Sympatry and interference of divergent Microbotryum pathogen species

Abstract 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.

In the current study, we analyzed DNA sequence identities of Microbotryum samples that were collected from a large number of natural populations, focusing on localities where multiple diseased host species were growing together. Our first goal was to determine whether localities with the disease on multiple host species represented sites of cross-species transmission or the sympatry of divergent pathogen lineages each specialized to their co-occurring host species. Our second goal was to investigate the movement of Microbotryum spores between sympatric host species by microscopic examination of spore deposition on flowers. Our third goal addressed a potential consequence of sympatry of pathogen lineages, experimentally investigating the potential for interference (direct or indirect) among divergently specialized pathogens, noting that the topic of hybridization in Microbotryum has been addressed elsewhere Gladieux et al., 2011;Petit et al., 2017;Shykoff et al., 1999). Specifically, we asked whether the ability of the endemic pathogen of S. vulgaris to infect its natural host was altered by prior exposure of plants with nonendemic pathogens from other host species. This study helps to reveal a complex source of interactions between relatively specialized pathogens on multiple host species and their potential impact on the occurrence of disease in natural populations.
F I G U R E 1 Anther-smut disease caused by fungi in the genus Microbotryum, here shown in Silene vulgaris. The pollen is replaced by masses of dark pathogen spores As with many diseases, the anther-smut life history plays a role in shaping where the pathogen is found in nature. Anther-smut fungi in the genus Microbotryum are obligate, biotrophic pathogens of perennial plants mainly in the Caryophyllaceae Thrall, Biere, & Antonovics, 1993), where infections cause host sterility, are systemic and persistent, and have the potential to competitively exclude subsequent invasion by other Microbotryum pathogens (Gold et al., 2009;Hood, 2003;López-Villavicencio et al., 2011). There are many host species for anther smut with broad and overlapping geographic distributions (Hitchcock & Maguire, 1847;Hood et al., 2010), and disease incidence can be remarkably high in some host species.
For example, the typical proportions of disease within host populations are between 0.10 and 0.30 for S. latifolia (Antonovics, 2004), ca. 0.30 for S. vuglaris in upper elevations (Abbate & Antonovics, 2014), or even >0.50 for Dianthus furcatus or Dianthus pavonius . Therefore, the potential for frequent contact between Microbotryum species on separate host species is high.

| Field specificity on sympatric host species
The fungus was sampled from natural populations as the spore contents of single diseased flowers, which are expected to contain one fungal genotype (Garber & Ruddat, 2002;Hood, 2003;Van Putten et al., 2005). Flowers were sampled at the bud stage whenever possible (i.e., to avoid possible contamination by pollinator visits) and stored under desiccation prior to use.
Sampling was carried out over several years during the course of fieldwork in many geographic regions and included diseased plants in the genera Silene, Lychnis, Atocion, Dianthus, Saponaria, Gypsophila, and Petrorhagia ( Figure 2). Sympatry of Microbotryum on two or more host species was defined as when the disease was found on another host within easy search distance (ca. 100 m) of the first-found diseased host. A collection of samples where the disease was on only one host species was also included to increase the inference of hostspecific pathogen lineages when occurring across localities.
To assessing disease transmission among sympatric host species within localities, we used DNA sequence identity for the pathogens' internal transcribed spacer (ITS) region of the nuclear rDNA. While ITS sequences have utility in resolving fungal lineages (Schoch et al., 2012; but see Hart et al., 2015), phylogenetic insights were secondary and need to be interpreted cautiously due to the use of a restricted amount of DNA sequence length and variation in statistical support among nodes. Nevertheless, to display the ITS sequence variation, neighbor-joining analysis (Jukes-Cantor distances) was performed using the MEGA software (Kumar, Tamura, Jakobsen, & Nei, 2001) with 1,000 bootstrap replications. The smut fungus infecting Persicaria bistorta (Syn: Polygonum bistorta) in the Polygonaceae was used as an outgroup because it is from outside the Microbotryum clade causing anther-smut disease on the Caryophyllaceae (Almaraz, Roux, Maumont, & Durrieu, 2002;Kemler, Lutz, Göker, Oberwinkler, & Begerow, 2009).
To prepare cultures for DNA extraction, the spores (i.e., teliospores, diploid) were plated on potato dextrose agar at 25°C. Upon germination, the fungus undergoes meiosis, and the resulting haploid sporidia can be grown as yeast-like cultures. Colonies derived from single haploid sporidia were obtained by microdissection or by dilution plating. All sporidial cultures were stored under desiccation at −20°C.
DNA was extracted from sporidial cultures using the DNeasy Plant Mini Kit (Qiagen). PCR was used to amplify the ITS region (primers: ITS1 and ITS4 from White, Bruns, Lee, & Taylor, 1990).
PCR products were sequenced using Sanger dye termination methods. For some sequences, where the chromatograms contained discrete regions of basepair ambiguities, individual ITS amplicons were cloned using the TA Cloning Kit (Invitrogen) and sequenced.
DNA sequences are available in GenBank under accession numbers KY084313-KY084399.

| Spore dispersal among sympatric host species
The potential for movement of Microbotryum spores from the diseased flowers of one host species to another sympatric host species was assessed in natural mixed plant communities. Using sites with two host species, but where only one was diseased, healthy flowers of the unaffected host species were collected in sealed coin envelopes. Distance to nearest disease plant of the other host species was recorded. Various host species combinations were examined (Table 1)

| Consequences of cross-species pathogen exposure
To assess whether the ability of Microbotryum to cause disease on its host-of-origin (i.e., endemic host-pathogen combination) was affected by prior exposure of the host to Microbotryum obtained from F I G U R E 2 The left side shows the neighbor-joining tree of the Microbotryum samples collected from multiple host species and localities. Collection sites where the disease was on more than one host species are indicated on the right side by vertically arranged dots, connected by vertical bars. Filled dots indicate sympatry of lineages found only on their respective (specific) host species. Open dots indicate instances of a single pathogen lineage infecting multiple sympatric host species. Brackets indicate groups of host species sharing phylogenetically similar pathogens and their source: samples are from Europe (EU) unless otherwise indicated as being from North America (NA) Proportions of plants that became diseased by the pathogen endemic to S. vulgaris were compared for each nonendemic pathogen treatment to the proportion in their water control treated plants.
The effects of host exposure to nonendemic pathogens on the ability of the endemic pathogen to cause disease were assessed using GLM procedures in SPSS v19 following arcsin square-root data transformation of the proportions.   where Microbotryum on S. vulgaris was identical in ITS sequence to the sympatric pathogen known to be specialized on S. latifolia or Silene dioica; two of these localities also contained other sympatric Microbotryum pathogens (Figure 2). These instances of cross-species transmission were likely independent because they were found to be consistent with slight regional differences in ITS sequence among Microbotryum from S. latifolia or were from different geographic locations. Despite evidence from prior reports of cross-species disease transmission by Microbotryum between S. dioica and S. latifolia (Gladieux et al., 2011), sympatric diseased populations of these two host species were not obtained in this study (Figure 2).

| Field specificity on sympatric host species
In some sections of the tree, Microbotryum from closely related host species were not seen to be host-specific (Figure 2

| Spore dispersal among sympatric host species
Microbotryum spores are readily found on the flowers of nondiseased species when they occur in mixed-host communities. Examination of flowers from the host species that was only healthy in a locality (target flowers) but co-occurring with another diseased host species (disease sources) revealed cross-species spore deposition in 96% of target flowers (Table 1)

| Consequences of cross-species pathogen exposure
The experimental prior exposure of plants to

Proportions of plants that became diseased by M. silenes-inflatae
were also lower when the inoculum was applied to older seedlings

| D ISCUSS I ON
In a broadly distributed multi-host/multi-pathogen system, this study revealed the co-occurrence of anther-smut diseases on sympatric host species and significant pathogen interactions that may impact disease and host distributions in natural populations. In a very early observation of disease ecology, Schröter (1877) suggested that host sympatry can be used to assess pathogen specialization, saying of anther smut that "Ustilago violacea [=Microbotryum] has been found on so many plants of the Melandrium [=Silene] family that one is tempted to assume that it lets itself be transferred to all its representatives," while "very often one finds large stretches The co-occurrence of multiple pathogen and multiple host species is reported to yield complex interactions and interference among pathogens that affect their community structure (Fenton, Streicker, Petchey, & Pedersen, 2015;Halliday, Heckman, Wilfahrt, & Mitchell, 2019;Johnson et al., 2015;Parker & Gilbert, 2018;Seabloom et al., 2015). We showed that cross-species movement of Microbotryum spores is very common in mixed-host communities and that prior exposure to incompatible, nonendemic pathogens reduced the abil- impact of cross-species spore movement on the Microbotryum community may be to limit the occurrence of multi-host/multi-pathogen assemblies. The mechanisms would be analogous to the "inhibitory host" model of Holt, Dobson, Begon, Bowers, and Schauber (2003).
This model describes the situation where a second host offers no contribution to a pathogen's reproduction but diminishes a pathogen's persistence on the endemic host species, either because the second host serves as an inoculum sink or reduces visitation rates in the case of vector-transmitted diseases (i.e., a dilution effect; Ostfeld & Keesing, 2000). In Microbotryum, however, our results suggest the inhibitory effect may be mediated by a "spill-over" type of cross-protection, such that Microbotryum pathogens may be less able to invade or to be maintained at as high a prevalence in host populations that are sympatric with anther-smut disease on other host species. Whether the protection is long-lasting, and whether it is due to competitive exclusion by asymptomatic infections or inducible host resistance mechanisms, is not yet known. Anther-smut inoculation has been shown to induce some changes to host growth even in the absence of symptoms at flowering , but the possibility of persistent asymptomatic infections has not been investigated. Intrahost competitive exclusion may also be important and is well documented among Microbotryum strains, including within-and among-species interactions (Fortuna et al., 2018;Gold et al., 2009;Hood, 2003). Studies have indicated heritable variation in anther-smut disease resistance (Alexander & Maltby, 1990;Cafuir, Antonovics, & Hood, 2007;Carlsson-Granér & Pettersson, 2005;Chung, Petit, Antonovics, Pedersen, & Hood, 2012), but the mechanisms of resistance and whether it is inducible have not been determined.
While such cross-species protection can potentially limit local pathogen diversity, the effects on the host diversity might be in the opposite direction. Where a diseased host is common, introduction of a competing host species would be facilitated because the diseased host (acting as an "inhibitory host") protects the introduced host against pathogen infection. In this way, the disease may contribute positively to host diversity, in a manner enhancing the densitydependent and species-specific feedbacks of the Janzen-Connell effect (Comita et al., 2014;Connell, 1970;Janzen, 1970). More extensive field surveys combined with experimental studies would be needed to directly investigate Microbotryum co-occurrence and its impact on host and pathogen assemblages.

Exceptions to the general pattern of host specificity of
Microbotryum included a number of cross-species transmissions to S. vulgaris from either sympatric S. latifolia or S. dioica. Such host-shifts to S. vulgaris have been confirmed by other genetic approaches and experimental studies (Antonovics et al., 2002;Hood et al., 2003;de Vienne et al., 2009). It remains to be determined whether S. vulgaris is predisposed to receiving host-shifts due to its susceptibility or whether the ecology of being weedy and very widespread creates more opportunities for host-shifts to occur and to be observed. Moreover, in the current and previous studies (Abbate & Antonovics, 2014;Abbate et al., 2018;Bucheli, Gautschi, & Shykoff, 2000;Le Gac et al., 2007), S. vulgaris from high elevations (>ca. 1,300 m) was found infected by multiple distinct, endemic, and apparently self-sustaining lineages of Microbotryum, which is also consistent with a greater propensity for host-shifts and perhaps new disease emergence onto this species. Additional exceptions to host specificity include historic host-shifts that have been inferred from the incongruence of the host and pathogen phylogenies (Refrégier et al., 2008). Also, in the current study and previous ones Le Gac et al., 2007;Petit et al., 2017), sequence variation indicated that several lineages of Microbotryum from Dianthus can share host species of this genus, suggesting less strict pathogen specificity on this recently radiated plant genus (Valente, Savolainen, & Vargas, 2010).
At a broader scale, this study supported patterns in the geographic overlap of Microbotryum lineages, including samples from European and North American host species. It was previously suggested that North America contains Microbotryum lineages that are highly divergent from those found in Europe (Freeman, Kellye Duong, Shi, Hughes, & Perlin, 2002;Hood, Rocha, 0. J., & Antonovics, J., 2001). However, on the west coast of North America occur members of both the previously described North American clade (i.e., Microbotryum from S. parryi) and members of the European clade (i.e., Microbotryum from S. lemmonii and S. douglasii; see also Lutz et al., 2005). It is remarkable that such great variation in Microbotryum would be found in North America because the history of the genus Silene has a Eurasian origin followed by migration with reduced species diversity into the Americas via the Beringian region (Popp, Erixon, Eggens, & Oxelman, 2005;Popp & Oxelman, 2007). Further studies (e.g., incorporating genomic-scale data; Branco et al., 2018) are warranted to address the large-scale phylogeographic diversification of Microbotryum, and the potential impact on the co-occurrence and interactions of pathogen lineages.
In summary, this study confirms that the host specificity seen in broad-scale phylogenetic and experimental inoculation studies of Microbotryum generally reflects specialization at an ecological level and that such specialization holds true even when host species are in close sympatry. This expectation needs to be tested in any particular case, as there are exceptions, especially in hosts such as S. vulgaris or Dianthus species. However, even in a community assemblage where there is apparent host specificity, cross-species exposure to multiple pathogen lineages is likely to occur, with consequences that may be overt or may be "cryptic," yet still may influence dynamics of the species assemblage. This study identified one such mechanism-inhibition of endemic pathogens by prior exposure to nonendemic ones-but further effects on occurrence and distribution of the multi-host/multi-pathogen communities are likely and deserve further investigation.

CO N FLI C T O F I NTE R E S T
None declared.

AUTH O R CO NTR I B UTI O N S
Hood and Antonovics conceptualized the project. Hood, Antonovics, Abbate, and Stern contributed to field collection and their assessment. Hood and Wolf contributed experimental studies. Hood, Antonovics, and Giraud wrote and revised the manuscript, with contribution also from Wolf and Stern.

DATA ACCE SS I B I LIT Y
DNA sequences: GenBank accession numbers KY084313-KY084399.