Research Article |
Corresponding author: Christian Wurzbacher ( c.wurzbacher@tum.de ) Academic editor: Kerstin Voigt
© 2016 Christian Wurzbacher, Norman Warthmann, Elizabeth Bourne, Katrin Attermeyer, Martin Allgaier, Jeff R. Powell, Harald Detering, Susan Mbedi, Hans-Peter Grossart, Michael T. Monaghan.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Wurzbacher C, Warthmann N, Bourne EC, Attermeyer K, Allgaier M, Powell JR, Detering H, Mbedi S, Grossart H-P, Monaghan MT (2016) High habitat-specificity in fungal communities in oligo-mesotrophic, temperate Lake Stechlin (North-East Germany). MycoKeys 16: 17-44. https://doi.org/10.3897/mycokeys.16.9646
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Freshwater fungi are a poorly studied ecological group that includes a high taxonomic diversity. Most studies on aquatic fungal diversity have focused on single habitats, thus the linkage between habitat heterogeneity and fungal diversity remains largely unexplored. We took 216 samples from 54 locations representing eight different habitats in the meso-oligotrophic, temperate Lake Stechlin in North-East Germany. These included the pelagic and littoral water column, sediments, and biotic substrates. We performed high throughput sequencing using the Roche 454 platform, employing a universal eukaryotic marker region within the large ribosomal subunit (LSU) to compare fungal diversity, community structure, and species turnover among habitats. Our analysis recovered 1027 fungal OTUs (97% sequence similarity). Richness estimates were highest in the sediment, biofilms, and benthic samples (189–231 OTUs), intermediate in water samples (42–85 OTUs), and lowest in plankton samples (8 OTUs). NMDS grouped the eight studied habitats into six clusters, indicating that community composition was strongly influenced by turnover among habitats. Fungal communities exhibited changes at the phylum and order levels along three different substrate categories from littoral to pelagic habitats. The large majority of OTUs (> 75%) could not be classified below the order level due to the lack of aquatic fungal entries in public sequence databases. Our study provides a first estimate of lake-wide fungal diversity and highlights the important contribution of habitat heterogeneity to overall diversity and community composition. Habitat diversity should be considered in any sampling strategy aiming to assess the fungal diversity of a water body.
Freshwater fungi, aquatic fungi, metabarcoding, LSU, GMYC, habitat specificity, Chytridiomycota , Cryptomycota , Rozellomycota , community ecology, lake ecosystem, biofilm, sediment, plankton, water sample, benthos, reed, fungal diversity
Aquatic fungi play an important role in the cycling of carbon and nutrients in ecosystems (
Estimates of total fungal diversity currently range from 1.5–3 M species worldwide (
Considering the multitude of available niches and fungal lifestyles in aquatic habitats (
We examined the fungal diversity of a temperate lake in North-East Germany (Lake Stechlin) using a high throughput sequencing and metabarcoding approach. Our first aim was to examine the effect of habitat specificity on the fungal community by measuring the extent to which different habitat types contained similar communities, or whether there was a pronounced taxa turnover among habitats. Our second aim was to test the morphotype hypothesis, specifically whether fungal groups present were related to the availability of major types of particulate organic matter (POM). We expected that the broad diversity of substrate size and structures sampled (e.g. plankton, macrophytes) would reveal a more heterogeneous fungal community than previously detected by traditional lake sampling strategies.
Lake Stechlin is a deep (maximum depth: 69.5 m), oligo-mesotrophic, dimictic hard-water lake in North-East Germany (53°10'N; 13°02'E). It has a surface area of 4.25 km2 and is divided into three distinct basins (Figure
Sampling sites in Lake Stechlin. Integrated water samples, above-sediment water, plankton (> 55 µm), and sediment were taken from pelagic locations. Surface water samples, reed plants (Phragmites australis), biofilm samples (from stone, wood and macrophytes) and benthic samples (detritus, macrozoobenthos) were taken from littoral locations.
We sampled eight different habitat types (Table
Overview of the total abundance of eukaryotic sequences and OTUs (97% similarity clustering) recovered for each lake habitat. Fungal contribution (reads, OTUs) was calculated as median percentage with standard deviation. Habitat abbreviations used in Figs
Habitat | POM type | n | Total reads/ total OTU | Fungal reads % ± SD | Fungal OTU % ± SD | Shared Chao (total) | Shared Chao (fungal) | |
---|---|---|---|---|---|---|---|---|
Pelagic | Pelagic water - Pel | FPOM | 3 | 19795/470 | 2.4 ± 0.4 | 5.8 ± 0.6 | 303–711 | 42–107 |
Above Sediment -AS | FPOM | 3 | 18701/592 | 2.2 ± 0.3 | 7.7 ± 2.5 | 467–1005 | 85–148 | |
Plankton - PT | FPOM | 3 | 17902/165 | 0.4 ± 0.2 | 3.7 ± 0.5 | 151–294 | 8–8* | |
Sediment - Sed | MIX | 3 | 79190/1759 | 5.1 ± 1.2 | 23.5 ± 0.9 | 612–3219 | 189–853 | |
Littoral | Littoral water - Lit | FPOM | 3 | 29128/521 | 1.5 ± 0.2 | 6.8 ± 1.0 | 363–883 | 54–116 |
Biofilm - Bio | MIX | 3 | 26302/1053 | 11.0 ± 6.8 | 25.1 ± 2.6 | 541–1971 | 225–640 | |
Benthos - Ben | CPOM | 3 | 59169/1070 | 63.1 ± 13.1 | 30.9 ± 8.4 | 580–2280 | 231–1323 | |
Reed | CPOM | 1 | 4277/179 | 88.7 | 35.8 | n.a. | n.a. |
Total DNA was extracted using the Power Soil kit (MoBio Laboratories, Carlsbad, USA) for Sediment samples; the Qiagen Plant kit (Qiagen, Hilden, Germany) for Reed, Biofilm, and Benthos samples; and the Qiagen Blood & Tissue kit for Littoral, Pelagic, Above Sediment and Plankton samples. Manufacturers’ instructions were followed with the following modifications: Reed and Benthos samples were homogenized with a mill (Pulverisette 9, rpm = self-optimize speed, 20 sec, Fritsch, Germany) and all other samples were subjected to a bead-beating step prior to extraction (MMX400, 2 × 2 min, f = 30 sec-1, Retsch, Germany). We added 20 µl Proteinase K (Qiagen, Netherlands) to the lysis buffer for Sediment, Reed, Biofilm, and Benthos samples, and incubated these for 1 h at 56 °C. DNA concentrations were measured using a PicoGreen assay (Invitrogen, USA). Approximately 20 ng of DNA was used as template for PCR.
DNA metabarcoding was carried out on all samples using the D1/D2 variable region of the ribosomal LSU with the eukaryotic primers NLF184cw (TACCCGCTGAAYTTAAGCATAT; modified from
Sequences were processed as briefly outlined in Suppl. material
Clipped reads were processed in Mothur following 4-5-4 SOP (
OTU count was positively correlated with read count (Pearson’s r = 0.90), thus we avoided single sample based richness estimates. For richness estimates of the habitats we applied a shared corrected Chao index (
POM and habitat types were compared by employing parametric statistics (ANOVA and Tukey honest significant difference) on “logit” transformed proportional read (read counts) or OTU (OTU counts) data. Plankton samples were excluded due to their skewing effect on the distribution caused by their low fungal proportion (including Plankton samples will still lead to a significant Kruskal-Wallis test, p<0.001, but renders PostHoc tests difficult to apply). Normality and homogeneity of variances were confirmed by Shapiro-Wilk tests and Levene’s Tests, respectively.
For all subsequent β-diversity analyses, an OTU table without singletons was used to account for noise in the data (e.g.,
As an alternative to the OTU-based RDP classification of our sequences, we performed two additional analyses with the aim to gain resolution for the taxonomic classifications of our sequences (see also Suppl. material
We recovered several Neocallimastigales (rumen fungi) sequences, as classified by SILVA NGS and also by the RDP classifier with a low probability (< 55%). We took 26 representative sequences and constructed a phylogenetic tree with an extension of the reference dataset from
A total of 54 sampling stations, representing eight habitat types, were sampled at three time points in spring of 2010 and analysed using pyrosequencing of the large ribosomal subunit (LSU) as a universal eukaryotic marker. Across all habitats, the total number of eukaryotic OTUs was 3695, as estimated using alignment-based clustering at 97%, 47% of which were singletons. The lower limit of shared estimated OTUs (shared corrected Chao index) varied considerably among habitats, with the highest values found in Sediment, Benthos and Biofilm habitats, and the lowest in Plankton and water samples (Table
Contribution of fungal phyla within each of the lakes habitat. Pie chart diameters are scaled by the contribution of fungal diversity to the total eukaryotic diversity in each habitat. Lower case letters group significantly different POM types in terms of OTU diversity and fungal reads (Tukey Post-Hoc tests, p < 0.01).
Fungal community composition was significantly structured into different habitats according to the NMDS clustering of OTUs (Figure
Habitat specificity of the fungal community in Lake Stechlin. NMDS plots based on the fungal OTU matrix (1027 OTUs). Ordination is based on Cao distances (
Only 23% of the fungal OTUs could be classified to the family or genus level, and around 20% of the sequences could not be assigned to the kingdom level at 0.8 confidence threshold (Figure
Distribution of fungal orders in Lake Stechlin habitats based on the mean percentages as determined with SILVA NGS. Fungal phyla are highlighted with brackets. Habitat codes are taken from Table
Only a small proportion of fungal sequences (0–6%) could be assigned to what we assume are forest taxa (Agaricales, Auriculariales, Boletales, Cantharellales, Gleophyllales, Hymenochaetales, Polyporales, Russulales; Suppl. material
In the following discussion, we first address methodological considerations and then discuss fungal diversity separately for the major habitats.
The occurrence of early diverging fungal lineages as well as members of the Dikarya renders a comprehensive assessment of the aquatic mycobiota challenging. This is due to the difficulty in finding a universally suitable marker (i.e., “DNA barcode”) with both sufficient coverage of evolutionary distant groups and meaningful resolution within any of the individual groups. We employed the D1 region of the LSU as a marker because of its high variability while still being conservative enough to amplify across the fungal kingdom (
All three water habitats (Pelagic, Littoral, and Above Sediment) had a low proportion of fungal sequences. Each were characterised by a predominance of Chytridiomycota. This was also the case for the habitats directly connected to processes in the open water (Plankton and Sediment). The proportion of fungal sequences in all samples classified as FPOM was significantly lower (Figure
There was a limited number of fungal taxa associated with water borne zoo- and phytoplankton samples (Plankton; > 55 µm), which presumably should represent attached or infective stages of fungi. 84–93% of these fungi belonged to Rhizophydiales, a group of well described phytoplankton parasites. By contrast, Rhizophydiales accounted for only 14–17% in the pelagic (open) water samples. This is insofar important because most microscopic studies on chytrids refer to infected algae of approximate the size of 50 µm or larger (e.g.,
The profundal sediment temperature in Lake Stechlin remains ca. 4 °C year-round while the upper sediment surface (~ 5 mm) is usually oxic. The sediment has a high water content (> 95%) and high organic matter content at the sampled sites because it receives sinking matter from pelagic organisms. Thus the sediments serve as a fungal spore bank. We therefore expected to observe elevated proportions of forest fungi that had probably blown in or been washed in as spores. The dominant fungal group was the Rhizophydiales (Chytridiomycota), as also found in the large plankton samples. Similar to their hosts, parasitic chytrids develop thick-walled resting spores (cysts), which can be found in sediment, while other parasitic species can actively infect algal resting stages in sediments (
Biofilm samples appeared to represent an intermediate fungal habitat between sediments and benthic samples by including a high diversity of early diverging lineages as well as elevated proportions of Dikarya (16–36%, Figure
In contrast to water samples, fungal sequences were dominant in CPOM (Benthos, Reed) samples and their relative proportions were significantly elevated in this POM type. Samples consisted mainly of submerged plant residues in addition to algae and benthic animals. Mitosporic ascomycetes lineages were predominant, followed by a small percentage of chytrids (mainly Cladochytridiales) and very few Basidiomycota. This appears congruent with our initial “morphotype hypothesis”. Mitosporic ascomycetes are effective plant decomposers in freshwater systems (
Fungi play an important role in the cycling of carbon and nutrients in a wide range of freshwater habitats (
Research was partially supported by the Leibniz SAW/Pakt for Research and Innovation project “MycoLink” and by an IGB Fellowship in Freshwater Science awarded to NW, HPG, and MTM. CW acknowledges a Marie Skłodowska-Curie post doc grant (660122, CRYPTRANS). This is publication 39 of the Berlin Center for Genomics in Biodiversity Research.
Accompanying data tables
Data type: PDF file
Explanation note: Two additional tables stating 1) the physicochemistry of the lake during the sampled time span, 2) the proportions of fungal-like organism and terrestrial fungi (“forest fungi”, see main text).
List of pyrosequencing primers.
Data type: PDF file
Explanation note: List of employed pyrosequencing fusion primers.
Detailed results of the clipping procedure
Data type: ZIP file
Explanation note: File archive in “zip” format. Decompress with e.g., “unzip” for inspecting individual files. Files describing the bioinformatic processing and the effects of the clipping procedure.
Krona charts of classified OTUs (RDP) and sequences (SILVA NGS).
Data type: ZIP file
Explanation note: File archive in “zip” format. Decompress with e.g., “unzip” for inspecting individual html files. Open html files with an internet browser (network connection must be enabled). Krona charts presenting the classified sequences based on a) OTUs classified by the RDP classifier, and b) based clipped reads processed by the SILVA NGS pipeline.
Rarefaction analysis
Data type: PDF file
Explanation note: Rarefaction curves based on the OTU table without OTU singletons for all eukaryotes (left panel) and for fungal OTUs (right panel).
Alternative ordinations based on binary data and taxonomy
Data type: PDF file
Explanation note: Two alternative NMDS accompanied by corresponding PERMANOVA statistics based on a) the presence/absence of OTUs (left panel) and the Jaccard index, and b) the taxonomic classification of SILVA NGS and the Cao index. The ellipses (coloured to POM type) are based on standard deviations around centroids with a confidence level of 0.95.
Comprehensive Blast analysis
Data type: PDF file
Explanation note: Blast results against the NCBI nt database (June 2015) for all eukaryotes presented in a Megan5 plot.
Phylogenetic tree of Neocallimastigales sequences
Data type: PDF file
Explanation note: Phylogenetic bayesian tree based on 10 million generations. Sequences that were classified as Neocallimastigales in the manuscript are coloured in red with the frequency of sequences in the dataset in black. The name corresponds to the representative read identifier in the dataset (accessible at ENA nucleotide archive: PRJEB14236). Node labels are posterior probabilities above 0.8. Statistically supported branches have an probability of 0.95-1.