SRP005391 Lekberg-454-melby Chaos belowground - 454 sequencing reveals strong priority effects and high resilience within arbuscular mycorrhizal fungal communities following disturbance Summary 1. Differences in life-history strategies have been shown to drive the abundance of plant species in various environments, but whether or not similar processes operate on associated soil microbial communities is less known. Based on the assumed trade-off between disturbance tolerance and competiveness, we hypothesized that a severe disturbance applied within a semi-natural grassland would shift the arbuscular mycorrhizal (AM) fungal community from dominant competitive fungi to fungi that are normally rare and possibly disturbance tolerant. 2. We used 454-sequencing of the large subunit rDNA region to characterize fungal communities in Plantago lanceolata grown in the field for four months and exposed to no disturbance, or severe disturbance where fungi from undisturbed soil were either allowed or prevented from re-colonizing the disturbed area. To identify the AM fungi that could potentially colonize the experimental plants, we also analyzed roots from adjacent, undisturbed vegetation. 3. We found 31 fungal operational taxonomic units (OTUs) distributed across five fungal families. Contrary to our expectations, disturbance did not significantly alter the community composition. Instead, OTU abundances were positively correlated across treatments; i.e. OTUs that were common in undisturbed soil were also common after the severe disturbance, which suggests a high resilience of resident fungi. However, the distribution of OTUs within and between plots was largely unpredictable and could only to a small extent be explained by differences in plant community composition. Instead, pair-wise scatter plots showed that the four most abundant OTUs were seldom abundant in the same sample, which could be indicative of competition. 4. Synthesis. Our results suggest that factors other than disturbance drive the relative abundance of OTUs in this grassland and question the long-held assumption that communities shift in a predictable manner after a disturbance event. The reassembly of fungal communities after disturbance in this grassland appeared to be driven by small-scale spatial distributions and strong priority effects among AM fungi possessing a similar – and high – degree of disturbance tolerance. Key-words: Arbuscular mycorrhizal fungi, community composition, disturbance, life history strategy, 454 sequencing, semi-natural grassland. Lekberg-454-melby-disturbance study 2009 Materials and methods FIELD SITE AND EXPERIMENTAL DESIGN The study was conducted within a grassland on the north coast of Zeeland, Denmark (56°01´N-11°59´E), which hosts a diverse grass and forb community (Rosendahl & Stuckenbrock 2004). On 2 July, 2008, 11 plots were selected within a 100 x 50 m area, and two disturbance treatments were applied within each plot; one that allowed re-colonization by surrounding AM fungi post disturbance (D), and another where a plastic barrier prevented re-colonization, and where colonization could only occur from fungi that survived the disturbance (DB). For both disturbance treatments, all shoots were cut off and removed from a 30 cm diameter area, and roots and soil were excavated with a trowel down to a depth of 20 cm. Roots were cut into 2 cm pieces and the soil was passed through a 10 mm sieve, and in order not to drastically change the depth distribution of AM fungi, the upper 10 cm of roots and soil were kept separate from the lower 10 cm. In the DB treatment, a 25 cm diameter plastic bucket was inserted into the hole before the soil was put back, which separated the disturbed area from the non-disturbed soil. The bottom of the bucket had been cut off at a depth of 20 cm to allow free drainage. This was considered to be sufficiently deep to prevent colonization from neighboring, undisturbed mycelium, because it was well below the rooting depth of most plants and also corresponded with the depth where large rocks started to appear. Two one-month old Plantago lanceolata seedlings were planted into each disturbed area immediately following the disturbance. Two plants were also planted into native soil in each plot as controls (C), and care was taken to disturb the soil and surrounding vegetation as little as possible during planting. All three treatments were within 1 m of each other. P. lanceolata was chosen because it occurs naturally in the grassland, and the seedlings had been grown from seeds (kindly provided by Dr. Pål Axel Olsson) in heat sterilized field soil under greenhouse ambient conditions and watered as needed with tap water. The non-mycorrhizal status of three randomly selected seedlings was confirmed prior to outplanting by examining the roots under a dissecting microscope after staining with trypan blue (Brundrett et al. 1996). Due to a persistent drought in July, the transplanted seedlings were watered twice weekly until 1 August, when the drought ceased. We surveyed the plant community within each plot non-destructively on 7 July by estimating the percentage coverage of individual plant species within a 1 m2 area that included the experimental plants. HARVEST One plant per treatment was harvested on 21 August by disturbing the surrounding area as little as possible. Plants and rhizosphere soil were placed in individual plastic bags and transported back to the laboratory and stored in +4°C and processed within 48 h. The rhizosphere soil from the C, D, and DB treatments were passed through a 2 mm sieve and frozen awaiting analyses. Shoots were separated from the roots and dried in 60 °C to constant weight and dry weight was recorded. Roots were washed free of adhering soil, cut into 2 cm pieces and thoroughly mixed in water. A small sub-sample was taken and stained in trypan blue as above for analyses of AM colonization using the gridline intersect method (McGonigle et al. 1990). Coarse mycorrhizal hyphae were scored separately from fine endophytes (FE). Both types were aseptate and stained blue, but the FE hyphae were much finer, displayed an irregular and knobby growth and a fan-shaped growth pattern within the root. The remaining roots were freeze dried and kept at -20 °C awaiting DNA extraction. On 18 November, 4.5 months after planting, the remaining plants were harvested and processed as outlined above. In addition to the C, DB and D treatments, native P. lanceolata that were growing in, or in close proximity (<3m), were harvested to determine if the AM fungal communities in experimental plants were similar to those of native plants. We found native P. lanceolata plants within, or adjacent to, four plots in our study, and these samples will hereafter be referred to as “N” as in native. Furthermore, 10 mycorrhizal plants of various species that were common within each plot were harvested and pooled to identify the AM fungal OTUs that were present and could colonize the experimental plants. These samples will hereafter be referred to as “S” for surrounding plants. We considered this approach appropriate given that no host preference has been documented in this site previously (Stukenbrock & Rosendahl 2005). Soil samples from the second harvest were analyzed for available P, NO3 and NH4 after extractions (1:10, w:v) in 0.01 M CaCl2 (Houba et al. 2000). Soil pH and electrical conductivity (EC) was measured in water (1:1, v:v), and soil organic matter (SOM) was determined based on loss of ignition. Soil samples from C and D treatments within four randomly selected plots were also analyzed from the first harvest for comparisons. DNA EXTRACTIONS AND MOLECULAR ANALYSES Due to the low AM colonization observed in the transplanted seedlings at the first harvest (<10%), DNA was only extracted from roots from the second harvest. DNA was extracted from approximately 25 mg of freeze dried, milled roots using chloroform and isopropanol according to Gardes and Bruns (1993) and stored in TE buffer at -20°C. Non-mycorrhizal pea roots were included as a negative control to ensure that no contamination occurred during the extraction. DNA was extracted from a total of 48 samples, including 11 D, DB, C, S, and 4 N. Samples were prepared for 454 sequencing in a two-step PCR procedure. In PCR1, the 3’ end of the large ribosomal subunit (LSU) was amplified with eukaryotic primers 0061 (van Tuinen et al. 1998) and LR5 (Vilgalys & Hester 1990). The PCR conditions included 30 cycles with an annealing temperature of 55 °C, resulting in PCR products of 900 bp. PCR products were purified by membrane filtration (Macherey-Nagel, Düren, Germany) prior to PCR2. In PCR2, target DNA was amplified with a novel forward primer glo454 (3’-TGAAAGGGAAACGATTGAAGT-5’) in combination with NDL22 (van Tuinen et al. 1998), which targets the nLSU-D2 region that was recently suggested to be suitable for high-throughput sequencing (Stockinger, Kruger & Schüßler 2010). This primer combination amplifies all known AM fungal clades, including the deep branching Paraglomus, but may also amplify some non-AM fungi, especially basidiomycetes. A preliminary cloning and sequencing of glo454-NDL22 products from the extracted root samples showed that sequences of AM fungal origin always dominated the amplicon pools (results not shown). The glo454 primer sequence included the 454 linker A while NDL22 included linker B (Margulies et al. 2005), which means that amplicons were sequenced from the glo454 primer end. Additionally, three different 10-base tags were included between linker A and the glo454 primer (Dowd et al. 2008), allowing all 48 samples to be distributed among sixteen 454-sectors with three samples in each sector. Secondary PCR included only 10 cycles with a 56 °C annealing temperature. Secondary PCR products were about 350 bp long and bands were gel purified using the QIAEX II kit (Qiagen, Hilden, Germany). The amplified fragments with adapters and tags were quantified using a Qubit™ fluorometer (Invitrogen) and qPCR (Mx-3000, Stratagene) as previously described (Larsen et al. 2010). The sample amplicons were mixed in equal amounts (5 x 107 copies per µl) to ensure equal representation of each sample. The three tag sequences used were tag1: TGTACCGATG, tag2: CTCACTTAGG and tag3: CGCCGTTATA. A two-region 454 sequencing run was performed on a 70_75 GS PicoTiterPlate (PTP) using a GS FLX pyrosequencing system according to manufacturer instructions (Roche). Sorting and trimming of sequences >150 bp was done by the Pipeline Initial Process at the RDP's Pyrosequencing Pipeline (http://rdp.cme.msu.edu/; Cole, J.R., Wang, Q., Cardenas, E., Fish, J., Chai, B., Farris, R.J., Kulam-Syed-Mohideen, A.S., McGarrell, D.M., Marsh, T., Garrity, G.M.) and Tiedje (2009).