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

Abstract

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.

Keywords: Microbotryum violaceum; Silene maritima; altitude; biogeography; endemicity; fungi; hybrid zones; population structure; speciation.

Figure 1

Maximum-likelihood tree showing the phylogenetic placement of Microbotryum lagerheimii (HQ832090), M. violaceo-irregulare (AY588104), and M. silenes-inflatae (JN223404), all parasitizing Silene vulgaris, among other Microbotryum species, based on internal transcribed spacer (ITS) sequences. Microbotryum from Polygonum bistorta was used as an outgroup. Bootstraps are indicated. Pictures show differences in teliospore color between the dark purple of M. silenes-inflatae and M. violaceo-irregulare versus the lighter lavender of M. lagerheimii, as well as differences in teliospore ornamentation (verrucose in M. violaceo-irregulare, fully reticulated in M. silenes-inflatae and M. lagerheimii) under 1000x light-microscope magnification.

Figure 2

Proportions of ancestry in K (from 2 to 10) clusters of Microbotryum spp. genotypes inferred with the STRUCTURE program. Each genotype is represented by a vertical bar, partitioned into K segments representing the amount of ancestry of its genome in K clusters. When several clustering solutions (“modes”) were found within replicate runs, only the major mode is shown with its corresponding proportion of runs. IS: Iceland, NL: Netherlands, UK: United Kingdom, CH: Switzerland, IT: Italy, F: France. For each region, genotypes are ordered by sampling elevation (represented at bottom). See Supplementary Figure S3 for a sorting by membership coefficient.

Figure 3

Proportions of ancestry in K (from 2 to 5) clusters of the Microbotryum violaceo-irregulare and M. lagerheimii genotypes inferred with the STRUCTURE program, using a sub-dataset excluding M. silenes-inflatae. In the sub-dataset we kept only the genotypes assigned to the blue cluster at K=5 in the output of the Structure analysis on the whole dataset (Figure 4). A single clustering solution (“mode”) was found among replicate runs. Each genotype is represented by a vertical bar, partitioned into K segments representing the amount of ancestry of its genome in K clusters. NL: Netherlands, UK: United Kingdom, CH: Switzerland, IT: Italy, F: France. For each region, samples are ordered by sampling elevation (represented at bottom; see Figure S5 for a sorting by assignment coefficient within regions).

Figure 4

Principal component analysis (PCA) on multilocus microsatellite genotypes for the dataset including the three Microbotryum species (A: principal component 1 vs principal component 2; B: principal component 1 vs principal component 3) for the sub-dataset including only M. lagerheimii and M. violaceo-irregulare (C: principal component 1 vs principal component 2; D: principal component 1 vs principal component 3). Scatterplots for the first three principal components are shown using a colour labelling of genotypes defined according to the assignment of multilocus genotypes to three species and six clusters within species using Bayesian clustering analyses. Colors indicate clusters as in Figures 2 and 3. Black outlines indicate the samples for which the ITS region has been sequenced.

Figure 5

Distribution maps of the different genetic clusters and species of anther-smut Microbotryum fungi on Silene vulgaris and S. uniflora, and maps of their co-occurrence. Pie charts are proportional to the number of samples. Colors correspond to those used in other figures.

Figure 6

Violin plots showing elevational distribution of species and clusters within species. All samples belonging to Microbotryum silenes-inflatae at K=5 (including all genotypes with low assignment power to the green, orange, pink and yellow M. silenes-inflatae clusters) are included in the leftmost M. silenes-inflatae column. Otherwise, only samples with high-confidence cluster assignment are shown. Samples belonging to M. lagerheimii and M. violaceo-irregulare were determined using the K=3 output from the sub-dataset excluding M. silenes-inflatae.