Anthropogenic nitrogen pollution threatens to alter the productivity and carbon storage of temperate and boreal forests. Soil nitrogen status is, for temperate and boreal forests, the dominant edaphic factor controlling forest productivity and shaping species composition . Anthropogenic nitrogen pollution has facilitated invasive species establishment in many forests of the temperate and boreal zone, and contributed to widespread species loss . There is ample evidence that moderate levels of ANP may significantly increase the net primary productivity of temperate forests .This is the core of the “nitrogen saturation hypothesis” developed by Aber et al. , and it is used by many as a theoretical framework to understand the ecological processes at work over the course of prolonged periods of ANP . According to the Nitrogen Saturation Hypothesis, the tipping point and proceeding drop in forest productivity is a result of soil acidification, and excess N inputs leaching out other essential nutrients, which then become limiting. This shift from nitrogen limitation to limitation or coSlimitation by phosphorous , potassium , or calcium due to prolonged ANP has already been observed in a number of forests in Eastern North America. Increased nitrogen availability decreases below ground carbon allocation . Decreased below ground carbon allocation involves decreased inputs of carbon into deep soil; carbon inputs which may lead to longer Sterm soil carbon retention than above ground litter . This decreased below ground carbon allocation also has profound effects on mycorrhizal relations. Anthropogenic nitrogen pollution has been shown in fertilization and deposition gradient studies to have large impacts on ectomycorrhizal communities.
N fertilization and deposition is commonly associated with a shift in ECM species composition with some species increasing in abundance while others disappear under high N fertilization or deposition . Many studies have also observed a decrease in both ECM diversity and colonization intensity with nitrogen additions.The effects of ANP on ectomycorrhizal communities may have serious negative implications for ecosystem integrity. Our knowledge of the respective ecological niches of ECM fungi is poor,vertical farming equipment but there is ample evidence that suggests discreet, nonSoverlapping niches of habitat preference and nutrient acquisition exist for some species. The potential loss of ECM species from nitrogen deposition reduces forest biodiversity and may represent a reduction in forests’ resiliency to future environmental change. ECM represent a very large sink for fixed carbon; studies have found more than 60% of recent carbon assimilation and net primary production may be allocated to ectomycorrhizal symbionts, though most estimates are closer to 15% . Ectomycorrhizal biomass may be much more recalcitrant than fine root biomass . Reductions in C allocation to ECM may significantly reduce soil C storage and serve as a positive feedback to global change. The great majority of studies on nitrogen fertilization and ECM have been conducted in conifer stands and have focused on the organic horizon, yet there is evidence that forests dominated by broad leafed angiosperms may react differently than coniferous gymnosperms to ANP . Very few studies examining the effects of ANP on ECM communities or even on ECM communities for any purpose have looked at the ECM community in the mineral soil. Over half of all ECM biomass may be in the mineral soil and ECM community composition varies significantly with soil horizon .
To examine the effect of N enrichment in broad leaved forests and in mineral and organic horizons we investigated the ECM community in a mixed broad leaved forest at the Harvard Forest Chronic N Enrichment research site. This National Science Foundation sponsored Long Term Ecological Research facility is the longest running nitrogen fertilization study in the US.Ectomycorrhizal sampling was performed in July, 2005. We randomly selected 4 5m X 5m subplots from the interior 16 subplots. We used 30cm X 2.5cm PVC pipe to collect soil cores. We sampled 4 subplots from each of the control, low N addition, and high N addition hardwood plots . Six cores were taken from each subplot.Because sampling was conducted over the course of a month during which time rooting dynamics may change, sample collection was divided evenly between different subplots and nitrogen treatments over time. Each core was divided into organic and mineral soil. There was typically a distinct border between the organic horizon and mineral horizon, and to prevent any crosscontamination 0.5S2 cm of soil at the interface between the cores was not kept. While the depth of the organic horizon varied somewhat, the organic horizon was generally around 5 cm thick, and the mineral horizon we sampled 25cm. After each core was divided, each portion of the core was washed over a 2mm sieve with distilled water. Fine roots were then collected and put into a petridish filled with water for ectomycorrhizal root tip sampling and quantification. Roots were examined under a dissecting microscope and roots that were not turgid or that appeared senescent were removed, as were any roots that were higher than second branching order. Any roots that appeared to be red maple were also removed. Red maple associates with arbuscular mycorrhizal fungi, not the ectomycorrhizal fungi that this study focused on. Fortunately, red maple roots are quite morphologically distinct as they have a much lighter color and a unique beaded morphology that makes them easy to distinguish form the ectomycorrhizal roots.
The live, ectomycorrhizal fine roots from each core were placed in a water filled petridish for community characterization and quantification. A subset of all samples was examined for percentage root length colonized before they were assessed for community composition. Petridishes were placed over a piece of transparency paper with a black grid. The dish was illuminated from underneath and the roots were examined under a dissection microscope at low magnification. Each line of the grid was followed visually and every intersection with a root was recorded as either mycorrhizal if the grid intersected the root at an ectomycorrhizal root tip or nonmycorrhizal if the grid intersected the root at a point on the root that was not an ectomycorrhiza. If the grid crossed a coralloid cluster of mycorrhizae each discrete intersection of the line with a seprate branchlet of the cluster was counted. All gridlines,what is vertical growing vertical and horizontal were counted for each dish and then the grid was rotated, the roots were mixed and the count was done again. For each sample 5 counts were done, and the percentage root length colonized for each sample was calculated as the average amount of total mycorrhizal intersections/divided by the average amount of total root intersections.The ITS region was selected for sequencing, and PCR amplification was done with the primer pair ITS 1F and ITS 4 . The ITS region is the most commonly sequenced region for fungal identification and has a large database of vouchered sequences. Its high variability makes it a suitable region for species identification . PCR products were treated with ExoSAP IT to remove primers and inhibitory salts. PCR amlicons were sequence directly without cloning using ABI Big Dye version 3.1 and pre sequencing cleanup was performed with the ABI recommended ethanol/EDTA precipitation. Single pass sequencing was conducted on an ABI 3100 16 capilary Sanger sequencing machine. Sequences were analyzed using Sequencher 4.2 . Sequences were edited to remove priming sites and poor quality portions of the sequences at the 3’ and 5’ ends. Only sequences with at least 200 clear distinct base pairs were used. Many were disqualified due to apparent contamination or cooccurrence of other fungal PCR product. Acceptable sequences were identified by comparison with the sequence database at the National Center for Bioscience Informatics using the basic local alignment search tool to identify rough phylogenetic identity. Sequences were then grouped according to these approximate phylogenetic groupings and clustered using Sequencher 4.2 with a minimum overlap of 50% and minimum sequence identity of 97%. High quality sequences were then selected from clusters for BLASTing against the NCBI database again. When matches at 97% or higher were found, the best match to a vouchered sporocarp sequence was used as the taxa name. In many cases no match at 97% or higher for a vouchered sporocarp was found and we felt that we could only reliably identify the genus of these taxa. The similarities between communities were assessed with ordination methods using the statistical software PCSORD . For ordination, each community consisted of all successfully identified ectomycorrhizal sequences from the 6 cores taken for a specific horizon in a specific subplot . The communities were compared across nitrogen treatments or between soil horizons . Mantel tests were independently conducted to assess whether the communities in different horizons or nitrogen treatments were significantly different. Differences were visualized using non parametric multi dimensional scaling . NMS is a suitable method for comparing complex microbial communities because it does not assume a normalized distribution of species or equivalent variance between communities . For NMS ordination an initial run was performed using the “medium thoroughness” default settings to identify the optimum dimensionality of the ordination. After that, the ordination was performed again . To account for unequal amounts of root tips between communities, the abundance of each taxa in each sample was divided by the total number of root tips in that sample such that the total abundance of all taxa within each sample equaled 1. Only the 45 most abundant taxa were used for community analysis. The effect of horizon and nitrogen treatment on the abundance of the 25 most common taxa was assessed using a two way ANOVA followed by post hoc comparisons using the student’s ttest or Tukey’s HSD test . The effect of horizon and nitrogen treatment on groups of taxa was assessed for monophyletic groups that had at least 3 species, each of which occurring on at least 3 subplots. For such groups of species the standardized species’ abundances were used with the additional standardization step of equalizing the total abundance of each species, so that the total abundance of each species was set to equal 1; this was done so that one very abundant species did not bias the whole genus or family. All ANOVA were done with JMP v5.0.1 .The 495 ectomycorrhizal sequences were grouped into 65 OTU’s, which we will henceforth refer to as species. They varied in abundance from 62 tips to a number of species for which only one tip was found. The rank abundance curve for the ECM community has the shape of a typical soil microbial community with a few abundant species and many rare species. The 5 most abundant species account for 42% of all tips. The most abundant genera were Lactarius, Russula, Cenococcum, Thelephora/tomentella, and Amanita, which comprised 23.2%, 22.6%, 12.5%, 8.2%, and 6.9% of the total number of tips sampled, respectively. A complete list of species, and their affinity for nitrogen treatment and horizon is detailed in tables 1 and 2. Four nitrophilic and 5 nitrophobic species could be identified amongst the most abundant species, as well as three species that had significantly higher abundance in the low N treatment . Four of the most abundant species exhibit a clear preference for the organic horizon, and 3 for the mineral horizon. There does not appear to be any interaction between horizon preference and reaction to nitrogen fertilization but the number of suitable candidate species was too low to allow significance testing. When we scale up and look at species groups at either the genus, family, or order level we see that the Agaricales , Russulaeae , and Calvulinaceae exhibit strong preference for horizon, while only the Clavulinaceae has a consistent reaction to nitrogen fertilization .Many studies have demonstrated that high levels of nitrogen addition significantly impact ectomycorrhizal communities, though very few of them have looked at deciduous stands, and none of those have looked at the mineral and organic horizons separately. In this study, we found that high levels of nitrogen fertilization have significantly altered the ectomycorrhizal community in the Harvard Forest NSF LTER Chronic N Enrichment study. There is no evidence that the community composition, diversity, or colonization intensity have been appreciably affected by “low” levels of nitrogen fertilization .