host species were differentiated, and these host races differed in amount of variation within populations and differentiation among populations. Anther smut from S. latifolia harboured significantly less microsatellite diversity and were more genetically differentiated from each other than those from S. dioica. Small effective population sizes, rapid population turnover, and less gene flow among populations could lead to this higher population differentiation and lower within population genetic diversity for anther smut populations on S. latifolia than on S. dioica. These results are in concordance with host ecology because S. latifolia grows in more disturbed habitats than S. dioica and may provide a shorter-lived host environment.     

Invasion biology and host specificity of the grapevine yellows disease vector Hyalesthes obsoletus in Europe

Within the past 10 years, the yellows disease ‘bois noir’ (BN) has become one of the commercially most important diseases of grapevine [Vitis vinifera L. (Vitaceae)] in Europe. Infection pressure is caused by phytoplasmas of the stolbur 16SrXII‐A group that are transmitted by a planthopper vector, Hyalesthes obsoletus Signoret (Homoptera: Auchenorrhyncha). Infestation happens as an accidental side‐effect of the feeding behaviour of the vector, as vector and pathogen proliferation is dependent on other plants. In Germany, the increase of BN is correlated with the use of a new host plant by the vector, increase in abundance of the vector on the new host plant, and dissemination of host plant‐specific pathogen strains. In this article, we investigate geographic and host‐associated range expansion of the vector. We test whether host‐plant utilization in Germany, hence the increase in BN, is related to genetic host races of the vector and, if so, whether these have evolved locally or have immigrated from southern populations that traditionally use the new host plant. The genetic population analysis demonstrates a recent expansion and circum‐alpine invasion of H. obsoletus into German and northern French wine‐growing regions, which coincides with the emergence of BN. No H. obsoletus mitochondrial DNA haplotype host‐plant affiliation was found, implying that the ability to use alternative host plants is genetically intrinsic to H. obsoletus. However, subtle yet significant random amplified polymorphic DNA (RAPD) genetic differentiation was found among host plant populations. When combined, these results suggest that a geographic range expansion of H. obsoletus only partly explains the increase of BN, and that interactions with host plants also occur. Further possible beneficial factors to H. obsoletus, such as temperature increase and phytoplasma interactions, are discussed.