Habitat, Snow-Cover and Soil pH, Affect the Distribution and Diversity of Mortierellaceae Species and Their Associations to Bacteria

Abstract

Mortierellaceae species are among the most frequent and globally distributed soil fungi. However, the factors shaping their diversity and distribution remain obscure. Several species have been reported to be associated to bacteria, but the kind and frequency of such associations were not addressed up to now. We hypothesized that such associations could be important for Mortierellaceae ecology. Therefore, our aim was to understand the driving factors responsible for the Mortierellaceae diversity, community composition and bacterial associations in alpine and subalpine habitats. For answering our question, we collected both snow-free and snow-covered soil at sampling sites from different habitats: bare alpine soil in a glacier forefield, alpine dwarf-willow habitats, and high-altitude Pinus cembra forests. The isolations were carried out by direct cultivation without any antibiotics to the isolation media. Altogether, we obtained 389 Mortierellaceae isolates representing 29 operational taxonomic units (OTUs). Many OTUs could be placed to the genera Mortierella sensu stricto, Dissophora, Entomortierella, Gamsiella, Linnemannia, and Podila, but others could not unambiguously be assigned to a genus. Our results demonstrate that both, the distribution as well as the diversity of the Mortierellaceae species, were significantly influenced by habitat, soil pH, and snow-cover. We noticed that >30% of our isolates were associated to a non-contaminant bacterium. The bacteria associated to our Mortierellaceae isolates belonged to seven different genera. Pseudomonas was the most frequently detected genus associated to the isolated Mortierellaceae species and it was found to be species-specific. Mortierellaceae-bacteria pairs, including those with Pseudomonas, were influenced by location, habitat, and snow-cover. The majority of the fungus-bacterium associations were potentially epihyphal, but we also detected potential endohyphal bacterial species belonging to Mycoavidus, Burkholderiaceae, and Paraburkholderia. Taken together, the non-random associations we detected suggest that fungus-bacterium associations are ecologically meaningful - an interesting path that needs to be investigated further.

Keywords: Mortierellomycotina; alpine; altitude; bacterial interactions; pH; psychrotolerant; sub-alpine; winter.

FIGURE 1

Culture characteristics of different species of Mortierellaceae isolated on PDA. (a) Podila verticillata, (b) Linnemannia elongata, (c) M. pseudozygospora (s.l.), (d) M. angusta (s.l.), (e) P. minutissima, (f) M. solitaria, (g) M. zonata (s.l.), (h) L. hyalina, (i) L. gamsii, (j) M. gemmifera (s.l.), (k) Entomortierella parvispora, (l) M. bainieri (s.l.), (m) M. globalpina (s.s.), (n) M. alpina (s.s.), (o) L. exigua.

FIGURE 2

Phylogenetic tree of the Mortierellaceae species isolated in this study (n = 389 isolates) (loglikelihood –3810.59) based on rDNA-ITS sequences. The isolates fall into 24 well-supported clades. For branches close to the origin, branch ≥70 is shown above the branches as Bayesian posterior probabilities/Parsimony-based bootstrap values. Sequences originating from type specimens are highlighted in red. In order to reduce the size of the tree, all isolates’ sequences were binned to 99% OTUs and a representative number of sequences for each OTU were shown. Blue rectangles on the right illustrate the total number of isolates represented by the sequences they cover (same height). The abundance of single sequences that are directly represented in the tree (n = 1) is not illustrated by rectangles. Rectangle areas are proportional to each other and to isolate abundance, which is also given in the rectangles.

FIGURE 3

Mortierellaceae species distribution across sampling sites (left) and habitats (right). Snowflakes and stars indicate whether the sample, from which the strain was isolated, was covered with snow or not. Species represented by one single species were excluded from the illustration. Bar width is proportionate to the sample size of the sample group in the overall data set. M. alpina, M. globalpina and M. antarctica represent Mortierella s.s., all other Mortierella s.l.; E., Entomortierella; L., Linnemannia; M., Mortierella; P., Podila.

FIGURE 4

Phylogenetic tree of a representative subset of Mortierellaceae-associated bacteria based on their 16S region (log-likelihood –3459.28). A total of 126 bacteria were associated to Mortierellaceae isolates. They were binned into operational taxonomic units (OTUs) based on their 16S sequence (99% sequence similarity). From each OTU, a representative number of sequences were chosen, from which the phylogeny was constructed. The detected bacteria fall into nine lineages. Relationships are generally well-supported. Support values [Bayesian posterior probabilities (BS)/Parsimony-based bootstrap values (BPP)] are provided for the nine major lineages besides the respective nodes. Thin lines represent relationships with BS and/or BPP < 80%. Sequences originating from type specimens are highlighted in red. Blue rectangles on the right illustrate the total number of isolates represented by the sequences they cover (same height). The abundance of single sequences that are directly represented in the tree is not illustrated by rectangles (n = 1). Rectangle areas are proportional to each other and isolate abundance, which is also provided as numbers in the rectangles.

FIGURE 5

Co-occurrences of Mortierellaceae isolates and bacteria. Each line represents an isolated Mortierellaceae strain with an associated bacterium. (A) Seasonal dependence of co-occurrences on snow-cover. Dark gray lines, isolate was obtained from snow-free soil; light gray lines, isolate was obtained from snow-covered soil. (B) Co-occurrences of isolated Mortierellaceae strains and Burkholderiales. (C) Co-occurrences of isolated Mortierellaceae strains and Pseudomonadales and Enterobacteriales. (D) Co-occurrences of isolated Mortierellaceae strains and Pseudomonas OTUs. For panels (B–D), line colors indicate the location of strain isolation: cyan, Hafelekar; royalblue, Haggen; orchid, Kühtai; springgreen, Obergurgl; purple, Patscherkofel; goldenrod, Pfitscherjoch; firebrick, Praxmar. Solid lines, isolate was obtained from snow-covered soil; dashed lines, isolate was obtained from snow-free soil. AG, M. angusta (s.l.); AL, M. alpina (s.s.); AN, M. antarctica (s.s.); BA, M. bainieri (s.l.); CL, Podila clonocystis; Ex, Linnemannia exigua; Exaff, L. aff. exigua; GA, L. gamsii; GL, M. globalpina (s.s.); HO, P. horticola; M. sp. 5, Mortierellaceae species complex 5; PA, Enteromortierella parvispora; PZ, M. pseudozygospora (s.l.); RI, L. rishikesha; SCaff, L. aff. sclerotiella; SC, L. sclerotiella; SO, M. solitaria (s.l.); VEHU, P. verticillata/humilis; ZO, M. zonata (s.l.).