Assessment of fungal spores and spore-like diversity in environmental samples by targeted lysis

Abstract

At particular stages during their life cycles, fungi use multiple strategies to form specialized structures to survive unfavorable environmental conditions. These strategies encompass sporulation, as well as cell-wall melanization, multicellular tissue formation or even dimorphism. The resulting structures are not only used to disperse to other environments, but also to survive long periods of time awaiting favorable growth conditions. As a result, these specialized fungal structures are part of the microbial seed bank, which is known to influence the microbial community composition and contribute to the maintenance of diversity. Despite the importance of the microbial seed bank in the environment, methods to study the diversity of fungal structures with improved resistance only target spores dispersing in the air, omitting the high diversity of these structures in terms of morphology and environmental distribution. In this study, we applied a separation method based on cell lysis to enrich lysis-resistant fungal structures (for instance, spores, sclerotia, melanized yeast) to obtain a proxy of the composition of the fungal seed bank. This approach was first evaluated in-vitro in selected species. The results obtained showed that DNA from fungal spores and from yeast was only obtained after the application of the enrichment method, while mycelium was always lysed. After validation, we compared the diversity of the total and lysis-resistant fractions in the polyextreme environment of the Salar de Huasco, a high-altitude athalassohaline wetland in the Chilean Altiplano. Environmental samples were collected from the salt flat and from microbial mats in small surrounding ponds. Both the lake sediments and microbial mats were dominated by Ascomycota and Basidiomycota, however, the diversity and composition of each environment differed at lower taxonomic ranks. Members of the phylum Chytridiomycota were enriched in the lysis-resistant fraction, while members of the phylum Rozellomycota were never detected in this fraction. Moreover, we show that the community composition of the lysis-resistant fraction reflects the diversity of life cycles and survival strategies developed by fungi in the environment. To the best of our knowledge this is the first time that the fungal diversity is explored in the Salar de Huasco. In addition, the method presented here provides a simple and culture independent approach to assess the diversity of fungal lysis-resistant cells in the environment.

Keywords: Extreme environment; Fungi; Lake sediments; Lysis-resistance; Microbial mats; Spores.

Fig. 1

Validation of the lysis-resistant enrichment method. Bar plots showing the DNA concentration in ng/µl from the direct DNA extraction (“Total” in blue) and after the application of the method to enrich lysis-resistant cells (“Lysis-resistant” in red) for mycelium, sexual spores, asexual spores and/or yeast stage of different fungal species. A Aspergillus niger; B Ulocladium alternata; C Coprinopsis cinerea; D Mortierella antarctica; E Candida albicans. For A, B, C, and E: Microscopic images on the right show the spores before (a) or after (b) the lysis-enrichment method prior to DNA extraction. Due to the small number of spores after the enrichment method in U. alternata and the low concentration in M. antarctica, no microscopic images could be obtained from them

Fig. 2

Fungal diversity at the Salar the Huasco. A Sampling zones indicating the position of the seven sediment cores collected from the main saline lake (right) and the ponds in which 20 microbial mat samples were collected (left). B Assessment of the fungal community composition per environment. Bar plots showing the fungal community composition at the phylum level, in the total community (DNA extraction from samples without enrichment) and lysis-resistant fraction (after lysis-resistant enrichment treatment) in the lake sediments (right) and microbial mats (left)

Fig. 3

Principal Coordinates Analysis (PCoA) of the total community and the lysis-resistant fraction and Venn Diagram of the fungal community in the lake sediments and microbial mats. A PCoA calculated based on the Bray-Cutis distances of the total community, grouped by environment, lake sediments (green) and microbial mats (brown). B PCoA calculated based on the Bray-Cutis distances of the lysis-resistant fraction, grouped by environment, lake sediments (green) and microbial mats (brown). C Venn diagram of the top 1000 ASVs for each fraction and environment, microbial mat (M. total and M. lysis-resistant) and lake sediments (S. total and S. lysis-resistant)

Fig. 4

Principal Coordinates Analysis (PCoA) and bar plots representing the enrichment of different fungal phyla in the total versus lysis-resistant fractions per environment. PCoA calculated based on the Bray–Curtis distances of the communities in A lake sediments and B microbial mats for the lysis-resistant fraction (red) and the total community (blue). Enrichment per phyla in C lake sediments and D microbial mats in the lysis-resistant or total fraction. Negative values show enrichment in the lysis-resistant fraction, positive values show enrichment in the total fraction. Values close to zero show equal abundance in both fractions

Fig. 5

Bar plots showing the relative abundance of the 50 most abundant ASVs per sampling point in the A lake sediments and B microbial mats. Left the total community and right the lysis-resistant fraction. Different colors represent different classes, in the legend on the right the classes are classified by phyla

Fig. 6

Enrichment of different fungal classes in the lysis-resistant or total fraction in the lake sediments and microbial mats and evaluation of the composition of the lysis-resistant community. Enrichment of the classes in the Ascomycota (A) and Basidiomycota (B) phyla in the lake sediments. Enrichment of the classes in the Ascomycota (C) and Basidiomycota (D) phyla in the microbial mats. Negative values show enrichment in the lysis-resistant fraction, positive values show enrichment in the total fraction, 0 indicates equal abundance in both fractions. The bar plots in the bottom of the Fig. 6 (E–F), show the fraction of the ASVs enriched in the lysis-resistant (enrichment index 0.8–1.0), the total community (enrichment index 0.0–0.2), or those shared between the two fractions in the lake sediments (E) and microbial mats (F). The enrichment index was calculated by comparing the abundance (sequence counts) in the lysis-resistant fraction over the total abundance (sequence counts in lysis-resistant fraction + total community)