Microb Ecol DOI 10.1007/s00248-016-0778-9
FUNGAL MICROBIOLOGY
Fungi Sailing the Arctic Ocean: Speciose Communities in North Atlantic Driftwood as Revealed by High-Throughput Amplicon Sequencing Teppo Rämä 1,2 & Marie L. Davey 3,4,5 & Jenni Nordén 3,6,7 & Rune Halvorsen 6 & Rakel Blaalid 3,8 & Geir H. Mathiassen 1 & Inger G. Alsos 1 & Håvard Kauserud 3
Received: 18 January 2016 / Accepted: 22 April 2016 # Springer Science+Business Media New York 2016
Abstract High amounts of driftwood sail across the oceans and provide habitat for organisms tolerating the rough and saline environment. Fungi have adapted to the extremely cold and saline conditions which driftwood faces in the high north. For the first time, we applied high-throughput sequencing to fungi residing in driftwood to reveal their taxonomic richness, community composition, and ecology in the North Atlantic. Using pyrosequencing of ITS2 amplicons obtained from 49 marine logs, we found 807 fungal operational taxonomic units (OTUs) based on clustering at 97 % sequence similarity cutoff level. The phylum Ascomycota comprised 74 % of the OTUs and 20 % belonged to Basidiomycota. The richness of basidiomycetes decreased with prolonged submersion in the sea, supporting the general view of ascomycetes being more extremotolerant. However, more than one fourth of the
fungal OTUs remained unassigned to any fungal class, emphasising the need for better DNA reference data from the marine habitat. Different fungal communities were detected in coniferous and deciduous logs. Our results highlight that driftwood hosts a considerably higher fungal diversity than currently known. The driftwood fungal community is not a terrestrial relic but a speciose assemblage of fungi adapted to the stressful marine environment and different kinds of wooden substrates found in it. Keywords 454 sequencing . Metabarcoding . Marine fungi . Marine wooden substrates . Diversity . Community ecology . Biosystematics
Introduction Electronic supplementary material The online version of this article (doi:10.1007/s00248-016-0778-9) contains supplementary material, which is available to authorized users. * Teppo Rämä
[email protected]
1
Tromsø University Museum, UiT The Arctic University of Norway, Tromsø, Norway
2
Marbio, UiT The Arctic University of Norway, Tromsø, Norway
3
Section for Genetics and Evolutionary Biology, Department of Biosciences, University of Oslo, Oslo, Norway
4
Department of Ecology and Natural Resource Management, Norwegian University of Life Sciences, Ås, Norway
5
University Centre in Svalbard (UNIS), Svalbard, Norway
6
Natural History Museum, University of Oslo, Oslo, Norway
7
Norwegian Institute for Nature Research, Oslo, Norway
8
Haukeland University Hospital, Bergen, Norway
Dead wood provides both the energy and habitat for a vast number of fungi, which are the primary recyclers of carbon and nutrients stored in wood [1]. Although most dead wood occurs in terrestrial habitats, a fraction ends up in the sea as driftwood, which can then be transported over long distances. Large amounts of wood from Russian Siberia drift through the polar latitudes and strand on the shore in northern coastal areas of Northwestern Europe [2, 3]. In this way, driftwood acts as a vector for many organisms, enabling long-distance dispersal of plants, animals and microorganisms [3]. Due to a cold and dry climate, the decomposition rates are low in this area, and driftwood can stay on shore for several decades, even a century [4, 5]. There, it provides habitats for specialized microorganisms, including fungi, which can tolerate the stressful conditions it presents, including high salt and sodium concentrations, low water potential, mechanical stress caused by waves, ice and sand scour, desiccation during emersion and low temperatures [6, 7].
T. Rämä et al.
The terrestrial fungal communities that initially colonise the wood obviously face a drastic change in environmental conditions if this wood ends up in the sea. Nevertheless, many fungi typically found in terrestrial habitats are frequently isolated from marine substrates [8, 9]. In addition, there is a suite of more strictly terrestrial wood-associated fungi that are apparently replaced by species adapted to the marine realm. The marine communities associated with driftwood have been poorly studied in comparison with wood-associated fungi in terrestrial ecosystems. It has been repeatedly demonstrated that driftwood fungal communities are dominated by ascomycetes [10, 11], and a broad selection of species have been isolated from marine wood substrates worldwide [12–14]. Altogether, 1112 marine fungi are known based on decades of taxonomic studies [15]. However, a far higher richness of marine fungi (at least 12,000 species) has been estimated to exist [16]. Our present knowledge of marine wood-associated fungal diversity is largely based on fruit body and culturing surveys (cf. [17, 18]). However, these methods do not detect nonfruiting and non-cultivable fungi. In addition, they are very labour intensive, which restricts the sampling effort that can be undertaken. In contrast, high-throughput sequencing (HTS) of DNA amplified from environmental samples is a powerful approach for screening fungal communities [19] with better capacity for detecting rare species, taxa present only as vegetative mycelia [20], and fungi that cannot be cultured. HTS approaches have successfully been applied to dead wood fungal communities in terrestrial habitats, e.g. [21], but they have not previously been applied to driftwood fungal communities despite presenting an excellent opportunity for profiling the whole fungal community inhabiting the marine substrate. In this study, we used 454 pyrosequencing of tag-encoded ITS2 amplicons to assess the fungal communities in stranded driftwood logs along the North Norwegian coast. We explored (i) the community richness and taxonomic diversity, (ii) whether the fungal communities are made up of only marine species or whether terrestrial wood-associated fungi are present and (iii) which environmental factors structure the fungal driftwood communities.
Materials and Methods Study Sites and Sampling Samples were collected from 47 shore-cast and two seabottom logs offshore in May–October 2010. The logs were located at 22 sites along the North Norwegian coastline between Bodø (N 67° 15′, E 015° 06′) and Vadsø (N 70° 04′, E 30° 06′) (Fig. 1). The selected shore-cast logs were situated in the sublittoral (4 logs), intertidal (34) or supralittoral (splash; 9) belts, and showed signs of submersion in the sea, as
Fig. 1 Map over the study area along the North Norwegian coast. Circles represent the study sites
indicated by cover of marine organisms such as algae and invertebrates. On three shores, four logs with dead terrestrial lichens, which indicate a shorter-lasting submersion in the sea, were sampled and included in the dataset. Sea-bottom logs that were located further away from the shore were trawled on board a vessel by use of an Agassiz trawl and sampled immediately on deck. Longitude and latitude was noted for each log. In addition, we systematically recorded other siteand substrate-specific explanatory variables (Tables S1 and S2). Six holes were drilled in each log; two 10 cm from the base, two in the middle and two 10 cm from the apical end. Individual holes were drilled from one side and almost through the log. One to two holes were drilled in each of the four sides of the log (see Appendix S1 for a schematic presentation of the sampling). Prior to each drilling event, the surface wood was removed with a sterilised knife. The drill was sterilised between logs by first cleaning it mechanically with a brush, then spraying it with ethanol that was subsequently burned off. The wood material from each log was pooled into air-tight plastic bags and kept cold on ice packs in an insulated bag, until they were frozen at −20 °C later the same day. The samples were later transferred to −40 °C for longer-term storage. DNA Extraction, PCR and 454 Pyrosequencing Each wood sample (n = 49) was thoroughly mixed, and a subsample of approximately 25 ml that included all grain sizes was taken with a sterilised teaspoon and homogenised with liquid nitrogen in a mortar. A total grinding time of 5 min was used, which resulted in a significant change in the grain size of all samples. However, samples in decay classes 1–2 (see Table S1 for the assignment of decay classes) still partly contained larger pieces of wood. The ground sub-samples were transferred into sterile 50 ml tubes and stored at −20 °C until DNA extraction. One millilitre of homogenised subsample containing all grain sizes was transferred with a sterile spatula into a 2-ml sample tube, and DNAwas extracted
Fungal Communities in North Atlantic Driftwood
using the Nucleospin Soil DNA extraction kit (MachereyNagel, Düren, Germany) according to the manufacturer’s instructions. Three mock isolations using milliQ water were included as negative controls. Samples were prepared for 454 pyrosequencing using a nested PCR approach, and all amplifications were performed on an Eppendorf Mastercycler EP Gradient thermocycler (Hamburg, Germany). In the first PCR (PCR1), the primer pair ITS1F 5′-CTTGGTCATTTAGAGGAAGTAA-3′ [22] and ITS4 5′-TCCTCCGCTTATTGATATGC-3′ [23] was used. The second PCR (PCR2) was performed using fusion primers that each included ITS3 5′-GCATCGATGAA GAACGCAGC-3′ or ITS4 [23], one of the eight unique 10base pair (bp) tags to allow downstream identification, and a 454-pyrosequencing adaptor. PCR amplifications were performed in reaction volumes of 20 μl containing 4 μl DNA template (PCR1). In PCR2, 2 μl of PCR1 product diluted 1:50 was used. Final concentrations in the PCRs were 0.16 mM dNTP mix, 0.2 μM primers, 0.4 units Finnzymes Phusion polymerase (Finnzymes, Espoo, Finland). The polymerase was added immediately prior to amplification to avoid any cleaving of the template DNA. A 20 + 15-cycle test run with eight samples failed to amplify three samples, so the total cycle number for the two PCRs was raised to 33 + 33. PCR conditions were the same in both steps, consisting of an initial 30 s denaturation step at 98 °C, followed by 33 cycles of 10 s at 98 °C, 30 s at 55 °C (annealing) and 30 s at 72 °C (extension), with a final extension step of 5 min at 72 °C. Amplification was confirmed by visualising the PCR products on 1 % agarose gels stained with GelRed Nucleic Acid Gel Stain (Biotum, Hayvard, USA). The concentration of all ITS2 amplicons was normalised using the SequalPrep Normalization Plate Kit according to the manufacturer’s instructions (Invitrogen, Paisley, UK). The amplicons were then combined in equimolar amounts to create eight libraries, each consisting of a mixture of eight uniquely tagged amplicons for a total of 64 samples. To demonstrate reproducibility, 12 samples were included in two different libraries to serve as technical replicates (see Appendix S1). Additionally, the amplicons generated from the three mock DNA isolations were included in the libraries in order to identify potential contaminants. The OTUs detected in the negatives (see Appendix S1) were later removed from the dataset. The Wizard SV Gel and PCR Clean-Up System (Promega, Madison, Wisconsin, USA) was used to clean the eight libraries according to manufacturer’s instructions. In the elution step, 50 μl of nuclease-free water was added to the samples which were then stored at −20 °C prior to sequencing. The eight libraries were sequenced in eight corresponding lanes on a 454 plate using the Genome Sequencer FLX (Roche, Basel, Switzerland) at the Norwegian Sequencing Centre, University of Oslo. The nucleotide sequence data reported are available in the DDBJ/
EMBL/GenBank databases under the accession number SRP071009. Bioinformatics The quality of the raw sequence data was controlled using the Qiime pipeline v. 1.3.0 [24]. Reads with lengths below 200 bp, above 1000 bp or an average quality score
