Arbuscular Mycorrhizal Community Recovers Rapidly Along a Tallgrass Restoration Chronosequence Mieke van der Heyde, Hongguang Liu, Brian Ohsowski, Miranda Hart Ecological Restoration, Volume 36, Number 2, June 2018, pp. 108-111 (Article)
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Arbuscular Mycorrhizal Community Recovers Rapidly Along a Tallgrass Restoration Chronosequence
Mieke van der Heyde (ARC Centre for Mine Site Restoration, School of Molecular and Life Science, Curtin University, Perth, Australia), Hongguang Liu (Jiangxi Institute of Soil and Water Conservation, Nanchang, China), Brian Ohsowski (Institute of Environmental Sustainability Loyola University Chicago, Chicago, IL) and Miranda Hart (corresponding author: Biology University of British Columbia, Kelowna, BC,
[email protected]).
T
allgrass prairies are one of the most devastated ecosystems in North America (Rodger 1998). Former tallgrass prairie rarely returns to its pre-agricultural state. However, efforts to restore tallgrass prairie are restricted to small isolated patches which can make restoration difficult. Studies suggest arbuscular mycorrhizal (AM) fungi may play a role in the restoration of tall grass prairie as plants as many grassland species are highly responsive to AM fungi (Hartnett and Wilson 1999, Richter and Stutz 2002, Zhang et al. 2012). AM fungi form root symbioses with most terrestrial plants and provide nutrients in exchange for carbohydrates (Smith and Read 2008). However, decades of intense agricultural practices may have denigrated the AM fungal communities (Boerner et al. 1996, Jansa et al. 2006). Such practices may select for reduced diversity of AM fungi including dominance by fungi in the Glomeraceae (Johnson et al. 1991, Helgason et al. 1998, Chagnon et al. 2013). The recovery of AM fungal propagules during tallgrass restoration has received some attention (Allison et al. 2005, Bach et al. 2010) but these studies did not consider fungal community composition. The absence of AM fungal taxa may impose a filter on the identity of plants able to establish Color version of this article is available through online subscription at: http://er.uwpress.org Supplementary materials are freely available online at: http://uwpress.wisc.edu/journals/journals/er-supplementary.html Ecological Restoration Vol. 36, No. 2, 2018 ISSN 1522-4740 E-ISSN 1543-4079 ©2018 by the Board of Regents of the University of Wisconsin System.
108 • June 2018 ECOLOGICAL RESTORATION 36:2
(Johnson et al. 2010, Busby et al. 2011). However, it is not known how much agriculture has changed AM fungal communities nor whether they are resilient post disturbance. Here we consider AM fungal community composition along a restoration chronosequence spanning 103 years. We hypothesized that the oldest restored communities would be more similar to a reference prairie remnant patch compared to more recently restored sites. Specifically, we predicted that older restoration sites would have higher fungal species richness and a fewer taxa belonging to the Glomeraceae. We sampled eight total sites representing a restoration chronosequence of 16 years plus two spontaneously regenerated sites (24 and 103 years) along the northern edge of Lake Erie in Ontario, Canada (Table 1). All restored sites had a legacy of conventional agriculture and were subsequently seeded with endemic plants: Rudbeckia hirta (black-eyed Susan), Artemisia spp., Panicum virgatum (switch grass), Elymus trachycaulus, (slender wheatgrass), Bromus kalmii (prairie brome), Schizachyrium scoparium (little bluestem), Quercus velutina (black oak), and Quercus rubra (red oak). The 24-year-old spontaneously regenerated endemic plant community was located along an abandoned railway corridor which was decommissioned in 1965. Track removal took place in 1987 after which it has remained undisturbed. The oldest site (103 years) is a conservation area previously a pine plantation until 1908 and spontaneously regenerated after that date. In September 2011, we collected five soil samples (15 mm × 100 mm) at each site from underneath a mature target plant, S. scoparium, and extracted DNA from roots using the FastDNA SPIN Kit (MP Biomedicals, Santa Ana, CA). DNA was amplified using AM fungal specific primers NS31/AM1 (Helgason et al. 1999). Five μL GoTaq Flexi buffer, 2μL MgCl2, 0.5μL dNTPs, 1μL BSA, 0.5μL NS31, 0.5μL AM1, 0.25μL Taq polymerase, and 10.25μL water was heated to 95°C for 3 min followed by 34 cycles of 30 sec at 95°C 30 s at 68.4°C 1 min 72°C and ending with 10 min at 72°C. Unique 10 base pair (bp) barcodes were attached for identification downstream. Samples were sequenced using Roche 454 pyrosequencing GS FLX+Titanium chemistry at the UBC Prostrate Center in Vancouver Canada. Sequence data was processed using QIIME (Caporaso et al. 2010) filtering for sequences between 450 and 600 bp and sorting them into Operational Taxonomic Units (OTUs)
Table 1. Site descriptions and soil properties of the eight sites representing the sampled chronosequence along the northern edge of Lake Erie in Ontario, Canada. Years indicate time since the initial restoration based on sampling in 2011. Restored sites were seeded post agriculture. The railway corridor and conservation reserve had spontaneous regeneration of a Schizachyrium scoparium (little bluestem) population in the plant community. History Tobacco then corn soybean rotation Tobacco then corn soybean rotation Tobacco then corn soybean rotation Tobacco followed by soybean Tobacco Tobacco & rye, burned several times since seeding Former railway corridor Private property before conservation reserve
at 97% similarity (UCLUST [Edgar 2010]). Taxonomy was assigned using the MaarjAM database (Opik et al. 2010) and singletons were removed to account for sequencing error. Denoising was not performed as it has been shown to affect community structure in AM fungal studies (Hart et al. 2015). After verifying no correlation between abundance of taxa and sequence depth the OTU table was normalized using the cumulative sum scaling method in the R package “metagenomSeq” (R Foundation, Vienna Austria). Changes in fungal species richness and community composition along the chronosequence were assessed using Duncan’s multiple range tests in the R package “agricolae.” (de Mendiburu 2016). Differences among samples were assessed using the Bray-Curtis dissimilarity metric (Bray and Curtis 1957). We tested differences among restoration years using a one-way permutational multivariate analysis of variance with 999 permutations (PerMANOVA [Anderson 2001]). We ran a follow-up pairwise PerMANOVA to determine which sites differed from one another. In summer 2015 we collected samples from eight sites (four replicates per site) for soil chemical analysis. These samples were sent to BC Ministry of Environment Technical Services Lab (Victoria British Columbia): soil phosphorous was extracted using the Bray-P1 method (Bray et al. 1945) and quantified using a spectrophotometer. Total carbon and nitrogen were quantified using combustion elemental analysis. Contrary to our prediction, species richness and diversity did not increase along the chronosequence (Figure 1). Instead, fungal species richness at the 0-year-old site (22.2 ± 1.46 OTUs) was not significantly different from the oldest prairie remnant site (27 ± 1.05 OTUs; p > 0.05). Other grassland studies using molecular identification have also found similar species richness among different sties (Lekberg et al. 2012). In total 65 Glomeromycotan OTUs were identified: three Acaulosporaceae, two Archaesporaceae, two Diversisporaceae, five Gigasporaceae, and 53 Glomeraceae. Some were rare (e.g., 11 were found in fewer than 10% of samples) but 16 OTUs were found in over 75% of the samples.
Restoration Practice Seed Mix Seed Mix Seed Mix Seed Mix Seed Mix Seed Mix Spontaneous Regeneration Spontaneous Regeneration
OTU richness
Site Age 0y 1y 2y 3y 5y 16y 24y 103y
45 40 35 30 25 20 15 10 5 0
P (mg/kg) 223.5 329.6 274.9 264.2 263.0 219.9 11.4 N/A
C (%) 0.712 0.811 1.024 0.615 0.689 0.678 1.562 N/A
N (%) 0.051 0.057 0.068 0.043 0.051 0.047 0.086 N/A
a bc c
0y
b
bc
bc
bc
d
1y 2y 3y 5y 16y 24y 103y Restoration chronosequence
Figure 1. Normalized AM fungal species richness (means ± SE, n = 5) along the restoration chronosequence along the northern edge of Lake Erie in Ontario, Canada. Shannon diversity showed the same pattern as richness. Different lowercase letters indicate significant differences among sites in OTU richness (α = 0.05).
The proportion of Glomeraceae did not decrease along the chronosequence as expected (Figure 2). The highest proportion of Glomeraceae were found in the 2-year (97.1% Glomeraceae) and 103-year sites (96.2% Glomeraceae) while the 1-year-old site had the lowest proportion (77.2% Glomeraceae). Fungal communities differed among sites (PerMANOVA; p = 0.001, Supplementary Figure 1). AM fungal communities associated with 2, 3, and 5-year sites were indistinguishable from the spontaneously regenerated 103-year site (p < 0.05) but different from communities associated with 0, 1, and 16-year restorations (p < 0.05; Table 2). The 1-year site, which had the lowest species richness diversity and proportion of Glomeraceae, was significantly different from all other sites (p < 0.05; Table 2). The 1-year site also had the highest Phosphorous (P) availability; levels at this site were more than 50 mg/kg higher than the site with the next highest p levels (Table 1). June 2018 ECOLOGICAL RESTORATION 36:2 • 109
100%
a
a
b
a
a
a
a
alter community composition although this usually results in increases in the proportion of Glomeraceae (reviewed in Jansa et al. 2006). The lack of directional response may also be due to the level of land use intensity (Hijri et al. 2006). The sites in our study were farmed using crop rotation or biannual fallow periods which may have allowed fungal communities to maintain high levels of diversity. More extreme forms of land use may have resulted in a more pronounced recovery lag. Furthermore, our results may also be limited to small scale disturbance; it is possible that our sites recovered quickly because they did not experience dispersal limitation and surrounding vegetation were able to maintain a large species pool of fungi. However, without a better knowledge of AM fungal dispersal limitations this remains to be seen. For this study tallgrass prairie restoration is was not hindered by AM fungal community recovery post-agricultural disturbance. This result is surprising given the large body of literature showing deleterious effects of agriculture on soil fungal communities. Such findings suggest that the use of AM fungal inoculum, which is standard practice in many restoration protocols (Ohsowski et al. 2012), may be unnecessary and represent an unnecessary expense. However, our results may be less relevant for severe soil disturbances, such as mining where top soil (and AM fungal spore bank) is removed. In such cases the use of AM fungal inoculants may represent the best restoration strategy.
Glomeraceae
a
Gigasporaceae
Proportion
Diversisporaceae Archaesporaceae Acaulosporaceae
50%
0%
0y
1y
2y
3y 5y 16y 24y 103y Chronosequence
Figure 2. Average AM fungal community composition at the family level along the chronosequence based on normalized data. Different lowercase letters indicate significant differences among sites in the proportion of Glomeraceae (square root-log transformed, α = 0.05).
Our results indicate that agriculture had little consequence for AM fungal communities over the long term. Whether these communities were resilient to disturbance or whether agriculture had little effect on natural communities is not clear. These results contradict studies which have found lasting effects of agriculture on fungal community composition (Johnson et al. 1991, Bach et al. 2010, Stover et al. 2012). For example, Johnson et al. (1991) identified “early” successional and “late” successional AM fungal species in an old field but their results were informed by spore identification which may not have reflected the entire fungal community. While our sites were different compositionally, these differences were not directional similar to Bach et al. (2010). The youngest site in our study (1 year) was the most different from other sites. This may be related to the phosphorous levels which were the highest at this site (Table 1). Phosphorous addition is known to reduce diversity and
Acknowledgements MH was funded by NSERC Discovery. Thank you to the Nature Conservancy of Canada for allowing the conduction of this field research on their property and The Ontario Aggregate Research Corporation (TOARC) funding and resource support. We would also like to thank Dr. Nicola Day for sampling assistance in the field.
Table 2. Pairwise PerMANOVA results comparing AMF communities along the chronosequence. * indicate significantly (p < 0.05) different communities among chronosequences. 0y
1y
2y
3y
5y
16y
24y
2
1y
R = 0.262* p = 0.004*
2y
R2 = 0.269* p = 0.008*
R2 = 0.447* p = 0.005*
3y
R2 = 0.318* p = 0.010*
R2 = 0.400* p = 0.014*
R2 = 0.187 p = 0.095
5y
R2 = 0.275* p = 0.010*
R2 = 0.425* p = 0.011*
R2 = 0.080 P = 0.680
R2 = 0.115 p = 0.574
16y
R2 = 0.349* p = 0.006*
R2 = 0.429* p = 0.010*
R2 = 0.199 p = 0.066
R2 = 0.284 p = 0.064
R2 = 0.244* p = 0.023*
24y
R2 = 0.168 p = 0.098
R2 =0.339* p = 0.013*
R2 =0.222* p = 0.009*
R2 =0.231* p = 0.006*
R2 = 0.215* p = 0.013*
R2 = 0.307* p = 0.007*
103y
R2 = 0.288* p = 0.010*
R2 = 0.452* p = 0.007*
R2 = 0.099 p = 0.559
R2 = 0.210 p = 0.062
R2 = 0.132 p = 0.330
R2 = 0.260* p = 0.016*
110 • June 2018 ECOLOGICAL RESTORATION 36:2
R2 = 0.254* p = 0.012*
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Evaluation of Clopyralid and Additives for Coronilla varia Suppression in a Remnant Prairie (Wisconsin)
Craig A. Annen (Corresponding author: Integrated Restorations, LLC, Belleville, WI,
[email protected]), Jared A. Bland (Integrated Restorations, LLC, Belleville, WI), Amanda J. Budyak (Integrated Restorations, LLC, Belleville, WI) and Christopher D. Knief (Integrated Restorations, LLC, Belleville, WI).
C
oronilla varia (crown vetch) is a perennial legume that has been extensively planted for erosion control along roadsides since the 1950s, and has become a widespread invasive species in grasslands throughout the Midwestern United States. Coronilla varia spreads through June 2018 ECOLOGICAL RESTORATION 36:2 • 111
