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1 USING STABLE ISOTOPES OF NITROGEN ( 15 N) TO EXAMINE THE SOURCES AND PATHWAYS OF FOREST NITROGEN CYCLES: A GLOBAL META ANALYSIS AND FIELD STUDIES IN ALASKAN BLACK SPRUCE FOREST By JORDAN RICHARD MAYOR A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010
2 2010 Jordan Richard Mayor
3 To all the ecologically important yet grossly understudied fungi of the world and my lovely fianc e whom has weathered life in Florida during this foolhardy endeavor.
4 ACKNOWLEDGMENTS I would first like to acknowledge the following people for assisting me at the University of Florida: my committee chair and advisor, E.A.G. Ted Schuur, for being supportive in my pursuit of an independent research track and for entertaining so m any impetuous ideas; Grace Crummer for steadfast laboratory assistance and management through thick and thin; the infamous Jason and Cathy Curtis for assistance modifying the mass spectrometer a feat that easily saved a year of my life; Andrea Albertin f or assisting me with the adoption of the bacterial denitrifier technique; Hak n Wallander for discussing hyphal ingrowth bags; Erland B th for personally showing me PLFA extraction techniques in Lund, Sweden; Erik Hobbie, Ayato Kohzu, and Bernd Zeller for generously provid ing their previously published raw data on fungal isotopes; Paulo Brando and Reinhold Kliegl for providing statistical a dvice on mixed effect models; Jason Vogel and Juan Posada for early discussions on dissertation topics; and last but n ot least, Hollie Hall for providing unfailing support through it all. With regards to work in Guyana, I would like to specifically acknowledge; the invaluable field assistance provided by the endearing Patamona Amerindians, Cathie Aime 's assistance with fu ngal identifications, Terry Henkel's steadfast support, the Gu yanese undergraduate researcher Clydecia McClure and graduate and undergraduate students from Humboldt State University. Work in Guyana was made possible by: the Guyana Environmental Protectio n Agency r esearch and export permits funding from the Working Forests in the Tropics Graduate Research Award supported by the National Science Foundation (DGE
5 0221599) to me, the National Geographic Society Research and Exploration Grant to Terry Henkel, and Mellon Foundation funds to Ted Schuur. With regards to work in Alaska I would like to specifically acknowledge the expert field assistance by Martin Lavoie and Emily Tissier, as well as the countless hours of lab assistance back at UF by Dominique Ardu ra, Rady Ho, Dat Nyguen, and Rachel Rubin. I would also like to thank Steve Allison and Kathleen Treseder for providing sporocarp 15 N data from the fertilized plots near Delta Junction. Work in Alaska was made possible by f unding from the National Science Foundation Doctoral Dissertation Award (DGE 0221599), the Forest Fungal Ecology Research Award of the Mycological Society of America, the International Association of GeoChemistry Student Research Grant, the Riewald Olowo U niversity of Florida Graduate Research Award, and multiple UF Graduate Student Council Travel Awards to me; DOE and NSF funding to Ted Schuur; and, the support offered by the Ecosystem Ecology Laboratory of Terry Chapin and the Bonanza Creek Long Term Ecol ogical Research site to Ted Schuur and Michelle Mack that supported the logistics of my research while living in Alaska.
6 TABLE OF CONTENTS page ACKNOWLEDG MENTS .................................................................................................. 4LIST OF TABLES ............................................................................................................ 8LIST OF FI GURES .......................................................................................................... 9ABSTRACT ................................................................................................................... 10 CHAPTER 1 INTRODUCTION AND OVERVIEW OF DISSE RTATION ...................................... 122 ELUCIDATING THE NUTRITIONAL DYNAMICS OF FUNGI USING STABLE ISOTO PES ............................................................................................................. 17Abstract ................................................................................................................... 17Introducti on ............................................................................................................. 17Methods .................................................................................................................. 21Data Asse mbly ................................................................................................. 21Guyana Field Site and Sa mple Proce ssing ...................................................... 22Data Anal yses .................................................................................................. 23Results .................................................................................................................... 26Discussio n .............................................................................................................. 29Implications of t he Global Pa ttern ..................................................................... 29Climatic Influence on Sporocarp 15N ............................................................... 33Climatic Influence on Sporocarp 13C ............................................................... 34Predictions of F ungal Ecol ogy .......................................................................... 35Summary ................................................................................................................ 383 SOURCE VS. PATHWAY: 15N PATTERNS IN CENTRAL ALASKAN BLACK SPRUCE FOREST REFLECT HIGH DEPENDENCY ON ECTOMYCORRHIZAL-DERIVED ORGANIC NI TROGEN ..................................... 46Abstract ................................................................................................................... 46Introducti on ............................................................................................................. 47Methods .................................................................................................................. 50Experimental Design ........................................................................................ 50Field and Laborat ory Anal yses ......................................................................... 51Statistical Analyses .......................................................................................... 56Isotope Mass Balance ...................................................................................... 58Results .................................................................................................................... 59Tree Biomass and Soil Fertility in Black Spruce Stands ................................... 59Black Spruce Foliar 15N, %N, and %P Patterns Across the Landscape ......... 60
7 Sporocarp 15N and Fungal Bi omass ............................................................... 6115N Patterns of Soil N Forms Across the Landscape ...................................... 62Modeling Fractionation of 15N in ECM Fungi and Transfer to Black Spruce ..... 63Discussio n .............................................................................................................. 65Black Spruce Elem ental Patte rns ..................................................................... 65Fungal Biomass and Sporocarp 15N values .................................................... 6915N Patterns of Soil N Moieties Across the L andscape ................................... 72Modeling N Transfer to Black Sp ruce ............................................................... 75Conclusi ons ...................................................................................................... 764 DETECTING ALTERED NITROGEN CYCLES IN BLACK SPRUCE FOREST FOLLOWING FERTILZATION USING SOIL, PLANT, AND FUNGAL 15N VALUES .................................................................................................................. 84Abstract ................................................................................................................... 84Introducti on ............................................................................................................. 85Methods .................................................................................................................. 90Site Descrip tion ................................................................................................ 90Field Sampling and La boratory A nalyses ......................................................... 92Statistical Analyses .......................................................................................... 94Mass Balance 15N Mixing M odels ..................................................................... 94Results .................................................................................................................... 96Responses of Black Spruce Element al Content to Fe rtilization ........................ 96Responses of Fungal Biomass and Sporocarp 15N to Fertilization ................. 97Soil Fertility Metrics .......................................................................................... 97Soil 15N Values ............................................................................................... 98Mass Balance Mixing Results ........................................................................... 98Discussio n ............................................................................................................ 102Effects of Fertilization on Soil Fertility and Soil 15N Values ........................... 102Effects of Fertilization on Plant %N, %P, and 15N ......................................... 106Effects of Fertilization on F ungal Biomass, Ingrowth, and 15N ...................... 109Fertilization Induced Decline in Bl ack Spruce Depend ency on ECM .............. 110Conclusion and Ecosystem Implicati ons ............................................................... 113APPENDIX A COLLECTOR BASED MI SSCLASSIFICATIONS OF FUNGI ............................... 121B CLASSIFICATION OF FUNGI WITH UNKNOWN ECOLOGY .............................. 123C ISOTOPE MASS BALANCE MIXING RESU LTS .................................................. 124LIST OF REFE RENCES ............................................................................................. 126BIOGRAPHICAL SK ETCH .......................................................................................... 151
8 LIST OF TABLES Table page 2-1 Summary of isotopic, climatic, and ecological data by study and site. ................ 40 2-2 Competing linear mixe d model results. ............................................................... 42 3-1 Black spruce and fungal elemental contents and biomass across 31 central Alaskan fore sts. .................................................................................................. 78 3-2 Soil fertility measurem ents made across 31 central Alaskan black spruce forests. ................................................................................................................ 78 3-3 High-ranking multiple regression models from 31 central Alaskan black spruce fo rest. ...................................................................................................... 79 4-1 Soil characteristics across bla ck spruce fertilization treatments SE. Each treatment corresponds to four plots .................................................................. 114 4-2 Mass balance mixing results to estimate the propor tional dependence of black spruce on ECM-derived N and the end member sources of N used across fertilization treatments. .......................................................................... 115 A-2 Collector based misclassifications of fungi ....................................................... 121 B-2 Classification of f ungi with unknown (unk) ec ological ro les .............................. 123 C-3 Supplementary table of mass balance mixing model results on a plot-by-plot basis ................................................................................................................. 124
9 LIST OF FIGURES Figure page 2-1 Dual isotope graphs of ectomycorrhizal (ECM), saprotrophic (SAP), and fungi of unknown (UNK) ecological role collect ed from 32 sites around the world ...... 43 2-2 Comparison of the mean 13C and 15N of ectomycorrhizal (ECM) and saprotrophic (SAP) fungi from each site ............................................................. 44 2-3 Regressions of site mean fungal 13C and 15N values with mean annual temperature, mean annual precip itation, and latitude ......................................... 45 3-1 Average elemental content (%) of full sun foliage ( N = 3) collected from black spruce (Picea mariana ) trees in 31 plots in central Al aska ................................. 80 3-2 Average ( SE) black spruce full s un needle, fine root, bulk organic soil, and ectomycorrhizal sporocarp 15N values across 31 plots in central Alaska. ......... 81 3-3 Relation among organic soil DO N content and fungal sporocarp 15N values .... 82 3-4 Relation among C:N ratios of the organic black spruce soils with the Phospholipid Fatty Acid (PLFA) based metrics of fungal biomass. ..................... 82 3-5 Mean ( N = 3, SE) soil N 15N values from dissolved organic N (DON), ammonium (NH4 +), and bulk organic soils across 31 plots in central Alaska. ..... 83 4-1 Foliar 15N values from black spruce trees were strongly correlated with %N across fertilizati on treatment s ........................................................................... 116 4-2 Black spruce and fungal response SE to five years of fertilization with nitrogen (N), phosphorus (P), both (NP), or none (C) ....................................... 117 4-3 Responses of soil N 15N values to five years of fertilization with ammonium nitrate (N), orthophosphate (P), both (N + P), or none (control) ....................... 118 4-4 Model of the patterns of N fluxes across our treatment types as informed by mass balance mixi ng models............................................................................ 119 4-5 Correlations between black spruce proportional dependence on ECM derived N () and other isotopic components in ex perimentally fertilized black spruce forest in centra l Alaska ..................................................................................... 120
10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy USING STABLE ISOTOPES OF NITROGEN (!15N) TO EXAMINE THE SOURCES AND PATHWAYS OF FOREST NITROGEN CYCLES: A GLOBAL META-ANALYSIS AND FIELD STUDIES IN ALASKAN BLACK SPRUCE FOREST By Jordan Richard Mayor December 2010 Chair: Edward A.G. Schuur Major: Botany Ecosystem ecologists are challenged by both the scal e and complexity of their discipline. Consistent methodologies and integrative metrics can ameliorate such challenges. Stable isotope ratios of nitrogen ( !15N) offer themselves as unique timeintegrated proxies of numerous biophysical processes. The key N cycling information contained in ecosystem !15N values, however, is obscured by multiple competing hypotheses. Here, I examine patterns in !15N values in fungi from around the world and in multiple ecosystem components along the soil -fungi -plant continuum in boreal black spruce forest of central Alaska. My objective was to determine both the causes of underlying !15N variability and the utility of these measurements. By combining previously published isotopic data with a novel dataset collected in poorly studied tropical rainforest, I was able to demonstrate the universal ability of dual isotope (!15N and !13C) datasets to discriminate ectomycorrhizal from saprotrophic fungi in >90% of 813 samples of fungi. Furthermore, I demonstrated that the isotope values
11 could be partially predicted by climate in a manner similar to that previously demonstrated in plants and soils. In Alaska I focused on severely N-limited forests dominated by a single species of ectomycorrhizal tree. By examini ng 31 plots varying widely in foliar 15N values, topography, and stand density, I aimed to under stand the fundamental controls over 15N variability in plants. Using 16 experim entally fertilized plots I examined how changes to soil nutrient fertility can alter 15N values, N sources, and pathways of cycling. Using a highly sensitive bacteri al denitrifier technique I overcame a methodological impasse of previous wo rk and demonstrated that black spruce 15N values were a product of the interaction of soil fertility and ectomycorrhizal activity, not merely a reflection of source N 15N values. In the fertilized plots I demonstrated that only the combination of N and phos phorus fertilization leads to a significant reduction of black spruce reliance upon ectomycorrhizal -derived N from 82 to 46% of total N nutrition. Phosphorus fertilization, in particu lar, also led to an unusual and previously undocumented response in soil N cycling.
12 CHAPTER 1 INTRODUCTION AND OVE RVIEW OF DISSERTATIO N My PhD dissertation work has sought to push the boundaries of stable isotope applications in ecology and ecosystem science by defining the ecological roles of fungi and through examining the function of boreal ecosystems from the perspective of N cycling. An advantage of my approach is that it is multidisciplinary and multivariate. For instance, I have used advanced analytical and chemistry techniques to examine stable isotope ratios, microbial biomass, and elemental abundances in a diversity of organisms and soils. These techniques involved extensive methods development, instrument modifications, and visiting of foreign laboratories. I have also applied modern multivariate and information theoretic statistical techniques to address global and local hypo theses. Lastly, my work has involved taxonomic ID and physiological knowledge of fungi and plants in both tropical and boreal ecosystems. Ecosystem ecologists are challenged by both the scale and complexity of their discipline. Because of difficulties i n large scale research, consistent methods and metrics are needed, and because of the complexity of ecosystem processes, integrators of space and time are needed. I chose to use stable N isotope ratios ( 15 N) as a proxy metri c of the form and pathway of the N cycle because it is naturally occurring, readily measurable, interacts strongly with biological organisms, and N is one of the primary mineral nutrients limiting ecosystem productivity. There have been numerous publicatio ns detailing 15 N patterns across a diverse range of organisms, soils, and environments. However, this body of literature, much of which is very recent, contains numerous examples where mechanistic interpretation of ecosystem 15 N patterns is muddied by confounding underlying causes. A major
13 difficulty has been making replicate 15 N measurements of bioavailable soil N pools at low field concentrations due to analytic al limitations. Adoption of the bacterial denitrifier method at UF was therefore one of the major contributions of my PhD research as it allowed me to overcome this methodological impasse and test previously intractable hypotheses. Fundamentally, my rese arch is an attempt at pinpointing specific causes of plant and fungal 15 N variability so that ecologists may feel confident in using it as a time integrated proxy of the form and pathway of N cycling. In Chapter 2 I used a m eta analytical approach to assign ecological roles to numerous species of fungi collected from around the world. The isotopic differences among fungi have been described from single sites again and again but the universality of the pattern was not explore d, particularly in tropical forests. By combining previously published isotopic data with a novel dataset collected in tropical rainforests of Guyana, I was able to demonstrate universal patterns of stable isotope values and relate this back to general ec ological functions and climatic controls over biogeochemical cycles. Why would an ecologist want to know the ecological roles of fungi? The simplest answer is because m ycorrhizal and saprotrophic fungi exist at the biogeochemical interface between soils and plants. They are essential to terrestrial element cycling due to their uptake of mineral nutrients and decomposition of detritus. Therefore, understanding their ecology is vital to the understanding of ecosystem function be it decomposition, soil r espiration, or mineral nutrient cycling. Furthermore, ectomycorrhizal fungi in particular have been shown to fractionate heavily against the stable isotope of N, 15 N; demonstrating the universality of this is informative to future modeling efforts and pre dictive efforts regarding N cycling.
14 My approach toward these goals was to use a discr iminant analysi s of stable isotope ratio s of nitrogen ( 15 N) and carbon ( 13 C) from 813 fungi across 23 sites. I was able to verif y collector based categorizations of individual sporocarps as either ectomycorrhizal (ECM) or saprotrophic (SAP) in >91% of the fungi, a nd provide probabilistic assignments for an additional 27 fungi of unknown ecological role s As sites ranged from boreal tundra to tropical rainforest, I was able to show that fungal 13 C and 15 N values could be roughly predicted by climate or latitude mirroring what was previously sho wn in plant and soil analyses. These results are applicable to biogeochemists, evolutionary mycologists, and ecosystem ecologists. This chapter was published in Ecology Letters as a Synthesis and Review Article and has initiated several collaborative opp ortunities. Chapter 3 continues my approach to understanding variability in, and applications of, 15 N now in the context of a severely N limited boreal black spruce forest of central Alaska where the influence of ECM fungi on 15 N patterns are expected to be particularly strong. The objectives of this work were to understand causes in black spruce 15 N variability with the belief that the large variability observed was not simply due to variation in the source N, as hypothesized elsewhere, but rather dependency on ECM fungi for N nutrition. I chose 31 stands that varied widely in foliar 15 N values, stand biomass, soil fertility, and topographic position with the g oal of differentiating the underlying causes of ecosystem 15 N variability across the range of black spruce growth conditions Multiple regressions were evaluated to explain variability in both elemental and biomass patterns of plants and fun gi using the information theoretic approach of model
15 selection. Both f oliar N and P content s were confirmed to reflect soil fertility; particularly soil dissolved organic N ( DON ) and resin exchangeable phosphorus B lack spruce foliar 15 N values were bes t explained by a multidimensional soil fertility metric (principle component) either alone or in combination with the 5 N values of soil N forms. Foliar and root 15 N values covaried, as expected, yet the magnitude of the difference converged under the least 15 N depleted conditions calling into question many previous modeling assumptions that assumed a constant difference Fungal sporocarp 5 N values were negatively correlated with the DON content of soils, but not negatively correlated with black sp ruce 15 N values as theoretically expected. In situ measurements of f ungal biomass increased with C:N ratio s of organic soil reflected an interplay between soil N content and plant C allocation S oil N 15 N values, when incorporated in to a series of mas s balance isotope mixing models, indicat ed that black spruce is highly dependent on ECM derived N (86 98% of to tal N nutrition) despite variability in foliar 15 N. Furthermore, in line with the predicted importance of organic N to boreal forest plants, DON was required to achieve mass balance in many of these mixing model solutions. Chapter 4 extends the previous chapter's natural gradient approach to an experim ental one. Notably, the same analytical approach was applied within a 5 year old full factorial N and P fertilization experiment, also in central Alaskan black spruce forest. Because climate induced nutrient mineralization may increase N availability, ex perimental manipulation of soil nutrient availability offers insight into a possible trajectory of future conditions and evaluates the use of 15 N as a proxy indicator of N cycling changes Combining similar measurements from Ch. 3 with mass balance
16 mixing models it was shown that N+P fertilization lead to a 36% decline in N dependency on ECM forming fungi. Specifically, we found that N fertilization, both singly and in conjunction with P, caused the 15 N values of foliage, fine roots, soil N, and fungal fruiting bodies to approach that of the fertilizer Surprisingly, P fertilization also influenced the N cycle leading to a 60 fold increase of resin exchangeable soil NO 3 pool and a 15 N enrichment of 17 relative to the control. Fertilization of nitrifying bacteria followed by f ractionation against 15 N during N volatilization could account for these findings Combined, our experimental approach illustrated that me asuring numerous ecosystem components, particularly source 15 N values, is necessary to understand how enhanced soil fertility in boreal black spruce forest can be detected and how it may influence ECM dependencies and access to DON In conclusion my PhD dissertation work has sought to push the boundaries of stable isotope applications to understand the ecological roles of fungi and the function of boreal ecosystems with regards to the productivity limiting N cycle. My rese arch is an attempt at pinpointing specific causes of plant and fungal 15 N variability so that ecologists may feel confidence in using it as a time integrated proxy of the form and pathway of N cycling. This work has laid the foundation for my upcoming postdoctoral work seeking to do the same in tropical forest.
17 CHAPTER 2 ELUCIDATING THE NUTR ITIONAL DYNAMICS OF FUNGI USING STABLE ISOTOPES Abstract Mycorrhizal and saprotrophic f ungi are essential to terrestrial element cycling due to their uptake of mineral nutrients and decomposition of detritus. Lin king these ecological roles to specific fungi is necessary to improve our understanding of gl obal nutrient cycling, fungal ecophysiology and forest ecology Using discriminant analyses of nitrogen ( 15 N) and carbon ( 13 C) isotope values from 813 fungi across 23 sites we verified collector based categorization s as either ectomycorrhizal (ECM) or sapro trophic (SAP) in >91% of the fungi, and provided probabilistic assignments for an additional 27 f ungi of unknown ecological roles. For sites that ranged from boreal tun dra to tropical rainforest, we were able to show that fungal 13 C (26 sites) and 15 N (32 sites) values could be p redicted b y climate or latitude as previously shown in plant and soil analyses. Fungal 13 C values are likely reflecting differences in C source between ECM and SAP fungi, whereas 15 N enrichment of ECM fungi relative to SAP fungi sugge sts that ECM fungi are consistently delivering 15 N depleted N to host trees across a range of ecosystem types. Introduction Fungi function at two fundamental biogeochemical interfaces between soil and plants. Decomposer fungi mineralize organic carbon (C) compounds in detritus and liberate mineral nutrients i n the process, while mycorrhiza forming fungi, as mutualistic root ex tensions, enhance mineral, and perhaps organic, nutrient uptake in exchange for plant photosynthate (Leake & Read 1997) Within the large guild of ectomycorrhizal (ECM) fungi there are potential, though rare, cheaters' (Egger & Hibbett 2004; Douglas
18 2008 ) and species with proteolytic capabilities that may blur these distinctions (Chen et al. 2001; Bu e et al. 2007) However, dividing fungi into saprotrophic (SAP) and ECM functional groups has proven useful to biogeochemical and ecological research despit e considerable variation among fungal species (Read & Perez Moreno 2003; Gadd 2006) Ectomycorrhizal fungi are a diverse assemblage of ~7,000 10,000 spp. that mutualisticaly associate with woody plant hosts in a number of families of dominant tree species ( e.g., Pinaceae, Fagaceae, Dipterocarpaceae, Myrtaceae subfam ily Leptospermoideae, and Fabaceae subfam ily Caesalpinio deae) in boreal, temperate, and to a more limited extent, tropical regions of the world (Halling 2001; Read & Perez Moreno 2003; Taylor & Alexander 2005) The formation of macroscopic sporocarps (mushrooms) by many ECM and SAP fungi allows for experimental tractability unavailable for most microbial organisms. As a result, there are well developed species concepts, a rapidly developing com prehensive phylogeny (Hibbett et al. 2007) and unique opportunities for advancing ecological research on forest nutrient cycles, anthropogenic impacts, and fung al interactions with host plant s (Wardle et al. 2004; Clemmensen et al. 2006; Hobbie & Hobbie 2 006; Bu e et al. 2007; Treseder et al. 2007) Assigning ecological roles to individual taxa is necessary to conduct research on the biogeochemical importance of fungi. Assignment has typically been based on the following methods: 1) fruiting on, and presu med decomposition of, dead plant tissue by SAP fungi, 2) exclusive co occurrence of sporocarps with ECM forming host plants, 3) phylogenetic distance to fungi with previously categorized ecological role 4) direct tracing of hyphae from sporocarp to ECM ro otlet, 5) molecular comparison of sporocarp
19 to ECM rootlet, and 6) dual isotope values of C and N to determine nutritional mode However, each of these methods ha s limitations: 1) fungal growth on well decomposed wood and/or aerial fruiting habits can confound categorization based solely on fruiting substratum (Henkel et al. 2006) ; 2) exclusive co occurrence of sporocarps with suitable host plants is often unresolve d due to inadequate field observation; 3) the evolutionary switching' by many ECM forming basidiomycete fungi makes assignment to ecological role based on phylogeny alone questionable (Hibbett et al. 2000; Matheny et al. 2006) ; 4) direct evidence, such as tracing hyphae from fruiting body to ECM rootlet, is difficult or impossible to obtain in most soil matrices; and, 5) while molecular comparisons certainly can link ECM root tips to sporocarps (Horton & Bruns 2001) their widespread adoption by ecologists remains technologically and financially constrained. In this stu dy, we explored the ability of the isotope based method to assign ecological roles to fungi by quantif ying the error associated with the technique. The 15 N: 14 N and 13 C: 12 C stable isotope rat ios (expressed as 15 N and 13 C in permil ( ) values relative to standards) of fungi provide time integrated biogeochemical information regarding the acquisition transformation, and export of C a nd N by fungi under natural conditions (Griffith 2004) Unique C and N cycling pathways in ECM and SAP fungi lead to different isotope fractionation effects (Hobbie & Wallander 2006) The 15 N enrichment of ECM relative to SAP fungi is thought to result from assimilation and transfer of 15 N depleted N to host plants, a process that cumulatively leads to fungal enrichment and host plant depletion (Hobbie et al. 1999; H gberg et al. 1999a; Hobbie et al. 2005) Thu s, the absence of N transference to plants by SAP fungi causes them to appear 15 N depleted relative to ECM fungi within a site Patterns of 13 C in fungi are
20 largely attributed to isotope differences in the substrate ( s ) used as an energy source Saprotrophic fungi use C from plant tissues and soil organic compounds comprised of diverse C source s each with distinct 13 C values often 1 6 different from more commonly measured bulk plant foliage or roots (Gleixner et al. 1993; Marshal et al. 2007; Bowling et al. 2008) whereas ECM fungi receive plant photosynthate C that is isotopically more homogeneous relative to plant tissue (Gleixner et al. 1993; H gberg et al. 1999b; Henn & Chapela 2001; Baldocchi & Bowling 2003) The resulting difference s in either fungal 15 N or 13 C ha ve been used to differentiate ECM from SAP fungi (Taylor et al. 1997; Gebauer & T aylor 1999; Hobbie et al. 1999; H gberg et al. 1999b) Recently, the simultaneous use of both isotopes has improved this approach (Kohzu et al. 1999; Hobbie et al. 2001b; Taylor et al. 2003; Trudell et al. 2004; Clemmensen et al. 2006; Hart et al. 2006; Z eller et al. 2007) However, global variability in C and N isotope values in plants and soils (Amundson et al. 2003) suggests that cross site comparisons may only be possible following som e form of site normalization to correct for differences in average isotopic baselines among sites (Henn & Chapela 2001; Post 2002) Isotopic baselines are the 15 N or 13 C values of basal C and N sources within a trophic system or food web, such as photosynthate or labile mineral nutrient sources (reviewed in Post 2002) If large scale site variability in baseline isotope patterns is partially attributed to climate, such as with plant and soil 15 N and 13 C values (Amundson et al. 2003; Craine et al. 2009) then site normalization of 15 N or 13 C values would clarify the influence of physiology and nutrition on ECM and SAP C and N isotope patter ns. The accumulation of multiple datasets from around the northern hemisphere has enabled us to address both the utility and cause of C and N isotope differences in ECM
21 and SAP fungi. In order to determine if fungal 15 N or 13 C patterns across ecosystems are similar to plants and soils, we assessed the explanatory capacity of mean annual temperature (MAT), mean annual precipitation (MAP), and latitude (LAT). Cross site comparisons were optimized by use of site based normalization to remove variability associated with changes in background isotope value s and uneven ECM and SAP sampli ng within sites. We then used a large number of published and unpublished fungal 15 N or 13 C values to statistically discriminate ecological categorization s (SAP vs. ECM) of fungi with suspected and unknown ecological roles. Isotope values in fungi provide a form of ecological information independent of phyl ogenetics, soil excavation, or molecular sequencing, and when combined with one or more of these other techniques, provides definitive evidence of the nutritional ecology of specific fungi and can inform biogeochemical and evolutionary research in many of the world's forested ecosystems Methods Data Assembly To test global predictions of ECM and SAP isotope patterns, we compiled 15 N and 13 C values from one novel and ten published data sets. Compiled data included 9 13 15 N and 813 13 C values from collector categori zed ECM or SAP fungi and 27 fungi of unknown ecological role together comprising 148 genera. Of the 32 study sites included, 30 were from temperate, boreal, subarctic, or arctic ecosystems and two from tropical rainforest (site descriptions available fr om referenc es listed in Table 1). The tropical sites included fungi from a dipterocarp dominated Malaysian rainforest (Kohzu et al. 1999) and a Guyanese rainforest dominated by a leguminous tree. Lowland
22 tropical rainforest sites with ECM trees, while un derrepresented in our analysis, are globally uncommon outside of dipterocarp or caesalpinioid dominated rainforests as well (Taylor & Alexander 2005) Fungal 15 N data from areas with high levels of atmospheric N deposition from anthropogenic sources were excluded to eliminate possible confounding of natural fungal 15 N patterns (Gebauer & Taylor 1999; Lilleskov et al. 2002b) For each site, MAT, MAP, and LAT were compiled from origin al manuscripts, or extrapolated from nearby climate stations when not reported. Guyana F ield S ite and S ample P rocessing F ungal sporocarps were collected during the 2003 2006 June August rainy seasons i n the Upper Potaro River Basin in the Pakaraima Mountains of Guyana (5 ¡ 18'04.8 N, 59 ¡ 54'40.4 W; elevation 710 m). The moist evergreen forests in this region receive 3855 mm yr 1 of rain, and occur on well drained, highly oligotrophic soils that are low in phosphorus (P), calcium, and magnesium, and high in iron and aluminum (Henkel 2003; Mayor & Henkel 2006) The fresh foliar N:P mass ratio of 25.5 ( N = 5 canopy sun leaves) for Dicymbe corymbosa Spruce ex. Benth (Caesalpini aceae ) and the highly weathered parent material are suggestive of P limiting conditions to primary productivity (G sewell 2004) Fungal sporocarps were collected from monodominant stands of the ECM forming canopy tree, D. corymbosa morphologically described while fresh, identified to species or morphospecies, and field dried with desiccants (Henkel et al. 2002) Additional herbarium specimens collected previously from this area within the last 10 years were also analyzed for isotope values to target taxonomically unusual fungi of unknown ecological role In the laboratory, equal portions of pilei and stipe or entire sporocarps
23 from 56 fungi were finely ground, dried at 60 C for 24 hours, and analyzed on a ThermoFinnigan continuous flow isotope ratio mass spectrometer coupled to a Costech elemental analyzer at the University of Florida. Stable isotope abundance s are reported as: 15 N or 13 C = (R sample /R standard 1) 1000, where R = 15 N/ 14 N or 13 C/ 12 C of the sample and reference standard (atmospheric N 2 and PeeDee belemnite C, respectively). Run err or rates were typically 0.2 for 15 N and 0.1 for 13 C. Voucher specimens for Guyana fungi are maintained at Hu mboldt State University (HSU). Data A nalyses Linear regressions were conducted on site mean 15 N and 13 C values to assess the individual ability of MAT, MAP, and LAT to explain ECM and SAP mean isotope pattern s. Best fit polynomial equations (linear versus second order) were chosen based on R 2 comparisons. Combined, 9 fungi from Tanigawa and Okinawa Japan (Kohzu et al. 1999) were removed from 15 N correlations as extreme statistical outl iers indicated by box plots. The presence of significant correlations among site mean fungal 13 C and predictor variables indicated that accuracy in discriminant categorization of ECM and SAP fungi could indeed be enhanced th rough a normalization procedure as previously suggested (Henn & Chapela 2001) To normalize datasets prior to discriminant analysis we separately calculated the mean 15 N and 13 C values of the ECM and SAP groups within each site Next, we averaged these two group means and subtracted this new unbiased mean from each individual fungal isotope value. This normalization procedure removed the overall sampling bias toward ECM fungi across sites (6 2% were ECM) and centered site
24 means to zero. Site normalization excluded isotope values from four fungi representing sites with only single ecological categories (Kagoshima, Okinawa, and Oodai, Japan) A comparable, yet more complicated site normalizati on procedure was previously used (Henn & Chapela 2001) on a subset of the data presented here. Their site normalization procedure was designed to examine the contributions of fungal physiology and ecology (e.g. substrate) on 13 C fractionation in fungi. Because of this objective, their site corrections involved removal of the difference in means between ECM and SAP fungi for C data' (Henn & Chapela 2001) to correct for substrate and highlight remaining isotopic differences caus ed by physiology. Our relatively more simple transformation procedure was designed to retain the ECM SAP differences, regardless of cause, while reducing cross site variability indicated by the abiotic proxies of climate and latitude. Therefore, our norm alization procedure was applied to both C and N isotope values without the assumptions needed for substrate corrections. We used standard discriminant multivariate analysis of both site normalized and actual fungal isotope values to: 1) statistically test for a global isotopic difference among ECM and SAP ( ECM SAP ) fungi, 2) assign collector based categorization error terms for fungi, and 3) categorize fungi of unknown ecological role from several of the sites using probabilities arising from the entire da taset In the discriminant analysis, p robabilities of categorical assignment were set proportional to occurrence, and because the assumption of equivalent covariance among variables was not met a pooled variance quadratic function was used instead. Dis criminant analyses have been described as circular processes because they use predefined groups to inform categorization of those same groups (Quinn & Keough
25 2005) To alleviate this concern we validated categorization with a second discriminant analysis using a 50% random subset of the data to categorize all remaining fungi, and a separate cluster analysis using only fungal isotope values to assign groups (Quinn & Keough 2005) The specific categorical assignments of individual sporocarps were compared a mong the original and subsequent analyses to test for consistency in sporocarp categorization The above analyses were conducted using JMP ¨ 7. 0.2 (SAS Institute Inc.). Following site normalization and discriminant analyses we sought to more fully examine the combined ability of MAT, MAP, and LAT, to explain fungal 13 C and 15 N patterns using linear mixed effect models. Mixed effect models were necessary because both fungal species and isotope values within site s are non independent clustered observation s, and therefore violate a parametric test requirement of uncorre lated error terms associated with each measurement (Faraway 2006) Modeling non independent variables as random effe cts is a well established statistical technique (Crawley 2007; Andersen 2008) that allowed each sample point in our dataset, as opposed to just site means as in linear regression, to inform linear model construction. Mixed effect models were constructed u s ing the lme4 (Bates 2008) and lattice (Sarkar 2008) packages in R ¨ (version 2.6.2; R Development Core Team 2008). All models had the dependent variables of sporocarp 13 C and 15 N described by ec ological group (ECM or SAP ) site and either species or genus, both with and without nested interactions. In addition, MAT, MAP, and LAT were compared in a factorial fashion as additive, multiplicative, or quadratic predictor variables to determine the most informative formulation These complex forms were justified by the possibility of
26 nonlinear and conditional interactions among predictors as indicated by visual assessment of scatter plot matrices. Centering of MAT, MAP, and LAT (subtraction of each value from overall mean) optimized model fitting procedures and, consequently, reduced multi colinearity among predictor variables Beginning with the most complex model mathematical formulations of MAT, MAP, and LAT were sequentially compared using maximum likelihood methods. Following convergence on the optimal fixed effect configuration (e.g. lowest Akaike information criterion (AIC) value) we evaluated random effects with either species or genera nested within site, using restric ted maximum likelihood methods (Crawley 2007) We assessed model quality using the common graphical diagnostics of residuals against fitted values, sample quantile against theoretical quantile plots, and regressions of predictor variables for interpretati on of effects (Quinn & Keough 2005) AIC was used to select final models because it is widely regarded as an unbiased estimator that assess relative model fit, penalizes over parameterization, and allows multiple working hypotheses to be simultaneously ev aluated (Andersen 2008) R esults Dual 13 C and 15 N graphs of fungal sporocarps illustrate a global divide in isotope values between different e cological categories of fungi (Fig ure 2 1 ). On average, ECM fungi were significantly 15 N enriched and 13 C depleted relative to SAP fungi ( 5 5 0. 16 vs. 0. 3 0. 16 and 25. 5 0. 06 vs. 23.0 0. 09 SE; P < 0.001, t test). Global ranges in sporocarp 15 N ( 7.1 to 21.8 ) and 13 C ( 31.7 to 19.0 ) values were broad and the range of MAT ( 8.5 to 26 C) and MAP ( 300 to 3866 mm yr 1 ) among sites represents much of global climatic variability in t errestrial ecosystems (Table 2 1).
27 Sorting fungi into either tr opical ( N = 2) or extra tropical ( N = 30 ) sites indicated that fungi from the tropical sites were significantly more 13 C depleted and 15 N enriched than extra tropical sites (SAP: P < 0.05, two tailed t test; ECM: P < 0.05, Wilcoxon rank sum test). Despite absolute differences among site mean 13 C and 15 N the magnitude of isotopic differences between ECM and SAP fungi was consistent across sites (Table 2 1) and not significantly different from a slope of one ( Fig ure 2 2; < 0.05). The large number of sporocarp dual isotope measurements used in the d iscriminant analysi s ( N = 8 13 ) allowed for reliable assignment of collector based categorization error (Appendix A 2 ), and consequently, confident categorization of fungi of unknown ecological role based upon >50% statistical probabilities (Appendix B 2 ). Collector based categorizations using normalized i sotope values were deemed valid in 91. 2 % of sporocarps indicating a high level of agreement with and among categorization methods used by collectors. Site normalization reduced collector categorization error by 0.7% relative to non normalized isotope valu es, and retained within site variability. Normalization appears to have only slightly reduced overlap between ECM and SAP groups (Fig ure 2 1B ) due to centering of all site means to zero. The increased accuracy of discriminant categorization could be due to increased model efficiency during extraction of eigenvector distances (Quinn & Keough 2005) Discriminant c ategorization of fungi was further supported by the additional discriminant analysis of a random 50% subset of the data, and cluster analysis of t he entire dataset. The random 50% subset increased overall discriminant categorization error by only 0.3% and was in agreement with individual categorizations based on the entire dataset The cluster analysis also identified ecological categories but inc reased
28 overall categorization error by an additional 1.5% These values are similar to error rates derived from the discriminant analysis using the full data set and are indicative of low a priori catego rical forcing of ecological groups due to categorica l assignment by collectors. Combined, these results suggest that sporocarp categorization is robust to a substantial reduction in data input and that collector knowledge, while accurate, is of little statistical importance in predicting fungal ecology rel ative to 13 C and 15 N values alone. Climate an d latitude serve as reasonably good predictors of fungal isotope values on a global scale. Linear regression of site mean ECM and SAP fungal 13 C indicated significant r elationships with MAT ( R 2 adj = 0.18 and 0. 63 respectively) and LAT ( R 2 adj = 0.49 and 0.67, respectively), but not with MAP, whereas 15 N was significantly correlated with MAT ( R 2 adj = 0.2 4 ) in ECM fungi only (Fig ure 2 3 ). Singly, MAP and LAT had no predictive power over mean fungal 15 N, but the inclusion of MAT, MAP, and LAT as centered quadratic variables in linear mixed models s ubstantia lly improved the ir explanatory power (i.e. I > 2; Table 2 2). Removal of LAT from mixed models was deemed useful because it produced comparable model fits as MAT and MAP and reduced obvious correlations among predictor va riables Remaining multicolinearity among centered MAT and MAP was low ( R 2 = 0.13) and both v ariables were retained despite potential similarity in information. Linear mixed models indicated that variability in sporocarp 13 C was best explained by MAT, LAT, an interaction between them ( MAT*LAT ) and sporocarp type ( ECM vs. SAP; Table 2 2). Substituting the random effect of genus for species also substantially increased model fit ( # i = 71.2; Table 2 2) Use of genus allowed for more informative mixed models due to the presence of the
29 same fungal genera across multiple sites while species were often confined to single locations. Sporocarp 15 N was best described by the fixed effects MAT, MAP, a nd their quadratic functions (Table 2 2 ). Altering random effects to include the interaction of species with site improved model fit, and, as with 13 C, substituting the random effect of genus for species produced a substanti ally more informative model ( # i = 83.0; Table 2 2). D iscussion Implications of the G lobal P attern The 13 C and 15 N based division in ECM and SAP fungi has been described from localized sites and is shown here to exhibit a consistent pattern across the global range of ecosystem types The magnitude of isotopic differences between ECM and SAP fungi was also similar across sites, as evidenced by a slope similar to one (Figure 2 2), and parallel relationships of site mean 13 C and 15 N values with climate and latitude (Figure 2 3 ) Site normalization eliminated correlations with climate and latitude but reduced discriminant categorization error by only 0.7%. Th e similar isotopic differences suggest comparable ecophysiological functioning of these two nutritional groups of fungi across sites that differ widely in climate, plant community, and baseline isotope values Because of the ubiquity of the pattern, the assembled dataset can be used to confidently (>90%) categorize the nutritional status of fungi based on 13 C and 15 N values of sporocarps or hyphae (Wallander et al. 2004) Furthermore, site normalization and collector knowledge increased prediction accuracy, but only by a small margin.
30 The observed 15 N enrichment of ECM relative to SAP sporocarps is attributed to 15 N discrimination ( 8 10 ) during the formation and d elivery of amino acid N from fungi to plants (Hobbie & Hobbie 2006) Additional cause s of ECM 15 N enrichment relative to SAP fungi could in clude preferential use of 15 N enriched forms of N as well as internal processing within the fungus irrespective of transfer to the host plants (Hobbie & Colpaert 2003; Brearley et al. 2005; Dijkstra et al. 2008) However, field and laboratory observations currently support the N delivery to host plant process as being most influential in terms of isotope fractionation. In addition, frequently observed 15 N depletion in ECM associating host plants relative to co occurring non ECM plants further supports thi s hypothesis (Hobbie & Hobbie 2008) Lower 15 N values in non ECM forming plants are expected because the assimilation and transfer of N by a rbuscular mycorrhizal (AM) fungi is thought to impart a smaller 15 N fractionation ( 0 2 ) in host plants (Handley et al. 1993; Craine et al. 2009) Therefore, a globally similar ECM SAP divide suggests that ECM fungi are delivering 15 N depleted N to host plants under both N limitation to plant productivity, such as might be found in temp erate and high latitude ecosystems, as well as in tropical forests that could be limited primarily by P (Palmiotto et al. 2004; Mayor & Henkel 2006) In support of these expectations, a recent analysis of foliar 15 N from 91 studies used the type of root symbiosis (ECM and ericoid mycorrhizal vs. AM or non mycorrhizal) to explain 29% of the variation in foliar 15 N globally (Craine et al. 2009) Because ECM and SAP fungi differ fundamentally in C source (i.e. plant photosynthate vs. detritus), sporocarp 13 C is expected to track the 13 C values of these two major C pools (H gberg et al. 1999b; Henn & Chapela 2001) Although the 13 C
31 values of fungi are thought to reflect patterns found in plant C pools they are typically 0.3 5 more enriched than host tissue (Gleixner et al. 1993; Hobbie et al. 1999; H gberg et al. 1999b; Hobbie et al. 2001b; Trudell et al. 2004; Kohzu et al. 2005; Hart et al. 2006) This offset from known (and suspected) substrates may be due to 13 C discrimination during decomposition by SAP fungi or during synthesis and translocation of various C pools from host plants to ECM fungi (Bowling et al. 2008; H gberg et al. 2008) Saprotrophic fungi tend to be relatively more 13 C enriched (relative to bulk leaves) than either ECM fungi and their plant sugar C source, or leaf lipids and proteins (Bowling et al. 2008) This differential o ffset from presumed C source suggests additional fractionation pathways may be contributing to SAP 13 C enrichment in particular (Kohzu et al. 2005) Regardless of physiological contributions to fungal 13 C differences in the 13 C values of C pools are considered responsible for the 13 C divi de among ECM and SAP fungi Our analysis illustrates that it is possible to reliably infer ecological roles of fungi over a broad range of conditions using a relatively simple site based normalization procedure of fungal 13 C and 15 N values. It has been suggested that the ECM SAP divide should become less distinct over large geographical areas an d that the isotopic signature of an organism is insufficient evidence to infer ecological role without first standardizing to an appropriate isotopic baseline (Henn & Chapela 2001; Hobbie et al. 2001b; Post 2002; Taylor et al. 2003) However, our normaliz ation slightly enhanced the resolution of the discriminant analysis through removal of mean isotopic differences among sites without comparison to a measured baseline. Given the small decrease in discriminant categorization error following site normalizat ion (0.7%) it is unlikely that
32 correcting to isotopic baseline measurements (leaves and mineral nutrients) could reduce categorization error further. For instance, isotopic corrections based on foliar samples could introduce variation based on sample posi tion in the canopy, taxonomic grouping growth form, or type of root symbiosis (Pate & Arthur 1998; Bustamante et al. 2004; Craine et al. 2009) Additionally, isotopic corrections based on soil 15 N could introduce variation depending on depth, disturbance, and fertility of the samples (Bustamante et al. 2004; Davidson et al. 2007; Dijkstra et al. 2008) I n addition to the discrimination of fungal ecological roles across multiple ecosystems, our dataset shows an ECM SAP difference ( ECM SAP ) in 15 N of 5.3 and 2. 3 for 13 C; values that will help constrain the magnitude of isotope fractionation s used in modeling efforts. Fractionation attributed to ECM mediated N assimilation and transfer to host plants were recently used to estimate that Alaskan tundra plants received 61 86% of their total N from ECM fungi and reciprocally delivered 8 17% of their photosynthetic C to ECM fungi (Hobbie & Hobbie 2006) The se simultaneous mas s balance calculations require accurate assessment of the fractionation magnitude associated with ECM delivered N in order to constrain additional estimated and interdependent, variables (Hobbie & Hobbie 2008) This quantification o f elemental cycling is a necessary step to the modeling of N cycles and the partitioning of below ground C allocation Values in o ur global analysis can refine th is approach because additional fractionation effects, such as during N uptake have been partially removed through the subtraction of co occurring SAP 15 N values. Better paramet er ization of this key 15 N fractionation step is necessary for expansion to, and testing of, C and N mixing models in additiona l ecosystems (Hobbie & Wallander 2006)
33 Climatic I nfluence on S porocarp 15 N F ungal 15 N patterns were expected to correlate with MAT, MAP, and LAT to the extent that soil and litter 15 N values correlate with those values I n previous global isotopic analyses, MAT and MAP were found to be good predictors of soil and plant 15 N due to t heir influence over soil N c ycling and the isotope ratios of ecosystem N inputs and outputs (Amundson et al. 2003; Craine et al. 2009) This is because w arm temperature and high rainfall conditions are conducive to high rates of N mineralization and nitrification leading to a loss of 15 N depleted N through den itrification or leaching and consequent 15 N enrichment of soil N (Amundson et al. 2003; Templer et al. 2007) Therefore, the integrated N pool in tropical soils is typically 15 N enriched re lative to hig h latitude ecosystems with low soil N availability and conservative N cycles (H gberg 1997; Amundson et al. 2003) S ignificant sporocarp 15 N enrichment was observed in fungi from tropical relative to temperate s ites but the predictive ability of individual variables to explain fungal 15 N patterns was only weakly correlated with MAT. The curved relationship among ECM 15 N values and MAT (Figure 2 3B) wa s driven by the coldest tussock tundra site near Toolik, A laska (Clemmensen et al. 2006) the exclusion of which removed the relationship. Causes for the higher than expected 15 N values in ECM sporocarps at Toolik, AK ( 12 ) could result from large proportional N transfer by ECM fungi to severely N limited plants (Hobbie & Hobbie 2006) or ECM and SAP access to anomalously 15 N enriched N sources (Lilleskov et al. 2002b) Whereas individual variables were poor predictors of f ungal 15 N, enhanced explanatory power in mixed effect models illustrate that MAT and MAP do hold predictive ability when properly formulated ( # i = 9.9; Table 2 2). In summary, the expectation that fungal 15 N
34 patterns would respond similarly to that seen for plants and soils was not supported suggesting that fungal physiology exerts primary influence over fungal 15 N patterns. However, t he inclusion of fungal and fo liar 15 N values from more tropical and subtropical sites will undoubtedly improve the predictive ability of discriminant and mixed models and help clarify the secondary influence of climate on sporocarp 15 N. Climatic I nfluence on S porocarp 13 C Mean 13 C values from fungi in the warm/wet sites were most similar to those from the cold/dry sites when viewed in relation to MAT and LAT as single predictor variables. As with 15 N we determined the influence of the coldest site (Toolik Lake, AK) by removing it, however in this case the relationship between fungal 13 C and MAT remained ( R 2 adj = 0.16 ECM, 0.56 SAP). Plant analyses of foliar 13 C generally provide reliable indices of plant water use efficiency (WUE) owing to 13 C discrimination during photosynthesis and its relation to stress induce d stomatal closure (Marshal et al. 2007) However, the weak relationship between fungal 13 C and MAP in our analysis indicated that this coarse climatic variable was of little utility in explaining fungal, and presumably plant, C isotope patterns across such diverse sites. For instance, the four driest sites in our meta analysis differed widely in te mperature ( 8.5 to 5 C), as did the four wettest (9.5 to 25 C). Therefore, partitioning individual climatic influences over fungal 13 C at these extreme conditions is confou nded by the joint possibility of greater water stress at dry sites and temperature induced photosynthetic inhibition at the cold est ones similarly altering plant, and indirectly fungal, 13 C (Allen & Ort 2001) Regional analy ses show MAP gradients are strongly correlated ( R 2 = 0.64 to 0.70) with foliar 13 C in Northern and Southeastern Australia respectively (Stewart et al. 1995; Austin & Sala 1999) but
35 not Hawaii (Schuur & Matson 2001) It is more likely that measures of actual or potential evapotranspiration would be more informative in future studies. Latitude explained the largest portion of mean fungal 13 C variability among sites ( R 2 adj = 0.49, ECM; 0.67, SAP) likely due to its integration of multiple effects on plant 13 C patterns, and combined with MAT and their interaction, substantially increased the explanatory power of mixed models ( # i = 26.8; Table 2 2). The proxies of MAT and LAT likely integrate the timing and form (snow vs. water) of precipitation among sites during growing season s, as well as other physiological stressors that can modify plant WUE and 13 C values, suc h as: 1) within species physiological variability (Pate & Arthur 1998; Schuur & Matson 2001; Kohzu et al. 2005) ; 2) species replacement particularly at low soil water contents (Swap et al. 2004) ; 3) relative N availability and C sink strength of ECM fungi (Hobbie & Colpaert 2003) ; and 4) compensatory effects of specific leaf area and leaf N concentration on plant WUE (Schulze et al. 2006) Similarly, a global meta analysis of 1,248 plants across 452 sites demonstrated the ability of MAT and LAT to explain patterns in foliar N and P at global scales, suggesting a reflection of both plant physiological adjustments and the relative shifts in nutrient limitations due to changes in the age of soils (Reich & Oleksyn 2004) Predictions o f F ungal E cology Despite the strength of the discriminant analyses, known error s in model categorization have been found in our dataset suggesting other, unknown error s are likely. Known SAP categorization errors are confirmed wood decay fungi that were categorized as ECM by the discriminant model with >90% probability. These apparent errors included the following individual sporocarp collections: Pleurotus ostreatus
36 (Jacq.) P. Kumm. from Chiba, Japan Microporus vern icipes (Berk.) Kunt. from Lambir, Malaysia (Kohzu et al. 1999) ; and Gymnopilus bellulus (Peck) Murrill, from Lamar Haines, Arizona (Hart et al. 2006) Including other apparent SAP errors with <90% mode led probabilities increases the SAP errors by six indi vidual samples (Appendix A 2 ). Error in the categorization of decomposer fungi could be partially caused by unique nutritional sources in addition to wood or litter. For instance, access to recent plant photosynthate with relatively low 13 C, or to accruing microbial and insect biomass with relatively high 15 N, could cause SAP fungi to appear' as ECM in the discriminant model. Discriminant model errors regarding ECM fungi are more difficult to discern because of the difficulty in determining the nutrition of terrestrial sporocarps. S everal individual sporocarps belonging to genera and species traditionally categorized as ECM we re categorized as SAP by the discriminant model with >90% probability. These presumed ECM categorization errors' included: Cortinarius sp. (Pers.) Gray Kyoto, Japan, Tylopilus sp. P. Karst. from Lambir, Malaysia (Kohzu et al. 1999) ; Cortinarius variosimi lis M.M. Moser & Ammirati from Deer Park Rd, WA (Trudell et al. 2004) ; and Cantherellus pleurotoides T.W. Henkel, Aime and S.L Mill. from Guyana (this study) Including other, typically ECM genera ( e.g., Amanita, Russula, Lactarius, Boletus ) with lower than 90% probabilities would increase this number by 23 individual samples (Appendix A 2 ). Causes for these discrepancies are unknown but warrant caution in accepting predictions based on dual isotope measures of single sporocarp collections in the absenc e of other evidence (Trudell et al. 2004) Triplicate analyses of six species in our Guyana dataset, collected across years, had standard deviations of 0.61 to 0.71
37 ( 13 C and 15 N, respectively) and were all assigned to the same ecological group by the discriminant model. When anomalous isotope values do occur for individual sporocarps across sites, particular caution of categorization error is warranted At low sampling number site normalized values are likely to be less accurate (e.g. not a true site mean) due to insufficient sporocarp numbers. This is particularly problematic if using archived specimens from distant or poorly documented sites. In contrast, when the same fungal species is ca tegorized incorrectly across multiple sites or collections it may offer insight into unique nutritional, kinetic, or physiological activities that run counter to traditional predictions in groups otherwise considered to have a narrow nutritional mode. Cl assification of fungi with unknown ecological roles using site normalized dual isotope measurements produced confident predictions (i.e. probability >90%) for 19 out of 27 fungi in our dataset, and weak to moderate predictions (50.2 to 89%) for the remaini ng eight (Appendix B 2 ). Such predictions could form the basis for research hypotheses about the evolution of the ECM and SAP habit (Hobbie et al. 2001b) and be combined in a phylogenetic context (Wilson et al. 2007) For instance, the inclusion of Phylloporus rhodoxanthus ( Schwein.) Bres., a lamellate genus included within an otherwise poroid hymeniphore forming family (Boletaceae), adds ecological support to the micro morphological, molecular, and observational data categorizing this genus as ECM. In addition, the previously uncertain ecological roles of Clavulina Schroet in Cohn, Helvella L., Coltriciella Murrill, and Tremellodendron G.F. Atk., now h ave strong isotopic evidence for the ECM mode of nutrition ; and in conjunction with phylogenetic af finity to other presumed ECM taxa (Henkel et al. 2005) now have additional support (Appendix B 2 ).
38 In agreement with natural history based methods, accurate identification of fungal genera is useful for predicting the ecological role of many fungi. Consi stent generic categorization in the discriminant model and improved linear mixed model predictive capacity using genus support this. The observation that 15 N patterns of fungal genera can exhibit either high or low 15 N (Trudell et al. 2004) and 13 C syndromes (Kohzu et al. 1999) suggest that future studies may seek to define a stable isotope based niche space similar to the metrics used to describe tidal food webs ( Layman et al. 2007) It is intriguing to speculate that specialization of some fungal genera with depth (Lindahl et al. 2007; Taylor et al. 2010) N source (Lilleskov et al. 2002b; Hobbie & Hobbie 2008) or other niche dimensions could be repres ented by an index of sporocarp 15 N and 13 C variability. If demonstrated, this could also contribute to how genus increased the ability of mixed models to predict fungal 15 N and 13 C patterns globally Summary The C and N isotopic difference between ECM and SAP fungi provides researchers with a reliable tool for the ecological categor ization of fungi regardless of site origin This time integrated, biogeochemical evidence offers insight into C and N cycles across most forested ecosystems and highlights the global importance of ECM fungi to host plant N nutrition and forest N cycling The ecophysiology associated with ECM and SAP nutritional roles was shown to exert primary control over fungal 13 C and 15 N values, however some of the variability was attributable to climatic a nd latitudinal proxies These findings offer a tool by which fungi can be integrated into our understanding of global elemental cycles and illustrates that fungal 15 N may be partially decoupled from soil and plant isotope pa tterns. As evidenced here, dat asets containing
39 fungi from tropical forests contributed to the reliability of stable isotope analyses to discriminate ecological roles of any sporocarp producing fungus and the future inclusion of fungi from subtropical and southern hemisphere sites will undoubtedly improve our confidence in this approach.
40 Table 2 1 Summary of isotopic, climatic, and ecological data by study and site. ( N ) ( C) (mm yr 1 ) ECM ( ) SAP ( ) ECM SAP Ref.* Site ECM SAP LAT MAT MAP 13 C 15 N 13 C 15 N 13 C 15 N 1 Aheden, Sweden 53 0 64 1.0 600 N/A 6.0 N/A N/A N/A N/A Betsele, Sweden 5 0 64 1.0 570 N/A 8.1 N/A N/A N/A N/A Flakaliden, Sweden 21 0 64 2.3 600 N/A 6.1 N/A N/A N/A N/A Kulbacksliden, Sweden 8 0 64 1.2 523 N/A 4.3 N/A N/A N/A N/A Norrliden, Sweden 1 0 64 1.6 595 N/A 5.4 N/A N/A N/A N/A Svartberget, Sweden 5 0 64 1.6 595 N/A 5.0 N/A N/A N/A N/A Vilan, Sweden 7 0 64 5.1 542 N/A 5.3 N/A N/A N/A N/A Study mean -5.7 ----2 Aishu, Japan 19 21 35 11.7 2353 24.6 5.0 22.6 1.6 2.0 3.5 Chiba, Japan 3 6 35 14.7 1550 26.2 0.8 23.7 0.6 2.6 0.1 Kagoshima, Japan 0 1 31 17.8 2236 N/A N/A 22.9 7.1 N/A N/A Kyoto, Japan 28 26 35 15.8 1814 24.6 4.8 23.1 0.6 1.5 4.2 Lambir, Sarawak Malaysia 17 14 4 26.0 2700 26.8 7.6 25.0 0.4 1.8 7.9 Miyajima, Japan 1 1 34 17.0 1546 24.6 9.8 22.1 1.5 2.5 8.3 Norikura, Japan 9 3 36 6.7 2766 24.3 8.4 21.6 0.1 2.7 8.5 Okinawa, Japan 1 0 24 23.0 1736 25.0 21.2 N/A N/A N/A N/A Ontake, Japan 9 8 35 6.7 2766 24.1 2.4 21.8 2.7 2.3 5.1 Oodai, Japan 0 2 34 15.7 1511 N/A N/A 22.2 0.2 N/A N/A Shirahama, Japan 1 2 33 16.8 1730 24.5 4.4 23.1 0.7 1.5 3.8 Tanigawa, Japan 2 6 36 5.2 1692 24.8 19.1 22.3 1.0 2.5 20.0 Study mean 25 8.2 22.7 0.7 2.1 6.8 3 Glacier Bay Alaska, USA 4 4 59 14.9 1830 25.4 4.5 22.9 1.9 2.5 6.4 4 Mixed conifer California, USA 18 25 N/A N/A N/A 25.8 9.0 22.5 0.1 3.3 9.1 5 Woods Creek Oregon, USA 20 25 45 11.0 1000 26.2 3.9 22.8 1.8 3.5 5.7
41 Table 2 1. Continued ( N ) ( C) (mm yr 1 ) ECM ( ) SAP ( ) ECM SAP Ref.* Site ECM SAP LAT MAT MAP 13 C 15 N 13 C 15 N ECM SAP 6 Aheden, Sweden 29 4 64 1.0 600 25.8 7.8 23.3 0.5 2.4 8.4 Stadsskogen, Sweden 110 13 59 5.5 541 25.7 5.8 23.1 1.6 2.6 4.2 Study mean 25.8 6.8 23.2 0.5 2.6 6.3 7 Deer Park Rd Washington, USA 64 23 47 9.0 1150 25.4 5.5 23.3 1.2 2.1 6.7 Hoh River Washington, USA 54 38 47 10.0 3450 25.2 4.7 22.9 2.3 2.3 7.0 Study mean 25.3 5.1 23.1 1.8 2.2 6.9 8 Snowbowl Arizona, USA 9 13 35 4.0 775 24.0 4.6 22.0 1.9 2.0 2.7 Lamar Haines Arizona, USA 12 13 35 5.0 775 24.0 3.2 21.9 2.4 2.1 0.8 Study mean 24.0 3.9 22.0 2.2 2.0 1.7 9 Heath tundra, Sweden 10 4 68 1.0 300 27.0 1.7 23.7 0.08 3.4 1.7 Tussock tundra Alaska, USA 3 5 68 8.5 350 26.4 12.0 24.7 3.0 1.8 9.0 Study mean 26.7 6.9 24.2 1.6 2.5 5.3 10 Breuil Chenue, France 33 14 47 9.0 1280 26.2 3.1 22.8 2.8 3.5 5.9 Spruce plantation, France 20 17 47 9.0 1280 24.1 3.7 22.6 0.6 1.4 4.3 Study mean 25.1 3.4 22.7 1.7 2.5 5.1 11 Upper Potaro River, Guyana 29 20 5 24.0 3866 26.0 5.7 24.9 1.6 1.1 4.1 Sum 605 308 Grand mean 25.3 6.4 22.9 0.3 2.3 5.3 Number ( N ) of individual ec tomycorrhizal (ECM) and saprotrophic (SAP) sporocarps collected from each site. The mean annual temperature (MAT) and precipitation (MAP) values correspond to either published or extrapolated measurements from nearby climate stations. Differences in mean isotope values ( 13 C and 15 N) from each site were subtracted from one another ( ECM SAP ) to illustrate variability in ECM SAP isotope differences among sites. References: 1, Taylor et al 1997 ; 2, Kohzu et al 1999; 3, Hobbie et al. 1999; 4, Henn & Chapela 2001; 5, Hobbie et al. 2001; 6, Taylor et al. 2003; 7, Trudell et al. 2003; 8, Hart et al 2006; 9, Clemmensen et al. 2006; 10, Zeller et al 2007; 11, this study. Values reported as gene ric means of 67 ECM and 29 SAP species. Tanigawa, Japan omitted as a statistical outlier.
42 Table 2 2 Competing linear mixed model results Model K Log like AIC i 13 C Species Null 3 1247.2 2504.3 102.4 MAT c +LAT c 5 1246.2 2506.4 104.5 MAT c +LAT c +(MAT c +LAT c ) 2 4 1239.9 2491.7 89.8 MAT c :LAT c 4 1237.1 2486.2 84.3 (MAT c +MAT c 2 )+(LAT c +LAT c 2 ) 5 1234.5 2485.0 83.1 MAT c +LAT c +MAT c :LAT c 6 1228.5 2473.1 71.2 Genus Null 3 1209.3 2428.7 26.8 MAT c +LAT c +MAT c :LAT c 6 1193.0 2401.9 0.0 15 N Species Null 3 2245.1 4500.1 2088.4 MAT c +MAT c 2 +LAT c +LAT c 2 7 2241.6 4497.3 2085.7 MAT c +MAT c 2 +MAP c +MAP c 2 7 2241.1 4496.2 2084.6 Site:Species MAT c +MAT c 2 +MAP c +MAP c 2 +SPECIES+SITE:SPECIES 9 1275.9 2565.7 154.1 MAT c +MAT c 2 +MAP c +MAP c 2 + SITE+SPECIES+SITE:SPECIES 10 1239.3 2494.6 83.0 Genus Null+SITE:GENUS 6 1204.8 2421.5 9.9 MAT c +MAT c 2 +MAP c +MAP c 2 +SITE+GENUS+SITE:GENUS 10 1196.4 2411.6 0.0 Models of 13 C and 15 N values in ectomycorrhizal (ECM) and saprotrophic (SAP) fungi were based on 913 15 N and 813 13 C fungal sporocarp measurements. The null mixed model contained th e fixed effect of sporocarp type (ECM or SAP) and the random effects of site and either species or genus as indicated. Number of model parameters ( K ), log likelihood (log like), Aikake information criteria (AIC) model selection results, centered mean annu al temperature (MAT c ), centered mean annual precipitation (MAP c ), centered absolute latitude (LAT c ). I = AIC i AIC min where AIC min is the minimum of the different AIC i values and represents the information lost using other models with higher AIC scores. As a rule of thumb, an I 2 have substantial support for being more informative over competing models (Anderson 2008).
43 Figure 2 1 D ual isotope graphs of ectomycorrhizal (ECM), saprotrophic (SAP), and fungi of unknown (UNK) ecological role collected from 32 sites around the worl d. A) Raw isotope values. B) S ite normalized isotope values with inset illustrating the approximate site locati ons of sporocarp collections
44 Figure 2 2 Comparison of the mean 13 C and 15 N of ectomycorrhizal (ECM) and saprotrophic (SAP) fungi from each site A) 13 C values. B) 15 N values. Both A and B linear fits (solid line) are not significantly different from a slope of 1 (dashe d line; <0.05 ) indicating a consistent isotopic difference between ECM and SAP fungi across sites. Tanigawa, Japan, was omitted as a statistical outlier. Equations of lines: (A) linear: = 7.6 4 + 0.77 slope constrained: = 2.32 + 1 ; (B) linear: = 5.32 + 0.7 slope constrained: = 5.32 + 1
45 Figure 2 3 Regressions of site mean fungal 13 C and 15 N values with mean annual temperature, mean annual precipitation, and latitude. Black circles are site means of ectomycorrhizal (ECM) fungi; grey circles are site means of saprotrophic (SAP) fungi. (A) 13 C values of fungi i n relation to MAT. (B) 15 N values of fungi in relation to MAT. (C) 13 C values of fungi in relation to MAP. (D) 15 N values of fungi in relation to MAP. (E) 13 C values of fungi in relation to latitude. (F) 15 N values of fungi in relation to latitude. Linear vs. quadratic best fit polynomial equations were compared and equations producing the best R 2 c oefficient are presented. Tanigawa and Okinawa, Japan, sites were removed from graphs B, D, F as statistical outliers.
46 CHAPTER 3 SOURCE VS. PATHWAY: 15 N PATTERNS IN CENTRA L ALASKAN BLACK SPRU CE FOREST REFLECT HIGH DEPENDENCY ON ECTOMY CORRHIZAL DERIVED ORGANIC N ITROGEN Abstract The productivity of many northern ecosystems is nitrogen limited but details of the N cycle remains little known. Recent analyses of global patterns in soil, plant, and fungal stable isotopes of N ( 15 N) show promise in provid ing an integrative metric of N cycling processes but differentiating the underlying causes of 15 N variability remains a research priority. We conducted a landscape sca le regression and modeling analysis of elemental patterns along the soil fungi plant continuum in 31 stands of central Alaskan black spruce. Because the stands varied widely in 15 N values, biomass, soil fertility, and topogr aphic position we encompassed much of the variety of low fertility black spruce stands and were able to specifically examine if fertility, soil N forms, or 15 N fractionation by ectomycorrhizal (ECM) fungi contributed to ecosystem 15 N patterns. B lack spruce foliar 15 N values were partially influenced by soil fertility and the 15 N value of N sources whereas %N was most influenced by soil dissolved organic N (DON) content. Incorporation of 15 N fractionation by ECM fungi into isotope mixing models indicated that black spruce is heavily dependent on ECM derived N (86 98% of total N nutrition), much of which is acquired from the DON pool. These findings indicate that the elemental content of black spruce needles integrate s both the source and pathway of N cycling as influenced by soil fertility and ECM fungal activity.
47 Introduction The productivity and dynamics of many ecosystems are governed by nitrogen (N) availability ; added N typic ally increases the biosynthesis of proteins and enzymes for photosynthesis (Galloway et al. 2004; Howarth & Marino 2006) Productivity of terrestrial ecosystems also typically increase s in response to increased N availability (Vitousek & Howarth 1991; Els er et al. 2007) Because many human activities can increase N availability through deposition, or increase N mineralization rates through climate warming and landscape modification, monitoring ecosystem responses to shifts in N availability is a research priority (Vitousek 1997; Mellilo & Cowling 2002) In N limited boreal forests, slight changes in N availability can alter ecosystem productivity, carbon storage, and diversity of plants and microbes (Helfield & Naiman 2002; H gberg et al. 2003; Toljander et al. 2006; Treseder et al. 2007) Assessing ecosystem N supplies, flux pathways, and overall budgets, h owever, remains difficult and error prone even with frequent measurements because N cycling processes vary in both time and space (Binkley et al. 2000 ; Schimel & Bennett 2004; Cleveland et al. 2010) Recent syntheses suggest that stable isotopes of N provide integrative measures of N cycling that can be used to infer N cycling processes at local, regional, and even global scales (Amundson et al. 2003; Pardo et al. 2006; Craine et al. 2009; Pardo & Nadelhoffer 2010) For instance, global 15 N patterns in soils fungi, and plants partially vary with mean annual temperature and precipitation (Handley et al. 1999; Amundson et al. 2003; Mayor et al. 2009) These large scale patterns are thought to be due to the indirect control of climate over microbial activity the openness of the N cycle, and the form of N lost from ecosystems (Gebauer & Schulze 1991; Nadelhoffer & Fry 1994; H gberg 1997; Martinelli et al. 1999; Schuur & Matson 2001; Pardo et al. 2006; Houlton
48 et al. 2007) At smaller scales, foliar 15 N is linked to multiple processe s in the N cycl e including : the total amount and distrib ution of N within a plant; the N concentration, depth, and retention capacity of soi ls; and the relative rates of N mineralization and uptake (Nadelhoffer et al. 1996; Falkengren Grerup et al. 2004; Houlton et al. 2007; Templer et al. 2007) For instance, high soil N mineralization rates are often correlated with greater losses of N by denitrification and leaching (Houlton et al. 2006; Kahmen et al. 2008) This loss of N forms (e.g. NO 3 N 2 O, N 2 ) that are 15 N light relative to their source pools causes 15 N enrichment of remaining soil N (Piccolo et al. 1994; Boeckx et al. 2005; P rtl et al. 2007) B ecause soil N sources can exhibit variable 15 N values depending on relative amounts of 15 N discrimination during ammonificati on, nitrification, and denitrification (L tolle 1980; Mariotti et al. 1981; Shearer & Kohl 1986) foliar 15 N values are often thought to reflect specialization on specific N forms or rooting depths (Schulze et al. 1994; Miche lsen et al. 1996; McKane et al. 2002; Houlton et al. 2007) Occasionally confounding this interpretation is the observation that the presence of ecto and ericoid mycorrhizal root symbionts can greatly modify foliar 15 N valu es from source to autotrophic sink during the production of 15 N depleted N transfer compounds (Michelsen et al. 1998; H gberg et al. 1999a; Hobbie & Hobbie 2008) This source of fractionation was highlighted globally in a regression model of >9,000 tree s where 29% of global variability in foliar 15 N values was attributed to mycorrhizal fractionation (Craine et al. 2009) Interestingly, the corresponding 15 N enrichment of many mycorrhizal fungi is thought to contribute to the common pattern of 15 N enrichment with soil depth (Lindahl
49 et al. 2007; Dijkstra et al. 2008; Etcheverr a et al. 2009; Hobbie & Ouimette 2009; Wallander et al. 2009) Des pite the many insights about N cycling based on ecosystem 15 N measurements, it remains difficult to differentiate the ultimate causes of plant 15 N values because or multiple mechanistic possibilities (Craine et al. 2009; Pardo & Nadelhoffer 2010) For instance the same plant 15 N patterns could be caused by increasing lability of N dependency on different forms of soil N or dependence on differing mycorrhiza l types Addressing these simultaneous possibilities has proved methodological intracta ble because of the difficulty involved with making replicate 15 N measurement of available soil N sources at field concentrations Such detailed and high throughput methods are now available and have been used in a diversity of terrestrial ecosystems (Ostle et al. 1999; Yoneyama & Tanaka 1999; Koba et al. 2003; Houlton et al. 2006; Houlton et al. 2007; P rtl et al. 2007; Takebayashi et al. 2010; Yano et al. 2010) Continued methodological refinements will undoubt edly refine understanding of the terrestrial N cycle. The objective of this research was to examine the underlying 15 N variability among soil N pools (DON, NH 4 + NO 3 ), soil fertility, and fungal biomass within the black spr uce ecosystem of central Alaska. By measuring 15 N values of source N moieties, modeling soil fertility parameters, and integrating isotope effects associated with ECM activity, we sought to evaluate the influence of both sou rce and pathway on the N cycle along major pools in the soil fungi plant continuum. If plants simply trace soil N sources then black spruce 15 N va lues should be well correlated with soil N 15 N values even if ECM based fractionation was taken into account. In contrast, if ECM fungi respond to
50 their autotrophic host's mineral nutrient requirements, then soil fertility should modulate the effected ECM based fractionation in relation to the total p roportion of black spruce N derived from them. A corollary of this prediction would then be that ECM biomass and activity should decline where soil fertility is greatest. Black spruce forests are ideal systems in which to disentangle causes of 15 N variability because they form monodominant stands over a broad range of N limiting growth conditions ranging from low productivity stands on shallow permafrost soils to high productivity stands on well drained deep soils (Viereck & Johnston 1990; Chapin III et al. 2006; Hollingsworth et al. 2006; Ping et al. 2010) Because a pproximately 65% of Ala ska is covered by boreal forests of which black spruce forest is the most abundant type covering approximately 40 million ha (Vancleve & D yrness 1983), extrapolation of our findings to a larger geographical area will inform the greater boreal forest research community. Furthermore, because the N cycle is closely coupled to C sequestration, providing baseline data and interpretation of ecosy stem 15 N patterns can inform global change research as well. Methods Experimental D esign During 2007, 31 mature black spruce plots in central Alaska were selected and sampled from a pool of 146 previously established, 1 ha circular plots (Hollingsworth et al. 2006; Hollingswo rth et al. 2008) The selected plots encompass an area approximately 14,000 km 2 and were selected based on the criteria that they were unburned or cleared and represented the full range of foliar 15 N values pre viously observed from analyses of 90 of the 146 plots (M.C. Mack, unpublished data).
51 Sampling in the plots occurred along a 30 m belt transect centered on a permanent plot marker and arrayed perpendicular to the slope or arbitrarily if flat Cation excha nge capacity (CEC), pH, stand diameter at breast height (dbh), and active layer depth for these sites were obtained using standard methods previously detailed (Hollingsworth et al. 2006) Black spruce biomass was estimated using updated equations derived from approximately 15 sites spanning the geographic range of or stands (Yarie et al. 2007) B lack spruce fine roots were confirmed to be uniformly colonized by ECM fungi (Ruess et al. 2003) during root sampling in each stand. Fie ld and L aboratory A nalyses Foliar 15 N, %N, and %P, were obtained from mature black spruce terminal branches at the peak of needle expansion during August and September, 2007, to reduce variability as sociated with changing concentrations during needle expansion (Chapin & Kedrowski 1983) although a small portion of terminal needles represented previous years' growth Five full sun branches were collected from five trees in each plot, composited by tree and stored at 4 o C for less than 48 hours until drying. Preliminary analyses indicated that only three trees need be analyzed to encompass within plot variability. Small diameter (<2 mm) terminal root samples were also excavated from the same trees samp led for foliage and stored at 4 o C for approximately two weeks before processing. The thin outer layer of secondary root tissue was removed in order to prevent inclusion of external ECM biomass in subsequent elemental and isotopic analyses (H gberg et al. 1996) although some internal hyphae may have been present in the cortical tissue remaining Both needles and roots were dried at 60 o C for 24 hrs, ground to a fine powder, and analyzed on a ThermoFinnigan ¨ continuous flow isotope ratio mass spectrometer co upled to a Costech ¨ elemental
52 analyzer at the University of Florida (UF) Stable isotope abundances are reported as: 15 N = ( R sample / R standard 1) 1000, where R = 15 N: 14 N ratio of the sample or atmospheric N 2 standard. Run error rates were less than 0.2 as compared to NIST standards. T o compare relative plant available soil NO 3 NH 4 + and PO 4 + concentrations among plots, ion exchange resin beads were incubated throughout the entire 2007 growing season in accordance wi th methods detailed elsewhere (Giblin et al. 1994; Schuur & Matson 2001) During June 9 21, five replicate nylon mesh bags containing either 3 g of anion (Biorad ¨ AG 1 X8, 500g, 20 50 mesh, Cl form, #140 1421) or cation exchange resins (Biorad ¨ AG 50W X8, 500g, 20 50 mesh, H+ form, #142 1421) were inserted below sub fibric organic soils at depths of 5 20 cm depending on the depth of undecomposed surface mosses. This placement was chosen to match the area of highest root density of black spruce forest o bserved in minirhizotrons (Ruess et al. 2003) Each of the five paired cation and anion resin bags were placed approximately 20 cm apart in equidi stant locations along a 30 m belt transect. Resin bags were constructed of 220 m polyester mesh bags (6.25 in 2 ), acid washed, and triple D.I. rinsed prior to deployment for the growing season. In addition, five hyphal ingrowth mesh bags were inserted along the opposite side of the belt transect immediately above the organic mineral interface to measure activel y growing fungal mycelia (Wallander et al. 2001; Nilsson et al. 2004) The ingrowth bags were constructed of 52 m nylon mesh bags containing an average of 7 cm 3 of acid washed triple D.I. rinsed moist quartz sand. Three additional bags were inserted ins ide buried PVC collars in each plot to account for saprotrophic fungal
53 biomass, but because negligible biomass was found within collared bags corrective accounting was unnecessary. Both resin and hyphal ingrowth bags were removed from the field at the end of the growing season in early September just prior to the first frost, refrigerated, and transported to UF for processing. Resin bags were extracted with 100 mL of an acidified salt (Giblin et al. 1994) and extracts frozen prior to colorimetric ion anal ysis on an Astoria ¨ auto analyzer. Hyphal ingrowth bags were slo wly dried, suspended in a petri dish with water and carefully scored under a dissecting microscope for degree of colonization (1 = no hyphae, 2 = light and diffuse hyphae, 3 = extensive hypha e, 4 = extensive hyphae and light rhizomorphs, 5 = extensive hyphae and extensive rhizomorphs). Visual examination has previously been shown to correlate with weight and biochemical proxies of fungal biomass (Nilsson et al. 2004) T o obtain total dissolved nitrogen (TDN) samples, 2 M KCl soil extractions were made at the height of the growing season (mid July 2007 ) Three cores were extracted with an SMS ¨ volumetric slide hammer (4.2 cm diameter) and composited from four zones along each 30 m transect in each plot (12 total cores per plot, 4 field replicates). The cores were located within 2 m of resin or hyphal ingrowth bag positions equidistant along belt transects. The depth s of the organic, mineral, and total core w ere recorded, green moss removed, and the horizons bagged separately. Only organic soils were used in isotopic analyses due to the logistic limitations posed by the large number of samples. Total volumes extracted from each plot averaged nearly 1 m 3 of wet soil. Eac h composited soil sample was stored at 4 o C for ~24 hrs prior to homogenization and extraction. TDN was extracted from 24 g (wet weight ) subsamples placed in a pre cleaned and acid washed 250 mL HDPE plastic cup with 120 mL of 2 M KCl in nano
54 pure (Barnstea d Thermo Scientific ¨ ) water. For each new batch of 2 M KCl prepared, four 60 mL blanks were taken to correct for 15 N contamination associated batch specific impurities (Knapp et al. 2005) Soil and extractants were shaken on a reciprocal table for 20 minu tes and left undisturbed for 18 to 30 hours. The resulting supernatant was vacuum filtrated through glass fiber filter papers (Whatman ¨ 1820 070) in 9 cm HDPE Buchner funnels in the E cosystem E cology L aboratory at University of Alaska, Fairbanks. Extract s were frozen in replicate 50 m L centrifuge tubes and shipped to the UF for subsequent isotopic analyses during the following year. Replicate 6 mL subsets of the salt extracted TDN and NH 4 + ions were oxidized to NO 3 using persulfate/thermo digestion (Cabr era & Beare 1993; Doyle et al. 2004) with an oxidant to sample ratio of 1:1.5. For each oxidation, standard solutions of 1, 5, and 10 mg of KNO 3 glycine, and 6 Aminocaproic acid (ACA) were used to asses s digestion efficiency. It was found that digestio n efficiency of 10 mg /L of AC A, the largest molecular weight and recalcitrant compound (MW = 131.17) at the highest concentration was improved to >87% at 0.5 M salinity (versus 2 M NaCl or KCl) presumably due to the interference of Cl with free radical generation (Peyton 1993) Accordingly, all samples were diluted to 0.5 M with nano pure water prior to oxidation. Following oxidation, 15 N values from the resultant NO 3 were measured using the highly sens itive bacterial denitrifier technique (Sigman et al. 2001; Knapp et al. 2005) This method involves : culturing of a naturally occurrin g denitrifying bacterial strain ( P. aureofasciens ); injection of small (~ 0.5 g N) amounts of NO 3 under anaerobic condit ions ; an overnight incubation period ; then in line cryo purification of the resulting gaseous N 2 O for isotopic analyses. All samples were analyzed on a gas sampler arm
55 coupled to a ThermoFinnigan ¨ continuous flow isotope ratio mass spectrometer at UF Ex tracted dissolved organic N (DON) was calculated using a mass weighted equation based on the original 2 M KCl concentration of ions ([N]) and resin extracted NH 4 + and NO 3 isotope values: 15 N DON = ( 15 N TDN [TDN] ( 15 N NH 4 + [NH 4 + ] + 15 N NO 3 [NO 3 ])) / [DON] ( 3 1) Using mineral N obtained by in situ exchange resins should be superior to salt extractions because the exchange resins are time integrated. I norganic and amino acid N pools have a very short mean residence time associated with episodic production and removal (Jones & Kielland 2002; Booth et al. 2005) These events' and their associated isotope discrimination patterns may be missed by single time point soil extractions that may vary from those from exchange resins (Koba et al. 2003) and which may change markedly within days to months (Evans 2007) For instance organic N extracted with i on exchange resins reduces the potentially severe isotope fractionation ( 16.5 ) of NO 3 associated with typical salt extraction s (Hales & Ross 2008) Furthermore, fractionation due to NH 4 + diffusion gradients around resins was unlikely because Biorad ¨ A G 50W X8 exchange resins have been shown to produce negligible effects on isotope fractions even in the presence of potentially interfering cations and dissolved organics (Lehmann et al. 2001) Phospholipid fatty acid analyses (PLFA) were conducted on or iginal frozen soil subsamples. From one to three 1 5 g wet weight subsamples were analyzed for PLFA's from the composited soil core replicates used to extract N. Nine of the 31 plots were run in triplicate. This process involved an initial lipid extract ion, fractionation, and successive elution (Frosteg rd et al. 1991) followed by conversion of the methanol
56 fraction into free methyl esters by mild alkaline methanolysis, and then analysis on a gas chromatograph with a flame ionization detector and a 50 m HP5 capillary column (Frosteg rd et al. 1993) The PLFA 18:2 6,9 was regarded as an indicator of total fungal biomass (Frosteg rd & B th 1996) and is likely to be comprised mainly of ECM fungi in spruce forest as the majority of the microbial biomass in boreal forest ecosystems is known to be predominantly ECM fungi (Allison et al. 2007; Lindahl et al. 2007; Taylor et al. 2010) We used the first PCA axis (contained 57% of the variability ) containing both fungal and bacterial PLFA's because of the capaci ty to incorporate additional microbial changes. Regardless, the first PCA axis was positively correlated with fungal:bacterial PLFA ratios ( R 2 = 0.83) and total mol% of the fungal PLFA 18:2 w 6,9 ( R 2 = 0.93) indicating that the variables were interchangeable. Of the thirty PLFA's retained for a PCA the PLFA's i 15:0, a 15:0, 15:0, 10 Me 16:0, i 17:0, a 7:0, cy 17:0, 10 Me 17:0, br 18:0, 10 Me 18:0, and cy 19:0 were used as an indicator of bacterial biomass and t he PLFA 18:2 6,9 as an indicator of total fungal biomass (Frosteg rd & B th 1996) Changes in the PLFA 18:1 9 was also used as corroborated evidence for changes in fungal biomass, although it was not used in calculation of a fungal:bacterial biomass inde x. Statistical A nalyses Linear regressions were constructed on the following response variables: foliar 15 N, %N, and %P; soil 15 N values of DON and NH 4 + ; standing aboveground biomass of black spruce; and belowground biomass of fungi. For each model we wished to reduce our explanatory variables to prevent inflation of Type I error rates (Harrell 2001) In particular, most soil fertility vari ables were reduced to single PC ax e s. This
57 partial variable reduction allowed specific variables of interest to be retained in subsequent models depending on the response variable being modeled. For instance, when modeling foliar N content, DON and resin extractable mineral N were retained as independent predictors whereas the remaining soil fertility variables were reduced to a single PC axis. Similarly, for foliar P, resin extractable PO 4 was withheld from it's PCA, and in the case of foliar 15 N, the 15 N values of the soil N forms were withheld. Fungal biomass was withh eld from all soil fertility PC analyses so that the explanatory power of this variable could be independently assessed. Pr edictor variables were standardized by subtracting from the mean and dividing by the standard deviation (Schielzeth 2010) Independence of explanatory variables was assessed with scatter plot matrices and variance inflation factors to assure a lack of mul ticolinearity and to detect severe outliers. PCA was conducted using JMP ¨ 8.0.2 (SAS Institute Inc., Cary, NC, USA). Second order bias corrected Akaike information criterion ( # i = AICc i AICc min ) was used to rank models along, with model probabilities ( i = exp[ $ # i ] / exp[ $ # ]), because it is generally regarded as an unbiased estimator that assess relative model fit, penalizes over parameterization, and allows multiple working hypotheses to be simultaneously evaluated (Burnham & Anderson 2004; Andersen 2008) In contrast, fitting all possible models or using stepwise model selection has potential to inflate Type I error rates and to fail selection of the most informative model (Whittingham et al. 2006; Andersen 2008) but see Murtaugh (2009) Graphical diagnostics of residuals against fitted values and sample quantile against theoretical quantile plots were used to assess underlying distributional assumptions for all high ranking models. Model fitting and
58 diagnostics were performed using the R statistical environment (2.9.2, The R ¨ Foundation for statistical computing 2009). Isotope M ass B alance Building upon the 15 N mass balance models described previ ously for arctic tundra (Hobbie & Hobbie 2006; Hobbie & Hobbie 2008; Yano et al. 2010) we estimated the proportional dependence of black spruce for ECM derived N iteratively within each of our sites using the following mathematically under determined equa tions: 15 N available = NH4 15 N NH4 + DON 15 N DON ( 3 2 ) 15 N available =(1 T r ) 15 N fungi + T r 15 N transfer ( 3 3 ) 15 N plant = 15 N available # (1 T r ) ( 3 4) 15 N fungi = 15 N available + # T r (3 5 ) w here NH4 and DON in equation ( 3 2 ) refer to the fraction of these soil N forms contributing to the available N pool T r in equation ( 3 3 ) refers to the proportion of total fungal N that is transferred to host plants, 15 N transfer refers to the 15 N value of the transfer compounds produced by ECM fungi and in equation ( 3 4 ) refe rs to the proportion of plant N supplied by fungi and # refers to the fractionation magnitude associated with transamination of soil N within ECM fungi (Hobbie & Hobbie 2008) We quantitatively constrained the mass balance solutions in the following wa y: ( a ) The fraction of plant N delivered by ECM fungi ( ) cannot exceed 100% of the trees' N supply; ( b ) Mixtures of the different N forms occur at 10% increments; ( c ) # was defined as 8 10 based on laboratory, field, and meta analyses as described in de tail elsewhere (Hobbie & Hobbie 2006; Hobbie & Hobbie 2008) ; and ( d ) similar to Yano et
59 al (2010) the proportional use of NH 4 + ( NH4 ) was initially assumed to contribute less or equally weighted contribution (e.g. 10 50%) of total N uptake by plants or fungi, however higher proportions were permitted in order to meet constraint ( a ) where necessary. High DON reliance by black spruce is justified because mineral N supplies in boreal ecosystems cannot typically meet annual tree N requirements (Ruess et al. 1996; Read et al. 2004; Valentine et al. 2006) The 15 N values from root tissue were used instead of t hose from foliage because this permits accounting of internal plant fractionation (Robinson 2001) In addition, IsoError ¨ 1.04 (Phillips & Gregg 2001) was used to estimate proportional mixing and to generate confidence intervals associated with a two sour ce pool (e.g. ECM fungal derived and direct uptake of N) mixture using data averaged across all plots. Results Tree B iomass and S oil F ertility in B lack S pruce S tands Black spruce exhibited a broad range of isotopic, elemental, and biomass values across the plots (Table 3 1). For instance, above ground black spruce biomass ranged from 2.8 to 100 Mg ha 1 in part due to changes in soil fertility. Soil fer tility variables such as active layer depth, soil moisture, pH, CEC, C:N, and mineral and organic nutrient contents (Table 3 2 ) were reduced to a single PC axis that accounted for substantial variation in black spruce biomass ( R 2 adj = 0.33, P = 0.003). Fungal biomass, in contrast was not retaine d in high ranking explanatory models (Table 3 3). Upon further inspection, much of the explanatory power of the soil fertility PC axis can be attributed to soil C:N values alone ( R 2 adj = 0.20, P = 0.0 08), not active layer depth, pH, or soil moisture, as might be expected under growth limiting conditions.
60 The pool of soil dissolved organic N (DON) ranged from 148 to 602 g g 1 in organic soils, a value six times greater th an in mineral soils (Table 3 2 ). Resin exchangeable NH 4 + and NO 3 concentrations were extremely low in many plots, ranging from 0.00 to 3.70 and 0.00 to 0.55 ng N g 1 resin d 1 respectively (Table 3 2). Resin exchangeable NO 3 levels in particular were undetectable in all but seven plots, and in those seven, compositing of replicates was necessary to provide sufficient N for 15 N measurements using the denitrifier method. NH 4 + in contrast, were at detectable concentrations in all but three of the plots. Resin exchangeable PO 4 concentration was nearly 12 times higher and 16 times more variable than NH 4 + (Table 3 2). The greater variability could be due to variation in depth of the relatively unweathered parent material and low PO 4 mobility in organic soils. Black S pruce F oliar 15 N, %N, and %P P atterns A cross the L andscape Foliar 15 N values in black spruce trees were strongly correlated with both foliar N ( R 2 = 0.46, P < 0.001) and foliar P content ( R 2 = 0.51, P < 0.001) across the 31 plots (Figure 3 1). Collectively, foliar 15 N values in this single s pecies varied by 6.5 a range that encompasses the lower third of foliar 15 N va lues observed from 9,757 plant samples world wide (Craine et al. 2009) Root 15 N values were on average 2.35 les s depleted than tree needles (Figure 3 2 ) and were positively correlated with foliar 15 N values although not strongly so ( R 2 = 0.30, P = 0.002 ). The slo pe of the relationship was not 1:1 as expected either ( 15 N foliar = 4.69 + 0.51 15 N root ). Instead root and foliar 15 N values converged at the least negative plant 15 N values (Figure 3 2 ). Multiple regr ession model selection indicated that foliar 15 N values were best explained by the 1 st soil fertility PC axis ( R 2 adj = 0.21), and in two of the three highest
61 ranking models, 15 N NH4 alone or with 15 N DON were also retained ( R 2 adj = 0.25 and 0.30, respectively; Table 3 3). These models did not include foliar N content (which doubles the R 2 adj ) because soil variables were of primary mechanistic interest. Foliar N cont ent, in turn, was best explained by models containing the DON content of organic soils ( g g 1 soil), either singly ( R 2 adj = 0.25 ) or in combination with either PLFA based fungal biomass ( R 2 adj = 0.35), resin extracted mineral N (ng g 1 resin, R 2 adj = 0.25), or a soil fertility PC axis excluding soil N pools ( R 2 adj = 0.31; Table 2 3). The model with the best explanatory power and weight included both the DON and fungal biomass, expressed as the first PC of the PLFA pattern ( R 2 adj = 0.35, i = 0.21). Variation in black spruce foliar P content (g g 1 ) was best explained by models containing both soil PO 4 + (ng g 1 of resin day 1 ) and either a soil fertility PC axis or an interaction with hyphal ing rowth biomass ( R 2 adj = 0.59 0.60; Table 3 3). The most parsimonious candidate model of lesser model probability ( i = 0.19 vs. 0.30) contained only the soil fertility principle component without losing substantial explanatory power ( R 2 adj = 0.56). Sporocarp 15 N and F ungal B iomass Using a discriminant model based on over 800 sporocarp 15 N and 13 C global patterns (Mayor et al. 2009) we confirmed that all sporocarps used in our analysis were likely to be ectomycorrhizal at >90% probability (data not shown). The majority of analyzed sporocarps were species of Cortinarius, Dermocybe, or Russula, based on macroscopic characters. On average the ECM sporocarps were 13.8 more 15 N enriched than black spruce foliage (Table 3 1), and, on average, similar to the mean for ECM sporocarps globally (Mayor et al. 2009) and elsewhere in Alaska (Clemmensen et al. 2008; Hobbie et a l. 2009) Sporocarp 15 N values were negatively correlated with g
62 DON m 2 in organic soil ( R 2 = 0.39; Figure 3 3), but not mineral N (as meas ured from ion exchange resins). Diagnostics of the two influential values were not indicative of gross outliers (Cook's distance <0.2, overall Leverage <0.5), although removal of the two values with the highest g DON m 2 reduce s the R 2 to 0.17. Sporocarp 15 N values were not explicitly modeled in a multivar iate context because of low sample size ( N = 17) limiting the number of predictor variables available to just two, although scatterplot matrices indicated no correlation with soil N 15 N v alues in a univariate context. The estimates of fungal biomass in organ ic soils, represented as a PLFA based principle component, were positively correlated with C:N ratios (g g 1 ) of the organic soil ( R 2 = 0.66, Figure 3 4) and were uncorrelated with hyphal ingrowth or other soil ferti lity metrics. Subsequent model selection confirmed that fungal biomass increased across the landscape in relation to soil C:N ratio alone ( i = 0.57). More complex models containing DON ( g g 1 soil) and CEC (meq) were less probable ( i = 0.19 or 0.18, respectively; Table 3 3). Of the four hyphal ingrowth bags with appreciable colonization, mean 15 N values indicated ~6 depletion (mean = 0.35 ) and on average 1.7% less N than ECM sporocarps. 15 N Patterns of Soil N Forms Across the Landscape As seen graphically, 26 of the 31 plots had more enriched mean 15 N DON values than 15 N NH4 yet no covariance was found and the few 15 N NO3 values that could be obtained lacked any pattern (Figure 3 5). Post hoc paired t tests indicated that average 15 N DON and 15 N NH4 values differed from each other and that of the 15 N NO3 and 15 N of bulk organic soil ( P < 0.001). Overall, the ranking pattern observed was: 15 N DON > 15 N NH4 > 15 N NO3 % 15 N soil (Figure 3 5). Despite overall differences in many soil N 15 N
63 values the variation across sites was unrelated to the parameter combinations used in the regression analysis and was uncorrelated with plant or fungal 15 N values (Figure 3 2 & 3 5). The predictor variables used were based on wha t were likely to be direct inputs to either the DON or NH 4 + pool measured in our system: the biomass of fungi, black spruce root s, and foliage for 15 N DON ; and 15 N DON for 15 N NH4 Because enrichment factors ( = 15 N foliar 15 N soil ) have been employed to standardize comparisons of foliar 15 N across sites (Garten Jr. & Van Miegroet 1994; Martinelli et al. 1999; Vervaet et al. 2002; Amundson et al. 2003; Kahmen et al. 2008; Takebayashi et al. 2010) we evaluated the following permutations: = 15 N foliar 15 N soil 15 N DON and 15 N NH4 Correlation among these factors and metrics of soil N availability (e. g. DON, resin NH 4 + and C:N) were then examined i n an attempt to differentiate N rich from N poor sites. Only the based on 15 N foliar 15 N NH4 exhibited a positive relationship with resin [NH 4 + ] ( R 2 adj = 0.16, P = 0.014; [ NH4 res ] = 2.42 + 0.133 15Nfoliar 15N H4 ). Modeling F ractionation of 15 N in ECM F ungi and T ransfer to B lack S pruce Using measured 15 N values of black spruce roots, soil N moieties, and ECM sporocarps (where available) from each plot left only two factors to define in equations 3 2 to 3 5 First, we iteratively set the fractionation effects attributed to ECM fungal transamination of transfer compounds to = 8 10 (Hobbie & Hobbie 2006; Hobbie & Hobbie 2008) and the proportional use of NH 4 + ( NH4 ) was a priori assumed minor in accordance with previous modeling efforts in Alaskan tundra (Yano et al. 2010) and for reasons related to the DON abundance and use in black spruce ecosystems previously discussed. However, NH4 was increased to achieve sensible values i n many plots (e.g.
64 if proportional supply of plant N from ECM fungi was greater than 100%). Owing to the extremely low availability of NO 3 and its detection in only seven plots, it was omitted from all but four subsequent mass balance models where soluti ons required its inclusion and values were available ( Appendix C 3 ). Where sporocarp 15 N values were unavailable we first attempted to use the average value of all the plots (6.5 ) as there was no trend from which to interpo late (Figure 3 2 ). However, in many instances this value was too low to achieve less than 100% proportional supply of plant N from ECM fungi. This constraint required assignment of ECM sporocarp 15 N values equivalent to the most enriched sporocarps collected from one of our plots (11 ) in 15 of the mass balance models ( Appendix C 3 ). Plant, ECM sporocarp, and soil DON, NH 4 + and NO 3 15 N value end members in mixing equations were measured in r eplicate from each plot but variability among replicate measurements of N sources was not propagated through the models because of mathematical uncertainty associated with three sources contributing to one mixture. If measurement variability was included then the range of potential solutions would likely increase, yet the average ECM derived N would decrease slightly in order to prevent upper confidence interval bounds from exceeding 100%. Plot specific solutions suggest that the fraction of tree N nutrit ion derived from ECM fungi ( ) ranged 0.6 1.0 (average = 0.9) and the fraction of fungal N that was transferred to host plants ( T r ) ranged 0.08 0.65 depending on the proportion of NH 4 + contributing to total N supply ( Appendix C 3 ). T hese plot specific values were uncorrelated with our metrics of soil fertility (DON, NH 4 + pH, moisture, CEC, active layer depth) or metrics of tree or fungal biomass. However, values were positively
65 correlated with 15 N NH4 ( R 2 adj = 0.16, P = 0.01, y = 0.85 + 0.017 # ) and negatively correlated with both sporocarp and root 15 N values ( R 2 adj = 0.39, P = 0.004, y = 1.07 0.025 # ; R 2 adj = 0.16, P = 0.02, y = 0 .74 0.03 # respectively). Next we sought to conduct a single mass balance model using IsoError ¨ End member 15 N values of the mixture were simply the 15 N root value from plot averages, whereas the two sources represented the 15 N value of soil available N derived from ECM fungi ( 15 N available 9 ) where 15 N available was estimated from equation ( 3 2 ) with a priori N contributions conservatively set to 70% NH 4 + and 30% DON. The contribution of NH 4 + to total plant N nutrition could not be below 70% without exceeding estimates of plant N depende ncy on ECM fungi being greater than 100% of total plant N nutrition. In addition, fractionation magnitudes associated with the formation of N transfer com pounds by ECM fungi had to be & 9 With these parameters, the average ( SE) fraction of total plant N nutrition der ived from ECM fungi was 98.9% 0.06 (C.I. 86 100%) with the remainder derived from direct plant root uptake. Discussion Black S pruce E lemental P atterns The 6.5 variation in foliar 15 N values in this study encompassed the lower third of foliar 15 N values observed from 9 757 plants (900 sites, 1103 taxa) collected world wide (Craine et al. 2009) Comparable magnitudes of variation in foliar 15 N values were recorded in an additional 40 plots from central Alaska (M.C. Mack, unpublished data) and along a latitudinal transect spanning 2 (~150 km) f rom the Yukon River to the north slope of Alaska (Hobbie et al. 2009) These studies suggest that the black spruce
66 15 N patterns observed in this study are representative of a much larger geographical area. In agreement with theoretical expectations, linear model results supported the use of both foliar 15 N and %N as indices of site fertility. The best explanatory variable(s) for foliar N content was DON (g 1 ) content in the organic layer, eith er alone or in combination with fungal biomass or mineral N. This finding provides further support that DON is likely an important N source for these boreal forest trees (N sholm et al. 2009) Foliar 15 N, in turn, was best explained by the first soil fertility PC axis, either with or without specific 15 N values of soil N moieties (Table 3 3). In both instances parsimony warrants the use of models with single predictor variables, but inclusion of fungal biomass increased the explanatory power of foliar N content from R 2 adj = 0.25 to 0.35 supporting the idea that ECM fungi are important for black spruce N nutrition and predicting additional plot variability may warrant the additional effort involved with the inclusion of PLFA data. Strong N limiting growth conditions of black spruce trees were indicated by a n average needle N:P ratio of 7 ( g g 1 ), as found in plants growing from other high latitude terrestrial ecosystems (G sewell 2004) It is therefore likely that the close correlation between 15 N P and N concentrations in needles (Figure 3 1) represents a stoichiometric relationship where plant P trails behind foliar N concentration Although P is seldom considered to limit plant growth other than in water saturated boreal and tundra ecosystems, there appear to be non flooded conditions under which soil mi crobes experience P limitation (Giesler et al. 2004) which in turn, may influence N acquisition by black spruce if extracellular enzyme production by ECM fungi is impaired
67 (Alvarez et al. 2009) Foliar %P model selection indicated that the resin exchange able PO 4 + was informative in two of the three highest ranking models and that complex models including a soil fertility PCA axis or an interaction with hyphal ingrowth biomass were also informative (Table 3 3). This finding may be due to the well document ed advantage fungal hyphae imbue on plant P supply owing to enhanced surface area for nutrient absorption (Smith & Read 2008) We observed black spruce needles becoming less 15 N depleted with greater N content (Figure 3 1). This pattern is explain ed by the fungal fractionation hypothesis (Hobbie et al. 2009) where reduced de pendency of black spruce on ECM derived N at relatively higher levels of N availability would reduce 15 N depleted N transfer compounds from influencing foliage. This pro cess of vary ing dependency on ECM derived N from fungi is supported by laboratory (Hobbie et al. 2001b; Hobbie & Colpaert 2003) greenhouse (Hobbie et al. 2001a) and field observations along successional chronosequences (Hobbie et al. 2005; Compton et al. 2007; Hobbi e & Hobbie 2008) The same correlative pattern has also been found in other high latitude ECM forming tree species such as Abies lasiocarpa and Picea glauca in British Colombia (Kranabetter & MacKenzie 2010) Picea glauca in northern Alaska (Schulze et al 1994) Pinus strobus in Rhode Island (Compton et al. 2007) and several other taxa in lower Alaska including species of Picea Populus Salix and Tsuga (Hobbie et al. 2000) However, similar correlative patterns have also been described from trees associating with arbuscular mycorrhizal fungi along gradients of succession (Davidson et al. 2007) precipitation (Schuur & Matson 2001) and soil age (Vitousek et al. 1989) among others (Pardo et al. 2006; Craine et al. 2009) The presence of these patterns in non ECM
68 associating trees suggests additional mechanisms may be at work Therefore, it seems prudent to first account for the type of root symbionts prior to assessment of additional underlying mechanisms of foliar 15 N patterns, such as soil N status or climate (H gberg 1997; Craine et al. 2009; Pardo & Nadelhoffer 2010) Internal fractionation, such as during N translocation from root to nee dle, were indicated in black spruce by large differences in root and foliar 15 N values (Figure 3 2). Internal fractionation in plants can occur during resorption of foliar N, incorporation into organic compounds, or during translocation of N from organ to organ, and it is important to consider these effects because they could obscure the 15 N signature of the N sources (Evans 2001; Robinson 2001) Complicating interpretation, however, was t he finding that observed differences were not consistent across plots; they ranged from 0 to 4 a nd converged where tissue 15 N values were the least 15 N depleted (Figure 3 2) and N content was highest (Figure 3 1). In genera l, the average magnitude of the isotopic fractionation ( # 2.35 ) was in line with expectations based on patterns observed for other plants growing in mesic conditions (Gebauer 1993; N sholm 1994; Yoneyama 1995; H gberg 1997; Evans 2001) although exceptions do exist for unknown reasons. For instance, i n Austrian spruce ( Picea abies ) plantations, no 15 N differences between root and needle were found but a 1.5 difference was found in beech (P rtl et al. 2007) These patterns il lustrate that internal plant fractionating processes are not necessarily cons istent within or across species and may diminish under conditions relative 15 N enrichment or enhanced N availability. For this reason assignment of a single internal fractionatio n magnitude in isotope modeling efforts may not be accurate under all conditions.
69 What cause d the observed reduction in (or loss of) internal 15 N fractionation in black spruce? In other tree species internal 15 N fractionation shift s in response to water stress and topographic position (Bai et al. 2009) or under conditions where N supply exceeds N demand, particularly when NO 3 is the main N source (Evans 2001) However, we found no correlation among soil moisture or soil N availability with either foliar or root 15 N values and NO 3 levels were exceedingly low in our stands, often be low detection limits (Table 3 2). Furthermore, in both black spruce and temperate oaks there is no evidence for 15 N discrimin ation during resorption of foliar N prior to abscission (Kielland et al. 1998; Kolb & Evans 2002) Eliminating these causes leaves varying N processing and/or internal allocation patterns as sources of observed 15 N fractionation in black spruce. Most pro cesses fractionate against 15 N, suggesting increased metabolic processing in the roots could cause the greater 15 N depletion observed in black spruce in the most 15 N depleted plots (Dijkstra et al. 2008) These processes could include, but are not limited to: greater incorporation of NH 4 + into glutamine via glutamine synthetase (Werner & Schmidt 2002) ; greater levels of transamination in roots relative to foliage during growth (Shearer & Kohl 1986; Evans 2001) ; and/or altered amounts of organic N transpor ted through the xylem or leaked from roots (Dijkstra et al. 2003; Yoneyama et al. 2003) Without compound specific phloem and xylem 15 N analyses, however, we cannot determine which process es are driving the observed patterns of shifting internal 15 N fractionation in black spruce. Fungal B iomass and S porocarp 15 N values We observed a tendency toward 15 N enrichment of ECM sporocarps under conditions of relatively low s oil DON content ( R 2 = 0.39), a pattern driven in part by two
70 high DON measurements (Figure 3 3). Assuming black spruce N demand increases in response to low DON content, 15 N enrichment of ECM sporocarps could result from greater retention of 15 N enriched N during the increased production and transfer of 15 N depleted N to black spruce (H gberg et al. 1999a; Hobbie & Colpaert 2003) This is a corollary to the hypothesis elicited above to explain the observed pattern of foliar and root 15 N Retention of soil DON content and fungal biomass in models explaining foliar N further support the role of soil DON content i n black spruce dependency for ECM derived N (Table 3 3). However, there was no negative correlation among sporocarp and foliar 15 N values (Figure 3 2 ) as would be expected to corroborate this explanation. We urge caution in accepting these patterns due to the small replication of opportunistically collected sporocarps from plots. The 15 N values are based on sporocarp samples that were replicated in only three of 17 sites. Single sporocarp 15 N values may be inaccurate representations of the 15 N values of the total fungal community if the sampled species accesses either a unique soil substrate or horizons (Dickie et al. 2002; Tedersoo et al. 2003; Lindahl et al. 2007; Wallander et al. 2009) or is of an unrepresentative age or exploration type (T rudell et al. 2004; Hobbie & Agerer 2009) It has also been suggested that organic N use itself, independent of the interaction with host trees, can contribute to 15 N enrichment of fungi relative to those constrained to only mineral N sources (Henn & Chap ela 2004; Trudell et al. 2004) This mechanism, however, has yet to be demonstrated in the field and likely wouldn't account for variation across the gradient if the majority of ECM taxa associated with black spruce access DON.
71 Variation in fungal biomas s was well explained and positively correlated with the parsimonious regression model containing only the C:N ratio of organic soil ( R 2 = 0.65; Table 3 3) ; an index that varied 20 units (25 to 45) largely in response to changes in soil N content. Positive relationships between fungal biomass and C:N ratios in surface soils have also been described from other forests as well (Nilsson et al. 2005; Smith & Read 2008; Wallander et al. 2009) Of the 180 hyphal ingrowth bags deployed throughout the growing season, very few produced enough tissue for 15 N measurement. This result could be due to the lack of growth limiting mineral nutrients contained within the acid washed sand and the relatively short incubation period (Wallander et al. 2004; Hendricks et al. 2006) In accordance with our observations, hyphae in other ecosystems dominated by ECM plants have also been observed to be 15 N depleted relative to co occur ring ECM sporocarps, possibly due to N being preferentially reallocated from evacuated' hyphae to protein and N rich sporocarps during development (Wallander et al. 2004; Clemmensen et al. 2006; Bostr m et al. 2007) This pattern was recently explained u sing two pool mass balance equations derived to account for 15 N patterns of differing ECM fungi exploration types (Hobbie & Agerer 2009) It is also possible that the small amounts of harvestable mycelium contained in our in growth bags could have been from fast growing saprotrophic fungi know to have 15 N values that are less enriched than ECM fungi (Mayor et al. 2009) F ungal biomass estimates range from 2.0 10 3 kg ha 1 in tussock tundra to 9.5 kg ha 1 temperate forest (Clemmensen et al. 2006) and 4.8 10 3 kg ha 1 in spruce to 5.8 10 3 kg ha 1 in mixed boreal forests (Wallander et al. 2004) These large pools of
72 ECM biomass are thought to contribute to observed 15 N enrichment of soil N with depth, forest maturation, and labile DON pools (Hobbie & Ouimette 2009; Wallander et al. 2009; Takebayashi et al. 2010) Microbial processing of these fungi derived N pools may itself lead to further 15 N enrichment as well (Kramer et al. 2003; Dijkstra et al. 2008) Foliar leaching of N and P is largely intercepted by feather and sphagnum mosses (Weber & Van Cleve 1984; Chapin III et al. 1987) leaving soil organic matter, and the resulting labile N, to be derived from slowly decomposing black spruce and moss litter (Smith et al. 1999) along with large quantities of 15 N enriched fungal biomass in spruce forests (H gberg & H gberg 2002) 15 N P atterns of S oil N M oieties A cross the L andscape Measuring specific forms of N in replicate across the landscape revealed substantial variability but different mean 15 N values of soil N moieties (Table 3 2, Figure 3 5). The extractable DON pool w as more 15 N enriched than resin extractable NH 4 + and bulk soil organic matter in 77% and 100% of the plots, respectively. Despite these overall differences, variability among 15 N values of N forms were not explained with the parameters used in regression models (Table 3 2) suggesting soil N isotope cycling may vary independently of plant N cycles. This is in agreement with models suggesting only inputs, hydrological leaching, and gaseous losses influence soil 15 N values (Amundson et al. 2003; Bai et al. 2009) As Pleurozium and Hylocomium mosses in eight of our plots had relatively consistent mean ( SE) 15 N values of 2.98 0.31 (unpublished data), inputs were l ikely from belowground sources. The 15 N values of individual soil N moieties were also uncorrelated with those from plant tissues or sporocarp s (Figure 3 2) indicating that source N cannot simply be traced without
73 accounting for fractionation along the way. These complex and uncorrelated 15 N patterns among the pools of cycling N support the claim that 15 N values of soil N, and especially bulk soil organic matter, may not approximate plant 15 N values in many N limited ecosystems (Evans 2001; Pardo et al. 2006) As mentioned many researchers have used factors to link observed plant and bulk soil 15 N values with the N status (Emmett et al. 1998) net nitrification (Garten Jr. & Van Miegroet 1994) or mineralization potential (Kahmen et al. 2008) In black spruce forest, only based on the difference between 15 N foliar and 15 N NH4 exhibited a positive relationship with resin NH 4 + availability ( R 2 = 0.19, P = 0.01). However, the low total variation explained and lack of correlation among based on bulk soil organic matter 15 N does not support continued use of enrichment factors to adjust foliar 15 N values across sites prior to elucidation of underlying soil N isotope patterns. We suggest that use of these corrections may mask natural complexity and limit accurate modeling of ultimate causes of plant 15 N variability in many ecosystems, part icularly those dominated by ECM forming trees. Black spruce soil N 15 N measurements ( Table 3 2) correspond with previously described patterns from tropical, temperate, a nd tundra ecosystems as follows : 15 N DON > 15 N amino acid > 15 N NH4 > 15 N bulk > 15 N NO3 (Houlton et al. 2007; Takebayashi et al. 2010; Yano et al. 2010) The 15 N DON may be enriched relative to other N forms due to inputs of 15 N enriched materials, such as fungal biomass discussed above, or preferential use and export of isoto pically light N compounds (Schmidt et al. 2006) However, this latter effect would require relatively 15 N depleted decomposition products to be removed from the system through leaching volatilization or stabilization in
74 underlying permafrost as previously hypothesized (Hobbie et al. 2009) The 15 N value of NH 4 + is generally considered isotopically lighter than its substrate due to enzymatic preference for 14 N bound poly meric N (Silfer et al. 1992; Nadelhoffer & Fry 1994) This pattern was supported in 77% of our 31 plots yet the magnitude of the differences varied appreciably (Figure 3 5). In addition to variability attributed to the source DON pool, NH 4 + is also being incorporated into organic N mainly via the glutamine synthetase pathway, which may be fractionating against 15 N (Werner & Schmidt 2002) In effect, increased demand for NH 4 + by soil microbes could increase the 15 N values of residual NH 4 + and as it cycles through microbial populations it may be subjected to multiple competing reactions, each with different fractionation factors (Evans 2007; Hobbie & Ouimette 2009) that could contribute to the complex soil patterns in black spruce forest. Other studies report amino acid 15 N values ranging from 8.7 to +8.1 (Melillo et al. 1989; Silfer et al. 1992; Ostle et al. 1999; Werner & Schmidt 2002; Bol et al. 2008; Yano et al. 2010) Furthermore, as soil organic matter increases in recalcitrance (e.g. aliphaticity) it has been observed to become gradually 15 N enrich ed ranging from 6 at low aliphaticity to 13 at high aliphaticity (Kramer e t al. 2003) This potentially broad range of DON substrate 15 N values suggests compound specific absorption could substantially influence black spruce 15 N. In tussock tundra of northern Alaska amino acid 15 N values were 15 N depleted relative to the total DON pool by as much as 10 (Yano et al. 2010). However, many species of ECM forming fungi can enzymatically degrade large molecular weight DON compounds such as chitin, proteins, and peptides (Abuzinadah & Read 1986; Abuzinadah & Read 1988; Lindahl & Taylor 2004; Read et al. 2004; Nygren et al. 2007; B deker et al. 2009; Talbot & Treseder 2010) that are
75 typically more 15 N enriched than amino acids (Werner & Schmidt 2002) Furthermore, black spruce forest soils have some of the highest protease activities of all taiga ecosystems (Kielland et al. 2007) and despite the high volume of protein binding tannins present in boreal soils (Joanisse et al. 200 9) it is likely that access to numerous DON compounds would smooth out substrate based isotopic differences. Therefore, regardless of the small proportion of directly absorbable amino acid N in the DON pool (Chalot et al. 2002; Jones & Kielland 2002; Jon es et al. 2005a) the 15 N DON in black spruce forest is likely to match the combined cumulative 15 N value of the numerous organic N compounds accessed by black spr uce and their associated ECM. Modeling N T ransfer to B lack S pruce Both mass balance methods, one based on detailed mechanistic modeling (Hobbie & Hobbie 2006) applied to plot by plot estimates, the other based on simple yet sensible a p riori two pool mixtures and fungal fractionation effects, illustrate a high dependency of blac k spruce on ECM derived N ( = 60 100%, averaging 89% or 98.9%, respectively ; Appendix C 3 ). Furthermore both modeling efforts illustrate the large contribution DON (30 49% on average) must make to meet N requirements for growth in these strongly N limited boreal forest ecosystems. The resulting dependencies on ECM derived N ( ) from individual modeling efforts indicated a positive correlation with the 15 N NH4 source and negative correlations with sporocarp and root 15 N values. The strongest negative correlation with sporocarp 15 N values appears at first glance to run counter to the fungal fractionation hypothesis where more enriched sporocarps might indicate greater proportional delivery of N and retention of 15 N enriched N. However, the particularly enriched sporocarp 15 N values may also
76 merely reflect specific peculiarities associated with those specific fungal taxa such as: access to enriched substrates, growth in deeper soil horizons, or, as previously mentioned, they could be generally unrepres entative of the greater ECM fungal communi ty in those plots. If the enriched DON 15 N values we measured were not representative of the proportion of DON that is actually bio available, and that smaller proportion is isotopica lly depleted relative to the total DON pool, then our estimates of ECM fungal delivery of N ( ) are exaggerated. This possibility exists because the actual 15 N available end member would be closer to that of the black spruce root 15 N value requiring less N transfer through ECM fungi to account for isotopic differences between source and sink. Recent partitioning of soil DON pool 15 N values (Yano et al. 2010) while a step in the right direction, does not directly address this issue because the question of what constitutes the bioavailable portion of DON is not easily determined in the se ECM dominated organic soils. Conclusions The mixing model and multiple regression data support the idea that ECM fungi are integral to meeting the N requirements of black spruce and DON contributes directly to black spruce N nutrition without complete mineralization prior to uptake. These findings are sensible given t he strong selective pressure for access to this abun dant N pool under such severe N limiting conditions (Neff et al. 2003; Schimel & Bennett 2004; Kranabetter et al. 2007; N sholm et al. 2009) T he variation in foliar 15 N and %N we re indicative of low N availability leading to a condition directly, via ECM, or indirectly, via soil biophysical proce sses, limits N bioavailability Because i nternal 15 N fractionation in
77 black spruce roots and foliage was not consistent along the gradient future research should seek to clarify mechanisms Finally, although factors based on 15 N foliar 15 N NH4 exhibited a positive relationship with resin [NH 4 + ], we caution against widespread use of factors without detailed soil isotopic evaluations.
78 Table 3 1 Black spruce and fungal elemental conten ts and biomass across 3 1 central Alaskan forests. PLANT & FUNGAL SUMMARY CHARACTERISTICS foliar %P foliar %N foliar 15 N foliar 13 C root 15 N fungi 15 N Hyphal ingrowth Fungal PLFA PC1 Total biomass (ton ha 1 ) mean 0.11 0.74 7.32 28.22 4.97 6.51 1.79 0.02 37.36 n 31 31 31 31 28 17 31 30 30 sd 0.03 0.13 1.49 0.6 1.5 2.54 0.47 0.42 27.69 range 0.08 0.47 6.55 2.53 5.98 9.48 1.66 1.73 97.36 Table 3 2 Soil fertility measurements made across 31 central Alaskan black spruce forests. DON org ( g N/g dry soil) DON min ( g N/g dry soil) NH4 res (ng N/g resin/day) NO3 res (ng N/g resin/day) DON 15 N NH4 15 N NO3 15 N Soil 15 N C:N org % PO4 res (ng N/g re sin/day) CEC (meq) soil moisture pH active layer depth (cm) mean 301 54.3 1.07 0.13 6.49 2.83 0.78 0.42 38 12.5 18.3 26.9 5.58 63 n 31 29 31 31 31 31 7 31 31 31 30 30 30 30 sd 109 36 0.95 0.14 2.86 2.61 6.34 0.91 5.78 16.7 6.85 5.6 0.7 25.9 range 455 163 3.70 10.3 10.4 10.3 15.1 3.87 22.8 58.7 27.3 22.32 2.9 126
79 Table 3 3 High rank ing multiple regression models from 31 central Alaskan black spruce forest. Model/Hypothesis df i i E i,j R 2 adj foliarN ~ DON org 29 0.00 0.22 1.00 0.25 foliarN ~ DON org + FunPC1 27 0.17 0.21 1.09 0.35 foliarN ~ DON org + MinN res 28 1.50 0.11 2.11 0.25 foliarN ~ DON org + N PC1 28 1.56 0.10 2.17 0.31 foliarN ~ DON org + MinN res + FunPC1 26 1.69 0.10 2.32 0.35 foliarP ~ P PC1 + log(PO4 res) 27 0.00 0.30 1.00 0.6 0 foliarP ~ log(PO4 res)*Hyphae 27 0.00 0.30 0.98 0.59 foliarP ~ P PC1 29 0.97 0.19 1.59 0.56 foliar 15 N ~ 15 N PC1 + 15 N NH4 27 0.00 0.33 1.00 0.25 foliar 15 N ~ 15 N PC1 28 0.07 0.32 1.04 0.21 foliar 15 N ~ 15 N PC1* 15 N DON + 15 N NH4 25 2.03 0.12 2.76 0.3 0 Spruce biomass ~ 15 N PC1 28 0.00 0.59 1.00 0.33 Spruce biomass ~ 15 N PC1 + foliarN 27 1.26 0.32 1.88 0.33 FunPC1 ~ C:N 28 0.00 0.57 1.00 0.65 FunPC1 ~ C:N + DON org 27 2.22 0.19 3.03 0.64 FunPC1 ~ C:N + CEC 27 2.29 0.18 3.14 0.64 df = degrees of freedom, i = bias corrected Aikake Information Criterion, i = model probability, E i,j = evidence ratio, R 2 adj = adjusted pearson's correlation coefficient foliarN = % (g g 1 ) N in black spruce foliage, DON org = concentration of 2M KCL extractable dissolved organic N from soils in the middle of the growing season, FunPC1 = PLFA based principle component axis representing fungal biomass, MinN res = mineral N measured from ion exchange resins, N PC1 = Principle c omponent axis containing soil fertility metrics without extractable N concentrations included; foliarP = % (g g 1 ) P in black spruce foliage, P PC1 = Principle component axis containing soil fertility metrics without extractable PO 4 concentration included PO4 res = resin extractable PO 4 concentration, Hyphae = hyphal ingrowth biomass metric; foliar 15 N = black spruce foliar 15 N value, 15 N PC1 = Principle component axis containing soil fertility metrics without soil N 15 N values such as 15 N NH4 or 15 N DON Spruce biomass = standing aboveground biomass of black spruce estimated from allometric equations applied to stand basal areas, C:N = carbon to nitrogen ratio of organic soils, CEC = cation exchange capacity of organic soils.
80 Figure 3 1 Average elemental content (%) of full sun foliage ( N = 3) collected from black spruce ( Picea mariana ) trees in 31 plots in central Alaska. Phosphorus % ( g 1 ): R 2 = 0.53, P < 0.001; Nitrogen %: R 2 = 0.46, P < 0.001.
81 Figure 3 2. A verage ( SE ) black spruce full sun needle, fine root, bulk organic soil, and ectomycorrhizal sporocarp 15 N values across 31 plots in central Alaska. Points are arbitrarily arrayed according to average foliar 15 N value s to illustrate the lack of strong covariance among components. Only foliar 15 N was significantly correlated with root 15 N (foliar 15 N = 4.69 + 0.51 15 N root R 2 = 0.30, P = 0.01). The slope for foliar 15 N (0.16) is nearly twice that of both the ECM sporocarps and black spruc e fine roots (0.09) although not statistically different Across all plots, ecosystem component s w ere different from one another (paired T test, P < 0.001) and the mean isotopic differences between foliage and root, foliage and soil, and foliage and fung al 15 N values were 2.35 7.74 and 14.12 respectively.
82 Figure 3 3 Re lation among organic soil DON content and fungal sporocarp 15 N values. R 2 = 0.39, f (x) = 0.9635 + 10.5225 with 15 degrees of freedom. Figure 3 4 R elation among C:N ratios of the organic black spruce soils with the Phospholipid Fatty Acid (PLFA) based metrics of fungal biomass represented as the first Principle Component (PC ) axis ( f (x) = 6.442E 2 + 2.458; R 2 = 0.65). The PC axis values are arbitrary but correspond to increasing fungal biomass at higher C:N ratios.
8 3 Figure 3 5 M ean ( N = 3, SE ) soil N 15 N values from dissolved organic N (DON), ammonium (NH 4 + ), and bulk organic soils across 31 plots in central Alaska. The N 15 N values on are arbitrarily sorted by 15 N DON values to examine covari ance among pools. The figure on the right is the mean ( SE) illustrating the differences among average pools (paired t test, P < 0.001).
84 CHAPTER 4 DETECTING ALTERED NI TROGEN CYCLES IN BLA C K SPRUCE FOREST FOLLOWING FERTILZATI ON USING SOIL, PLANT AND FUNGAL 15 N VALUES Abstract M any northern forests are limited by nitrogen (N) availability, slight changes in which can have profound effects on elemental cycling and diversity of plants and microbes. Because climate induced nutrient mineralization may increase N availability, experi mental manipulation of soil nutrient availability offers insight into possible future ecosystem conditions. However, because N uptake occurs largely in an opaque soil medium it remains difficult to assess ecosystem N supply and cycling pathways without fr equent measurements and the assembly of complete N budgets. Here we use detailed measurements of ecosystem stable isotope ratios of N ( 15 N) to examine the form and pathways of N cycling following five years of NH 4 NO 3 and/or super triple phosphate fertilization of mature black spruce forests in central Alaska. N fertilization reduced soil dissolved organic N (DON) content and when combined with P fertilizer, reduced black spruce dependency on ectomycorrhizal fungi by 36%. N fertilization also reduced the 15 N values of fungal sporocarp and black spruce needles, but not soil N or roots, to approach that of the fertilizer. Surprisingly, P fertilization altered the soil N cycle in these stands as e videnced by increase d NO 3 concentrations and substantial 15 N enrichment of resin exchangeable soil NO 3 relative to the control plots Fractionation against 15 N during denitrification could account for the isotopic enrichment in the NO 3 pool Combined, our experimental approach illustrated that fertilization altered soil fertility the pathways of N cycling, and the role of fungi in black spruce forest.
85 Introduction Increased terrestrial N availability is becoming a global issue with impacts extending beyond industrialized regions of North Eastern North America and Northern Europe (Matson et al. 2002; Galloway et al. 2008) In most boreal forests anthropogenic deposition is less of a problem, but lands cape modification and accelerated decomposition resulting from climatic warming can increase in situ N mineralization in otherwise severely N limited boreal ecosystems (Nadelhoffer et al. 1991; Mack et al. 2004; Hyv nen et al. 2007; Allison & Treseder 2008) As a result, productivity and ecosystem dynamics can be profoundly alter ed (Tamm 1990; Vitousek & Howarth 1991; Nordin et al. 2005; Elser et al. 2007) Therefore, determining baseline patterns in and key integrative sign als of, the N cycle are necessary to detect when natural N cycling conditions have been altered by climate, disturbance, or deposition. This research need is particularly strong in high latitude ecosystems where plants are heavily dependent on mycorrhizal fungi for access to slow cycling N pools. Boreal ecosystems subjected to i ncreased N availability may respond with greater carbon (C) fixation (H gberg et al. 2003) altered C allocation patterns (Nadelhoffer 2000; Vogel et al. 2008) accelerated nutrien t cycling (Mack et al. 2004) and shifts in plant community diversity (Shaver et al. 2001; Mack et al. 2004) Enhanced N availability can alter microbial communities in uncertain ways, perhaps owing to the inherent difficulties in detecting changes (Wall et al. 2010) For instance, the biomass of ectomycorrhizal ( ECM ) and ericoid mycorrhizal fungi can increase or decrease in response to N add ition (Clemmensen et al. 2006; Treseder 2008; Ishida & Nordin 2010; Janssens et al. 2010) and ECM, saprotrophic, a nd arbuscular mycorrhizal fungal community composition and diversity are typically altered or negatively affected
86 largely with unknown functional consequences (Wallenda & Kottke 1998; Egerton Warburton & Allen 2000; Lilleskov & Bruns 2001; Peter et al. 200 1; Avis et al. 2003; Nilsson et al. 2007) Changes in f ungal species assemblages, for instance, may affect elemental cycling and plant community structure through direct and indirect mechanisms related to C allocation, residual litter quality, and enzymatic capacity of different fungal species (Lilleskov et al. 2002a; Lilleskov et al. 2002b; Lucas & Casper 2008; H gberg et al. 2010a) Furthermore, reduced belowground C al location under increased nutrient availability may alter plant dependency on ECM derived N (Treseder & Vitousek 2001; Nilsson & Wallander 2003) further exacerbating shifts in community diversity and function (van der Heijden et al. 2008) Black spruce ( P icea mariana ) is the most prevalent tree in boreal forests of Alaska and Canada (Viereck & Johnston 1990) In these boreal forests N limitation is severe because cold temperatures limit both growing season length and organic matter decomposition These c onditions, combined with high ly recalcitran t moss and spruce litter slow the N cycle to the point where the majority of labile N is in organic, rather than mineral form. The resulting dissolved organic N (DON) pool typically outweigh s inorganic N by as m uch as 90% (Van Cleve & Yarie 1986; Jones & Kielland 2002; Yu et al. 2002) As growth requirements in Alaskan black spruce forest typically exceed annual N mineralization rates (Shaver & Chapin 1991; Shaver et al. 1992) a strong selective pre ssure for access to this DO N is believed both common and necessary (Lipson & Nasholm 2001; Neff et al. 2003; Schimel & Bennett 2004) Whereas, amino acid uptake occurs in numerous ecosystems, plant species, and mycorrhizal types (Wallenda & Read 1999; N sh olm et al. 2009; McFarland et al. 2010)
87 use of larger molecular weight organic N requires ECM fungal mediated depolymerization via exoenzyme (Talbot & Treseder 2010) or peroxidase production (B deker et al. 2009) Black spruce is extensively colonized by ECM fungi (Ruess et al. 2003) and both the autotrophic host and heterotrophic fungi can directly absorb a portion of the rapidly cycling amino acid pool (Jones & Kielland 2002; Allison et al. 2007; Kielland et al. 2007; Treseder et al. 2008) The E CM fun gi however, can also access a larger portion of organic N including proteins, peptides, and chitin (Abuzinadah & Read 1986; Abuzinadah & Read 1988; Lindahl & Taylor 2004; Read et al. 2004; Benjdia et al. 2006; Nygren et al. 2007) Furthermore, the observ ation that black spruce forests have the highest soil protease activities (e.g. the rate limiting step) recorded in central Alaskan boreal forest (Weintraub & Schimel 2005; Kielland et al. 2007) suggests that sustained productivity likely rests on continue d access to the DON pool by ECM fungi Stable isotope ratios of N ( 15 N: 14 N represented as 15 N ) measured in different ecosystem N pools may offer clues for detecting and quantifying the dependency of black spruce on different forms and pathways of N absorption. These 15 N values however, often have multiple underlying causes that must be disentangled (Craine et al. 2009; Pardo & Nadelhoffer 2010) For instance, global m ean annual temperature and precipitation correspond well with 15 N patterns in plants, and to a lesser degree, in fungi an d soils (Handley et al. 1999; Amundson et al. 2003; Mayor et al. 2009) At smaller landscape scale s, however, the main control s over plant 15 N appear to be N status (measured as foliar %N) preferred N forms (DON, NH 4 + NO 3 ) and the associated type or p resence of mycorrhizae (arbuscular, ericoid, or ectomycorrhizal) (Michelsen et al. 1998; H gberg et al. 1999a; Miller & Bowman 2002; Hobbie & Hobbie
88 2006; H gberg et al. 2010b) Because plant 15 N patterns are fundamentally influenced by isotope fractionation during microbial processing or losses of specific soil N forms as they leak' from an ecosystem (Gebauer & Schulze 1991; Nadelhoffer & Fry 1994; Nadelhoffer et al. 1996; H gberg 1997; Martinelli et al. 1999; Schuur & Matson 2001) measuring 15 N values in distinct soil pools is necessary to definitively resolve underlying mechanisms (Houlton et al. 2006; Pardo et al. 2006; Templer et al. 2007; Kahmen et al. 2008) Within a site, the types of soil N sources available may control plant foliar 15 N. Individual soil N moieties differ in 15 N values because of 15 N discrimination during ammonification, nitrification, and denitrification within soil (L tolle 1980; Mariotti et al. 1981; Shearer & Kohl 1986) Assuming incomplete conversion, the expectations are that as the N cycle progresses, N moieties become successively 15 N depleted so that 15 N DON > 15 N NH4 > 15 N NO3 > 15 N N2 These differences, with some caveats, can then theoretically be traced to plants (Chapin et al. 1993; Pate et al. 1993; Nadelhoffer et al. 1996; Robinson 2001; Miller & Bowman 2002; Sah et al. 2006; Miller et al. 2007) once the methodological challenge of measuring 15 N values of soil N moieties at field concentrations has been overcome. It is believed that plants may shift preference among N forms depending on factors such as forest disturbance (Pardo et al. 2002) climate induced changes to the most abundant N forms (Houlton et al. 2007) or in response to the application of fertilizer (H gberg 1997; H gberg et al. 2010b) Also, isotopic differences between mineral forms of N have been used to infer plant preferences of NH 4 + versus NO 3 in high latitude coniferous forest (Hobbie et al. 1998) temperate grasslands (Kahmen et al. 2008) and among co occurring shrubs in tundra
89 and alpine ecosystems (McKane et al. 2002; Miller et al. 2007) Recently, the addition of DON to these analyses has more accurately extended the sources of N that can contribute to plant nutrition (Houlton et al. 2007; Takebayashi et al. 2010; Yano et al. 2010) Further complicating the tracing of soil N to plants using 15 N values is the possibility that N can be taken up directly from soil through black spruce roots or transferred w ith heavy 15 N fractionation through ECM fungi (Hobbie & Colpaert 2003) Furthermore, small isotope effects may be incurred during N translocat ion from roots to foliage (Robinson 2001) Each of these steps and pathways must therefore be quantified in ord er to accurately model N flux. The degree of 15 N depletion in a given host plant is therefore expected to result from not only the sources of N, but also the proportional dependency on fungal derived N (Hobbie et al. 2000) followed by the translocation ef fects as N moves from root to needle. This sort of accounting has recently been demonstrated in tussock tundra (Hobbie & Hobbie 2006; Yano et al. 2010) and boreal forest undergoing forest succession (Hobbie et al. 2005) enabling the modeling of proportion al N and C flux with associated ECM fungi (Hobbie & Hobbie 2008) Expanding upon these pioneering approaches with inclusion of 15 N values from organic N pools illustrated that tussock tundra plants may depend on ericoid and ECM fungi for 30 60% of their total N requirements (Yano et al. 2010) and that black spruce may depend on ECM fungi for 86 99% N of their N uptake requirements (Mayor et al. Chapter 3 ). Here we examine the N cycling patterns using detailed measurements of 15 N in plants, fungi, and soil across 16 fertilized interior black spruce plots following 5 years of
90 mineral nutrient fertilization. The objective was to assess the utility of using 15 N values to understand the form and pathway of N cycling in black spruce forest changes under elevated N and phosphorus (P) avai lability in a full factorial manner. By combining detailed measure ments of ecosystem 15 N pattern s with metrics of fungal biomass and growth, we explicitly sought to examine hypothesized relationships between soil mineral nutrients, ECM fungi, and plant 15 N val ues. Given that productivity of black spruce is severely N limited, we hypothesized that N fertilization: causes plant 15 N values to become less 15 N depleted and ECM sporocarps to become less 15 N enriched as a result of a g eneral reduction in reliance on ECM derived N by black spruce and increased use of the fertilizer N by both black spruce and associated fungi. We also hypothesized that fungal biomass declines in response to alleviation of plant N, but not P demands, lead ing to a reduction of below ground C allocation as payment' to the fungi for N. In contrast, we expected P fertilization to not influence plant or sporocarp 15 N values or modeled dependency upon ECM fungi. With regards to soil N pools, we hypothesized that soil N 15 N values to become less variable under N fertilization and to match that of the mineral fertilizer following 5 years of N additions. We expected P fertilization to have no influenc e on soil N 15 N values in the absence of potential pH effects. Methods Site D escription Boreal forest is the second largest terrestrial biome in the world (Whittaker 1975) and b lack spruce ( Picea mariana (Mill.) BSP) dominated forest is the most abundant forest type in boreal North America (Viereck & Johnston 1990) encompassing some
91 40% of i nterior Alaska alone (Vancleve & Dyrness 1983) Its success in the landscape is attributed to extreme freezing tolerance, the ability to grow in shallow permafrost soils with impeded drainage, as well as the ability to grow on well drained productive upla nd sites (Chapin III et al. 2006) The experimental site was located approximately 15 km south of Delta Junction, AK, and consist s of 16 plots arrayed in four blocks of four 10 10 m 2 plots. The black spruce forests are mat ure (~80 years old) and classified as dry nonacidic blac k spruce forest (Hollingsworth et al. 2006) featuring relatively xeric c onditions with low rainfall (~30 0 mm yr 1 MAP ), cold conditions ( 2 o C MAT ), and a relatively shallow organic layer (6.3 cm O horizon, Table 1) compared to many black spruce forests The plant community overstory is dominated by black spruce but two of the blocks also contain a minor component of Populus tremuloides The understory consists of mi nor components of Betula glabra, Salix spp., Vaccinium vitis idaea, V. uliginosum, and Rhododendron groendlandicum ((Oeder) Kron & Judd) shrubs, along with 30 50% moss (mainly Pleurozium schreberi or Hylocomium splendens ) or lichen ( Cladina, Cladonia and Cetraria spp. ) cover (Treseder et al. 2004; Mack et al. 2008) Each treatment plot was fertilized in the early spring for 5 years prior to and including the 2007 summer field season when sampling for this study was conducted I n 2002, each plot received single broadcast doses of NH 4 + NO 3 (N) ortho PO 4 (P) both together (N+P) or none (control) annually at a level of 200, in year 1, or 100 kg ha 1 yr 1 in subsequent years, per nutrient
92 Field S ampling and L aboratory A nalyses Foliar 15 N, N, and P content ( % ) of needles were obtained from four mature black spruce trees in each plot. Five terminal full sun branches were collected from each tree and composited by tree at the peak of needle expansion during August 29 30, 2007. In addition, three ~2mm diameter roots were also carefully excavated from each of the same trees, composited, and refrigerated until outer secondary root tissue could be carefully removed approximate ly three weeks later. This step was necessary to prevent potential inclusion of fungal biomass in subsequent isotopic analyses (H gberg et al. 1996) although a minor component of ECM hyphae were likely present in the remaining root cortex Needle and ro ot tissue were dried at 60 o C for 24 hrs, ground to a fine powder, and analyzed on a ThermoFinnigan continuous flow isotope ratio mass spectrometer coupled to a Costech elemental analyzer at the University of Florida. Stable isotope abundances are reported as: 15 N = ( R sample / R standard 1) 1 000 (4 1) R = 15 N/ 14 N refers to the ratio of the sample and reference standard of atmospheric N 2 Run standard error rates were typically less than 0.2 During the entire 2007 growing season, metrics of bioavailable NH 4 + NO 3 and PO 4 were obtained from field incubated anion or cation exchange resins (Giblin et al. 1994) In addition, seven hyphal ingrowth mes h bags were also inserted throughout each plot at the organic and mineral soil interfaces to measure actively growing fungal mycelia (Wallander et al. 2001; Nilsson & Wallander 2003) The construction, deployment, and analyses of the resin and hyphal ingr owth bags are described in detail elsewhere (Mayor et al. Chapter 3).
93 Extractable soil N forms were obtained either from the exchange resins or from a 2 M KCl extract made at the height of the growing season, early August 2007. In each plot, three cores were extracted and composited from within three zones of each 10 10 m plot (12 total cores for each of 16 plots) with an SMS ¨ volumetric slide hammer (4.2 cm diameter). The cores generally consisted of 5 cm of a mineral soil plug' with the remaining be ing compressed organic soil ( ~15 cm maximum) Green moss or lichens were removed, depth was recorded and the horizons separated. Green moss was removed from the surface of each core prior to homogenization for subsampling. Each comp osited soil sample w as stored on ice in the field or under refrigeration in the lab for approximately 24 h ou rs prior to salt extraction filtration, and freezing Because 80% of black spruce roots are typically found in the organic horizons (Ruess et al. 2003) and the logist ical limitations of high throughput denitrifier samples only the organic soils were extracted for 15 N values of the N moieties. Total dissolved N was extracted from ~24 g (wet weight) subsamples in 2 M KCL in nano pure D.I. water, oxidized with persulfate/thermodigestion, and then coupled to the denitrifier method for 15 N measurements as previously described in detail elsewhere (Knapp et al. 2005; Houlton et al. 2007) inc luding specific modifications to the oxidation method ( Chapter 3 ). The 15 N value of the extractable dissolved organic N (DON) pool wa s then determined from the mass weighted equation ( 4 2 ) below. S porocarp sample sizes vari ed from 12 to 29 a cross treatments with the fewest collected from the N+P and N treatments ( N = 12 and 13, respectively), and the most in the P and control treatments ( N = 22 and 29, respectively). Phospholipid fatty acid analyses (PLFA) of original froze n soil subsamples were performed on three 1 5 g ( wet
94 weight ) subsamples of the same organic soils as a metric of fungal biomass. This process involved an initial : lipid extraction, fractionation, and successive elution (Frosteg rd et al. 1991) ; followed b y conversion of the methanol fraction into free methyl ester s by mild alkaline methanolysis; and then analysis on a gas chromatograph with a flame ionization detector and a 50 m HP5 capillary column (Frosteg rd et al. 1993) The content of the specific PL FA 18:2 6,9 was regarded as a proxy for total fungal biomass (Frosteg rd & B th 1996) and to mainly comprise ECM forming fungi in boreal forest (Allison et al. 2007; Lindahl et al. 2007; Taylor et al. 2010) Statistical A nalyses Prior to ANOVA comparisons, parametric assumption s were assessed using Levene's t est for homogeneity of variance and Shapiro Wilk's test for normality ( # = 0.05 ) using R ¨ (2.9.2, The R Foundation for statistical computing 2009) Fertilizer treatment effects were analyzed using Dunnett's test of treatment against control or Tukey's HSD test on specific variables of interest. All ANOVA tests were performed with the inclusion of the experimental blocki ng effect in accordance with the factorial design of the experiment using JMP ¨ 8.0.2 (2009 SAS Institute). Mass B alance 15 N M ixing M odels The 15 N value of black spruce represents the 15 N value of all N sources taken up and the fractionation effects during their assimilation and transfer (Emmerton et al. 2001; Robinson 2001) To estimate proportional contribution and pathway of N flux to black spruce across treatments we used the following 15 N based mass balance mixing models developed for arctic tundra ecosystems (Hobbie et al. 2009; Yano et al. 20 10) :
95 3 pool N source: 15 N DON = 15 N TDN [TDN] KCl ( 15 N NH4 [NH 4 + ] KCl + 15 N NO3 [NO 3 ] KCl )) / [DON] KCl ( 4 2) 15 N available = DON 15 N DON + NH4 15 N NH4 + NO3 15 N NO3 ( 4 3) 15 N available =(1 Tr ) 15 N fungi + Tr 15 N transfer ( 4 4) 2 pool plant mixture: 15 N root = 15 N available # (1 Tr ) ( 4 5) 2 pool fungal mixture: 15 N fungi = 15 N available + # Tr ( 4 6) w here 15 N DON in equation ( 4 2 ) is derived from a mass weighted equation based on the original 2 M KCl concentration of N ions ([N] KCl ) and 15 N values measured from resin extracted NH 4 + and NO 3 detailed elsewhere ( Chapter 3 ). Equation ( 4 3 ) solves for the 15 N value of the effective availab le N ( 15 N available ) to plants and fungi based on proportionally weighted 15 N values of the three soil source pools. Inclusion of the extractable DON pool as a potential N source enhances th e bi ological realism of these N based mixing models (Houlton et al. 2007; Yano et al. 2010) because DON can comprise grea ter than 90% of DON in high latitude ecosystems and inorganic N fluxes are typically unable to account for annual plant N requirements in t hese cold, high latitude ecosystems (Ruess et al. 1996; Neff et al. 2003; Valentine et al. 2006; N sholm et al. 2009) In the remaining equations (4 4 to 4 6), Tr refer s to the proportion of total fungal N that is transferred to host plants, 15 N transfer refers to the 15 N value of the transfer compounds produced by ECM fungi, refers to the proportion of plant N supplied by fungi, and refers to the fractionation magnitude associated with transamination of soil N within ECM fungi (Hobbie & Hobbie 2006; Hobbie & Hobbie 2008).
96 We placed quantitative restraints on the source mixtures (DON, NH 4 + NO 3 ) and pathways (ECM vs. direct uptake) of N flux to black spruce. Fractionation magnitudes associated with the transformation of soil N to 15 N depleted transfer compounds by ECM fungi ( ) were estimat ed at # 9 1 based on laboratory and field analyses as described in detail elsewhere (Hobbie & Hobbie 2008) Based on the extremely low NO 3 concentrations in c ontrol plots (Table 4 1; 0.08 0.33% of 2 M KCl extractable TD N was NO 3 ) and because ECM fungi strongly discriminating against NO 3 under n atural conditions (Rygiewicz et al. 1984; Clemmensen et al. 2008) we omitted 15 N NO3 values from c ontrol plot mixing models yet retained the possibility of a minor contribution (10%) in the P treatments because of a notable increase in soil [ NO 3 ] adsorbed to resins relative to the c ontrol (Table 4 1; Figure 4 1). Plant, ECM sporocarp, and soil DON, NH 4 + and NO 3 15 N value end members were measured in replicate from each plot and means were used as soil N end members Results R esponse s of B lack S pruce E lemental C ontent to F ertilization Nitrogen fertilization, alone and in combination with P, increased foliar N con centration by rough ly 50% relative to the control (Figure 4 1 & 4 2 a ; P < 0.00 1 Dunnett's test) and caused 15 N enrichment (Fig ure 4 2 b P = 0.018 and P = 0 .005 respectively, Dunnett's test ). Similarly, P, alone and in combination with N, increased foliar P concentration relative to the control (Fig ure 4 2 c ; P = 0.00 2 and P = 0.0 6 respectively, Dunnett's test ) while foliar 15 N values were unaffected (Figure 4 1 b) Root N concentration and 15 N values were not affected by fertilization (Fig ure 4 2b ). Foliar C content was higher and 1 3 C values lower in the N trea tmen t relative to the
97 control (data not shown), although the effects were marginal ( P = 0. 1 5, 0.055, respectively; Dunnett's tests ). R esponse s of F ungal B iomass and S porocarp 15 N to F ertilization Fertilization did not alter fungal biomass as measured by PLFA 18:2 $ 6,9 (Fig ure 4 2 d ), but measured hyphal ingrowth was greater in N+P rela tive to N fertilized treatments (Fig ure 4 2 e P = 0.045, Tukey' s HSD). Neither metric of fungal standing biomass or seasonal growth varied from the control ( # = 0.10, Dunnett's test). Sporocarp 15 N values were less 15 N enriched in the N treatm ent relative t o the control ( P = 0.046, Dunnett's test ) and P fertilized plots (Fig ure 4 2 f, P < 0.10). Soil F ertility M etrics Soil O horizon depth and bulk densit y of soils were uniform among treatments ( n = 12 cores per plot ; Table 4 1). Soil pH values were higher in the N treatments relative to the control ( P = 0.02, Dunnett's test ; Table 4 1 ). 2 M KCl extractable TDN in both the c ontrol and P fertilized plots were dominated by DON ( 96% ) In contrast, the ex tractable TDN pool in plots fertilized by N and N+P was only 13 and 14% DON, respectively owing to the increases in resin exchangeable [NH 4 + ] and [NO 3 ] and a halving of extractable [DON] relative to the control (Dunnet t 's test P < 0.05 ; Table 4 1). The [NH 4 + ] in the N treatment was more tha n twice as high as in the N+ P treatment while [NO 3 ] was comparable (Table 4 1). Similarly, P fertilization increased resin exchangeable [PO 4 ] in soil solution ( P < 0.001, Dunnett's test on Box Cox Y transformed var iable) although values were highly variable across plots ( SE = 379 to 540; Table 4 1). Soil C:N was higher in the P treatment ( P < 0.1) largely due to lower
98 soil N in the organic matter of the P relative to N+P treatment only ( P = 0.11, Tukey's HSD) Soil 15 N Values The 15 N values of bulk soil organic matter did not vary across treatments despite five years of N addition (avgerage = 0.5 0.14 ; data not shown) Similarly, average 15 N values of the large salt extractable DON pool were unaltered by fertilization although both N and N+P fertilization increased the variability of 15 N DON values across plots ( P = 0.006, Levene's test ) The 15 N value of resin extractable N H 4 + became enriched in the N treatment relative to the control ( P = 0.07, Dunnett' s test) and in the N relative to the P treatments ( P < 0.1, Tukey's HSD). This NH 4 + enrichment in N treatment meant the relative ranking of isotope pools was shifted from the typical pattern in the control of 15 N DON values b eing more enriched than 15 N NH4 (6.1 vs. 4.41 respectively; Table 4 2). The 15 N value of resin extractable NO 3 was enriched in P relative to all treatments ( P < 0.001, Tukey's HSD) and the N+ P treatment relative to the control ( P = 0.07, Dunnett's test; Figure 4 3). Mass B alance M ixing R esults Average solutions to mass balance mixing models, along with associated 15 N end members, are reported on a plot by plot basis (Table 4 2). Black spruce was e stimated to receive 68 94% (average = 82%) of total N from ECM fungi (denoted as in equation 4 5 ) in control plots The contribution of ECM to black spruce N nu trition was also estimated to increase along with greater estimated contributions of DON ( DON in equation 4 3) in 15 N available calculations. Similarly, black spruce in both P and N fertilized stands were estimated to rely on ECM derived N for 74% and 76%
99 respectively, of their N (Table 4 2), but o nly the P treatment followed the same pattern of greater with greater DON In contrast to the other treatments, b lack spruce trees in the N+P treatment were estimated to be less reliant upon ECM derived N relative to the control (46%, P = 0.03, Dunnet t 's test; Table 4 2). Unlike the trends in control and P plots, no pattern with specific soil N sources and estimates was observed. In the N+P treatment, two plots had increasing values with greater contributions of soil NH 4 + ( NH4 ). Collectively, t hese findings are depicted graphically in a qualitative diagram that i llustrate s the complicated patterns in N acquisition and pathways of N flux across treatment types as informed by mass balance mixing models ( Figure 4 4). A High proportional N flux through ECM fungi was estimated in both t he control and P treatments ( red arrow in Figure 4 4a ) and this required a high dependency on DON (large black arrow in Figure 4 4a) to achieve mass balance. In contrast, there was overall a general lack of NO 3 contributing to modeled N uptake. The N treatment, in contrast, shows a switching of soil N sources to include more NO 3 at the expense of DON (Figure 4 4b) yet continued reliance upon ECM. Lastly, the N+P treatment illustrates a reduced reliance upon E CM derived N (diminishing red arrow in Figure 4 4c) with a corresponding increase in direct uptake of soil N by black spruce. Estimated values subsequently compared with multiple isotope and nutrient pools in the stands. The values were negatively c orrelated with both NH 4 + and DON based enrichment factors ( R 2 = 0.4 3 P = 0.0 1, Figure 4 5a; and R 2 = 0.4 1 P = 0.0 1, Figure 4 5b ) and foliar 15 N foliage ( R 2 = 0.44, P = 0.01 Figure 4 5c ) whereas they were positively correl ated with extractable [DON] g 1 pools ( R 2 = 0.75, P < 0.001 Figure 4 5d).
100 Despite the averaged estimated values being well constrained across plot s ( average SE = 0.025), mass balance was not achievable in two plots because subsequent mixture values did not fall within the available end member sources ( n/a in Table 4 2). In addition, i n order to produce sensible results in one plot, a higher proportion of NO 3 (0.7) was required despite a priori assumptions limiting proportional NO 3 contribution to 0.5 Valid solutions in these plots were impaired by either particularly depleted sporocarp 15 N or enriched 15 N NH4 end member value s (Plot 14 N and 12 P respectively; Table 4 2). For the control and P treatments root 15 N values were used as plant end members in mass balance equations instead of foliage because this permitted accounting of a variable internal plant fractionation demonstrated in black spruce of central Alaska ( Chapter 3 ). In contrast in the N and N+P treatments, only foliage responded to fertilization implying roots 15 N values were poor integrators of plant N sources. Because of this lack of root response foliar 15 N values were deemed better end member values for N fertilized plots Furthermore, u sing these two different end member values for black spruce was necessary to achieve mass balance and was justified by the belief that the roots measured were not representative of the fertilizer N acquisition for the following reasons: 1) internal assimilation and/ or translocation of N from roots to foliage produce negative fractionations not positive as would be nece ssary here (Robinson 2001; Kolb & Evans 2002) ; 2) root N content and 15 N values were not altered by N fertilization, yet foliage was, suggesting no increased N allocation to roots; and 3) a presumed low N demand in roots because non 1 st order ECM roots (e.g. >2 mm) have long, yet poorly constrained, turnover times likely exceeding one year (Ruess
101 et al. 2006) and are less active physiologically than the smallest diameter fine roots (Pregitzer 2002) When solving mass balance mixing models proportional mixtures of soil N were iteratively adjusted for each plot under the following conditions: (a) the proportion of black spruce derived from ECM fungi ( ) could not exceed 100%; (b) mixtures of the different N forms o c curred at 10% increments, allowing a maximum of 10 different values from 0 to 100%; (c) proportional mixtures in the control and P plots were constrai ned to a 2 source mixing model with a priori DON proportions constrained between 20 70%; and, (d) for the eight plots receiving N fertilizer, we adjusted the range of 3 source contributions to include low (0%) to high (50%) proportional DON concentrations with the remainder equally distributed across the mineral N fertilizer with NO 3 contributions restrained to equal or lesser proportional contributions than NH 4 + These conditional rules were justified because NO 3 availabilities were extremely low under natural conditions (Table 4 1) and because partitioning of soil DON into bioavailable components from other studies indicate 100% of the DON pool is not available yet a minimum of 20% is entirely feasible based on multiple lines of evidence (Jones et al. 2 005b; Yano et al. 2010) In P fertilized plots, where 15 N NO3 contributions were permitted at 10%, inclusion of NO 3 only acted to increase dependency on ECM but was not necessary to achieve mass balance Given that, models were mathematically underdetermined the results are presented as ranges of likely mixtures.
102 Discussion Effects of Fertilization on Soil Fertility and Soil 15 N Values Fertilization with mineral N, both with and without P, caused a decline in DON but no change in average 15 N DON or bulk soil 15 N values (Table 4 2, Figure 4 3). Mineralization of NH 4 + from DON has been shown to cause large shifts in 15 N values (e.g. 17 ) in single compound studies in the lab particularly during phenylalanine ammonia lyase activity (W erner & Schmidt 2002) H owever, many isotope studies in terrestrial ecosystems indicate that N isotope fractionation during mineralization is relatively small (Amundson et al. 2003; Evans 2007; Hobbie & Ouimette 2009) The similar 15 N values measured from DON and NH 4 + pools in control, P, and N+P fertilization treatments support the later interpretation. It was surprising that fertilization with N alone caused no difference among average soil 15 N DON and bulk soil 15 N values relative to the control given the plots had received cumulatively 600 kg ha 1 of N fertilizer over the course of the 5 year experiment Only the N treatment led to 15 N enrichment of the NH 4 + pool ( P < 0.1; Figure 4 3). Although other N addition experiments of similar magnitude and duration have caused isotopic changes in soil total N (Pardo et al. 2007) a general lack of changes here may indicate long turnover times of the soil organic N re sulting from climatic extremes limiting microbial activity (Van Cleve & Alexander 1981) The variability of the DON pool, in contrast, suggests that mineralization of some fraction of the DON pool did occur during the preceding 5 years. Because t he reduct ion in extractable DON under N and N+P fertilization (Table 4 1) also corresponded with increased variance of 15 N DON values ( P = 0.03, Levene's
103 test ; Figure 4 3) it is likely these two observations are mechanistically relate d. For instance, the decline in DON suggests reduced production and/ or accelerated decomposition under substantially higher mineral N availability Reduced DON production could result fro m the release of mineral N requirements for fungal growth, thereby limiting the need for exoenzyme production. N fertilization has been shown to decrease lignolytic activity (Neff et al. 2002; Waldrop & Zak 2006; Lucas & Casper 2008) In contrast, N fert ilization increases proteolytic enzyme activity in fung i (Lucas & Casper 2008) Reduced lignolytic activity combined with increased proteolytic activity would cause the more labile proteins in the DON pool to decline rapidly over the course of the study a nd recalcitrant complexes, including bulk soil N, to be retained longer Following this line of reasoning, the remaining recalcitrant DON pool would be comprised of a greater proportion of recalcitrant compounds with correspondingly distinct 15 N values and bulk soil N would remain relatively unaffected. The invariance in bulk soil 15 N values clearly illustrates the isotopic disconnect from plant available N in this ecosystem. Surprisingly, P add itions also influenced the N cycle in these otherwise N limited boreal forests. When N fertilization was combined with P, 15 N NO3 values became 15 N enriched relative to the control (Figure 4 3). However, P addition alone cau sed an even larger 15 N enrichment of the smaller residual NO 3 pool. The P treatment led to a 60 fold increase in adsorbed [NO 3 ] and 14 fold increase in adsorbed [N H 4 + ] relative to the control although only [NO 3 ] was significantly higher given high variability in [N H 4 + ] (Figure 4 3). In contrast, 2 M KCl extractions taken at the height of the growing season and used to extract total dissolved N, did not indicate any increase in standing [NO 3 ] or
104 [NH 4 + ] pools (dat a not shown) Combined, these findings suggest that P fertilization accelerated the rate of mineral N cycling but not necessaril y the standing labile N pool size Accelerated N cycling caused by P fertilization could result from multiple processes. Fo r instance, a stimulating influence on nitrifying bacteria could be the cause either directly through elevated P bioavailability (Purchase 1974; Mahendrappa & Salonius 1982) or indirectly via pH influences on enzyme activity (Persson & Wiren 1995; Sinsabaugh et al. 2008) or through stimulation of mineralization rates and subsequent relief of NH 4 + substrate limitations to nitrifiers (Munson & Timmer 1991) As both pH and [NH 4 + ] were statistically indistinguishable in the P fe rtilized plot from that in the control (Table 4 1), the hypothesis that direct P fertilization of nitrifying bacteria is most plausible given the evidence available. Furthermore, a higher C:N ratio in the P fertilized treatments (Table 4 1) also indicates an accelerated N cycle, where soil N (but not soil C) was gradually reduced in response to P fertilization of decomposers. Alteration of the N cycle from P addition in boreal forest ecosystems is not well understood M ost studies in N limited ecosystems have instead examined or assumed that N fertilization, but not P fertilization, benefits plant growth and that microbial growth responds to alterations of below ground C allocation by these plants (H gberg et al. 2003; Janssens et al. 2010) However, b ioa vailability of both organic and inorganic P in boreal soils can be limited by aluminum iron complexes in organic material (Giesler et al. 2004) and N mineralization rates in other ecosystems have been observed to accelerate following P additions (Munevar & Wollum 1977; Ross & Bridger 1978; Haynes & Swift 1988; Sinsabaugh et al. 1993) Of the abundance of N fertilization
105 studies in spruce forest, only a few factorial P addition studies were found, and of these, only one of three measured increased labile so il NO 3 concentrations and mineralization rates following PO 4 addition (Mahendrappa & Salonius 1982) This study occurred in a 55 60 year old black spruce stand fr om New Brunswick, Canada and the fertilizer was also super triple phosphate administered at comparable levels to our study In contrast, two other P fertilization experiments, one in a Norway spruce plantation in Denmark, the other in a Sitka spruce plantat ion in North Wales, UK, measured a decline in mineral N following P fertilization (Stevens et al. 1993; Vesterdal & Raulund Rasmussen 2002) Determining the mechanism for these findings is made difficult, however, because P fertilization occurred together with other mineral nutrients, although increased N demand of spruce following release of spruce P limitation is plausible. Apart from increasing soil NO 3 concentrations, P additions also induced a strong isotopic influence over 15 N NO3 values, causing 17 enrichment relative to the control. Fractionation effects of this direction and magnitude likely reflect losses of 14 N NO 3 from the system In Delta Junction soils NO 3 losses to leaching are unlikely given the extremely low rainfall (290 mm yr 1 ) limiting the leaching potential even during the June, July, and August growing season which receive s 65% of MAP (Mack et al. 2008) Several lines of evidence suggest gaseous losses lead to the observed NO 3 enrichment instead. First, d enitrification has been shown to discriminate against 15 N by as much as 27.2 2.8 calculated as: % denitrification = 15 N product $ 15 N substrate (P rtl et al. 2007) and gaseous NO x and N 2 O losses during nitrification can be even higher, estimated at 34.7 2. 5 (Mariotti et al. 1981; Hobbie & Ouimette 2009) The realized degree of enrichment, however, depends on the proportion of substrate consumed. The
106 second line of evidence comes from the 15 N/ 14 N and 18 O/ 16 O ratios of the remaining NO 3 pool, a pattern demonstrated in forest groundwater where denitrifica tion contributes appreciably to N losses (Aravena & Robertson 1998; Mengis et al. 1999; Houlton et al. 2006) As expected if denitrification caused the observed enrichment, 15 N and 18 O values of NO 3 were strongly correlated only within the P treatment ( R 2 = 0.78, N = 4, P = 0.11; data not shown). A strong correlation between the particular denitrifier gene nir K and available P in a Central European spruce forest also suggest nutrient sensitivity of denitrifying bacteria (B rta et al. 2010) Denitrification in many high latitude N limited systems is thought to be quantitatively unimportant owing to the low NO 3 levels (Stehfest & Bouwman 2006; Hobbie & Ouimette 2009) but the mechanism s governing gaseous N losses (e.g. N 2 O and N 2 ) in arctic soils are generally not well understood (Chapin 1996; Wolf & Brumme 2003; Siciliano et al. 2009) E ffects of F ertilization on P lant %N, %P, and 15 N Nitrogen addition caused a doubling of black spruce foliar N concentration and, as a result of the uptake of N fertilizer, approximately 3 15 N enrichment of folia ge (Figure 4 1 & 4 2 b ). Beginning with some of the first field applications of 15 N measurements in agricultural systems it has been known that fertilization with N causes actively growing plant tissue to more closely approximate the isotopic val ue of the fertilizer, typically near the atmospheric standard of 0 2 (Kohl et al. 1973; Shearer & Legg 1975; Vitoria et al. 2004) Given black spruce is one of the most 15 N depleted trees globally (Craine et al. 2009) absorption of near 0 fertilizer N would expectedly lead to relative 15 N enrichment. Whereas short duration fertilization experiments have documented this expected trend in temperate (Magill et al. 1997; Davis et al. 2004) and
107 boreal forest (Eriksson et al. 1996; Bergholm et al. 2007) prolonged fertilization can also lead to large losses of soil N and successive 15 N enrichment of residual soil N pools well beyond that of the fertilizer (Pardo et al. 2007) Under these conditions, plant tissue is expected to be substantially more 15 N enriched as a result of plant absorption of the resultant 15 N enriched residual N pool as seen in red spruce in Vermont ( Pardo et al ., 1998; McNulty et al ., 2005 ) and in pine plantations in temperate and boreal forests (Hogberg 1991; Choi et al. 2005; H gb erg et al. 2010b) Unlike black spruce foliage, fine root N concentration and 15 N values were surprisingly unaffected by fertilization (Figure 4 2b). Whereas fertilization can increase above ground production (Mack et al. 2004) root production was unaffected by fertilization in Alaskan tundra (Nadelhoffer et al. 2002) and can decline in other ecosystems. For instance, six years of N fertilization in a Norwegian spruce plantation led to a 69% relative increase in total N pools but a 30% decrease in fine root N pools (Bergholm et al. 2007) This preferred allocation of new fertilizer N to foliage, combined with old root N reallocation to new roots could account for the observed patterns (Kolb & Evans 2002) Because NO 3 c an be biochemically assimilated by either the root or shoot tissue, and NH 4 + can only be assimilated by the root (Andrews 1986) it has been hypothesized that intra plant variation in 15 N can result from differential assimila tion of NO 3 (Evans 2001; Kolb & Evans 2002) However, the isotopic effects of such differences could not cause the patterns we observed in black spruce. For instance, nitrate reductase activity discriminates against 15 N, so root assimilated fertilizer NO 3 would actually cause relative enrichment of root tissue concurrent with the translocation of unassimilated isotopically light NO 3 from root to shoot. Whereas this mechanism
108 could explain foliar patterns, it would no t explain the lack of a root response observed here. Instead, it appears that fertilizer N is preferentially allocated only to growing needles, leaving old root N as the only retained N source to new roots. This discovery reinforces the idea that all pla nt tissue 15 N values do not necessarily reflect N source 15 N values. Black spruce P concentrations in needles also nearly double d in response to P fertilization, which indicates either substanti al excess consumption or a stoichiometric relationship among N:P ratios ( gren 2008) N:P ratios of needles were higher in N fertilized stands (17) compared to all other treatments and those in N+P treatments were higher than just P alone (9 versus 5; P < 0.05, Tukey's HSD). The control and P treatment N:P ratios were statistically indistinguishable. Relative ranking of treatment N:P ratios was therefore: N (17) > N+P (9) > C (7) > P (5). Because C and P treatments were equivalent, yet P increased more in the N+P relative to P treatments, black spruce foliar elements may indicate achievement of a stoichiometric balance and co limiting growth conditions under N+P fertilization (Elser et al. 2007) Preliminary above ground plant biomass and N tissue conc entration data from the fertilized plots don't indicate any changes in the plant community apart from mosses. Mosses had higher N concentrations under both N and N+P fertilization ( P % 0.01, Dunnett's test), yet N fertilization reduced moss biomass overal l ( P = 0.07, Tukey's HSD; M.C. Mack, unpublished data ). No changes in eith er black spruce biomass or %N were seen in P fertilized plots according to preliminary analyses of stem diameter growth data (M.C. Mack, unpublished data ).
109 Effects of Fertilization on Fungal Biomass, Ingrowth, and 15 N Fungal biomass and hyphal ingrowth trended to decrease in N fertilized plots although not significantly so relative to the control (Figure 4 2 d,e ). Declining biomass of mycorrhizal fungi is expected under increasing N availability as previously demonstrated in natural forest ecosystems (Nilsson et al. 2005; Treseder 2008; Wallander et al. 2009) and after single N fertilizatio n events (H gberg et al. 2010a) Reduced ECM fungal biomass following fertilization is thought to result f rom a reduction in below ground C allocation to ECM fungi in response to a decline in mineral nutrient requirements by the autotrophic host plant (H gberg et al. 2003; Hobbie 2006; H gberg et al. 2010a) The lack of declines in fungal biomass or ingrowth in our study may be due to low statistical power resulting from a logistically limited number of PLFA analyses and a short incubation period of the i ngrowth bags. Although microbes are generally considered C limited mycorrhizae are unique in that they have access to relatively abundant C supplies from their autotrophic hosts. Therefore, stimulation of mycorrhizal ingrowth in response to N+P fertili zation may indicate mineral nutrient limitation s of the exploring hyphae (Clemmensen et al. 2006; Treseder et al. 2008) Apart from standing biomass and ingrowth responses, abrupt increases in N bioavailability can also reduce the diversity of ECM fungi (Lilleskov et al. 2001; Lilleskov et al. 2002a; Frey et al. 2004) Although ECM diversity was not measured in our plots, a decline in saprotrophic species in our N fertilized plots was detected (Allison et al. 2007) Declining or altered belowground dive rsity may have unknown but potentially negative consequences to soil C stabilization and mineral nutrient cycling that deserves more attention, especially in light of our changing climate (Courty et al. 2010)
110 Similar to the black spruce autotrophic hosts N fertilization caused sporocarp 15 N values to shift down toward the applied N fertilizer (Figure 4 2f). The near 50% loss of 15 N enrichment under N fertilization suggests fungal use of the N fertilizer. Alternatively, de clining sporocarp 15 N values could also represent reduced processing or retention of 15 N during the transfer of 15 N depleted N to host plants. However, this later explanation is unlikely given that no similar patterns were f ound in the N+P treatments where proportional N transfer by ECM fungi was modeled to be lower (see next section). As ECM fungal genera differ in their levels of 15 N enrichment (Trudell et al. 2004; Hobbie & Agerer 2009) changes to the ECM fungal communit y under N fertilization (Lilleskov et al. 2001) could have also contributed to the pattern of 15 N depleted ECM sporocarps in the N treatment. Fertilization Induced Decline in Black Spruce Dependency on ECM P lot specific estimates of black spruce dependency on ECM derived N ( ) were, on average, lower in the N+P treatment relative only to the control (Table 4 2 ; Tukey's HSD, P = 0.027 ). Why was a similar decline in dependency on ECM derived N not also found in N treatments as well (Treseder & Vitousek 2001) ? One possibility is that increased P demands in the N treatment causes continued N delivery resulting from the inability of ECM fung i to selectively deliver only one mineral nutrient at a time. This hypothesis can account for the observation that ECM dependency declines only when both N and P fertilization are combined. Under this interpretation, sporocarp 15 N values become depleted under continued N fertilization because of mineral N dilution of otherwise enriched 15 N values, not declining 15 N retention and transfer of N. Why there were no changes in sporocarp 15 N values following N+P fertilization, however, is
111 unknown and may indicate an additional mechanism for ECM sporocarp 15 N enrichment. To understand why only the combination of N+P fertilizers led to a decline in we regressed it again st several other potentially meaningful site variables (Figure 4 5). Examining the relationships among variables that were either directly or indirectly input into the mass balance mixing models to solve for allows for an examination of relative relatio nships among variables. We found that the N+P treatment plots were among those with: the smallest isotopic differences between black spruce foliage and resin exchangeable NH 4 + and DON (Figure 4 5a,b); the most enriched black spruce needle 15 N values (Figure 4 5c); and, the lowest extractable [DON] pools (Figure 4 5d). There were no relationships found with estimates of and bulk soil 15 N soil C:N ratios, PLFA based fungal biomass, or soil DON, NH 4 + and NO 3 concentrations. What could these correlations indicate about changes to the functioning of these forests under fertilization? Declining foliar 15 N val ues in the same tree species along successional chronosequences have been hypothesized to represent declining dependency on ECM fungi for N (Hobbie et al. 2000; Hobbie et al. 2005) Here foliar 15 N values were positively cor related with estimates, suggesting declining dependency on ECM derived N is reflected in foliar 15 N values (Figure 4 5c). However, foliar 15 N values were uncorrelated with estimates from 30 additional black spruce sites spanning a diversity of soil fertilities in central Alaska ( Chapter 3 ) As mentioned previously, t he same pattern could result from 15 N enrichment resulting from use of the applied mineral N fertilizer (Figure 4 2 b ). This i ssue was recently addressed in a Swedish pine forest while assessing the return of ECM dependency following
112 abandonment of N fertilization 6 years prior (H gberg et al. 2010b) These authors suggested it was not the fertilizer signature that led to subseq uent depletion of Pinus sylvestris needle 15 N values, but rather a return of 15 N retention by associated ECM fungi during their assimilation and deliver of soil N to their autotrophic hosts. Their determination was based lar gely on indirect evidence that their fertilizer was 15 N depleted relative to endogenous N sources and that ECM biomass was correlated with foliar 15 N (H gberg et al. 2010b) Declining # DON and # NH4 with low in part reflect s the foliar 15 N values used to calculate these metrics but the smaller differences at low values also could indicate a reduction in isotopic discrimination resulting from ECM deliver y of N A relationship with and 15 N NH4 ( R 2 adj = 0.16, P = 0.01, y = 0.85 + 0.017 & ), ECM sporocarp and root 15 N values ( R 2 adj = 0.39, P = 0.004, y = 1.07 0.025 & ; R 2 ad j = 0.16, P = 0.02, y = 0.74 0.03 & respectively) was also observed across a black spruce soil fertility gradient (Chapter 3 ) suggesting 15 N NH4 is a useful integrator of ECM activity when compared with host plant 15 N values in boreal spruce forest. We must use caution when over interpreting these results because these values were involved, either directly or indire ctly, in isotope mixing models used to estimate and could be interpreted as auto correlated. Lastly, the correlation of low N+P plots with low soil [DON] and highly variable 15 N DON values suggests that as ECM fungi are r elied upon less for N (e.g. low ), they also presumably use less of the now depleted and more recalcitrant DON pool. Oddly, no pattern was observed with proportional contributions of DON in the four N+P plot mixing model solution s ( perhaps due to the var iable 15 N DON values) but this pattern
113 was seen in both the control and the P treatments as well as in the majority of 30 mixing model solutions in another study of black spruce 15 N patterns (Cha pter 3). Conclusion and E cosystem I mplications Fertilization with N caused no changes in 15 N values of bulk soil or DON pools but did cause black spruce foliar 15 N values to become more enriched and foliage to contain twice as much N g 1 leaf. N fertilization also caused fungal biomass and ingrowth to decline slightly, as expected, while also causing ECM sporocarp 15 N values to decline, but these trends were not observed in the N+P treatment. N fertilization, with or without P, also caused a reduction in [DON]. P fertilization similar ly doubled foliar P concentration and had surprising influence on the N cycle, causing a spike in exchangeable soil [NO 3 ] and marked 15 N enrichment of the same pool likely caused by increased gaseous losses. When plots were fertilized with both N and P s oil [DON] also declined nearly 50% as in the N fertilized plots, presumably due to alterations to enzyme production by the decomposer community. Furthermore, N+P fertilization caused shifts among both the form and pathway of N cycling through the soil fun gus plant continuum with significant declines in the dependency for N delivery by ECM fungi. Collectively, experimental alterations to soil fertility led to higher foliar N and P concentrations that will likely incre ase N cycling rates (Magill et al. 1997) a reduction in labile [DON], and presumably reduced C allocation to ECM fungi. These can effects can have important consequences for soil carbon storage in boreal forests (Neff et al. 2002; Mack et al. 2004) Lastly, we demonstrated that ecosystem 15 N values contain interpretable signals regarding forest N cycling even with complex sources of 15 N fractionation associated with ECM fungi.
114 Table 4 1 S oil charac teristics across black spruce fertilization treatments SE Each treatment corresponds to four plots. C = control, N = nitrogen addition, P = phosphorus addition. ** = difference from control ( P < 0.05, Dunnett's test ), = P < 0.1. Treatment O horizon depth (cm) Bulk density (g cm 3 ) DON (ug g 1 soil) DON ( g N m 2 ) NH 4 ( g g 1 resin day 1 ) NO 3 ( g g 1 resin day 1 ) PO 4 ( g g 1 resin day 1 ) C:N pH (H 2 O) C 5.80 0.52 0.16 0.03 313.97 35.39 2.95 0.61 1.08 0.49 0.08 0.01 3.86 1.86 24.96 1.56 4.75 0.06 N 6.31 0.60 0.12 0.02 148.86 09.97 ** 1.09 0.20 ** 382.32 96.66 ** 399.30 130.24 3.19 1.33 24.77 0.94 4.99 0.06 ** N+P 7.09 1.08 0.14 0.03 135.94 40.81 ** 1.23 0.37 ** 168.11 68.23 463.69 151.10 ** 839.05 539.66 25.36 0.60 4.87 0.06 P 5.97 0.35 0.15 0.01 279.89 19.70 2.42 0.07 14.31 7.95 67.43 29.15 1008.14 378.60 29.15 1.28 4.74 0.08
115 Table 4 2 M ass balance mixing results to estimate the proportional dependence of black spruce on ECM derived N and the average ( SE) end member sources of N used across fer tilization treatments. Block Plot Treatment of ECM N 15 N DON 15 N NH4 15 N NO3 15 N Plant 15 N fungi 1 1C Control 0.68 0.23 7.29 3.77 0.18 2.43 11.55 2 5C Control 0.90 0.00 4.73 5.19 2.73 5.18 11.31 3 10C Control 0.94 0.02 6.99 3.81 1.16 2.6 7.85 4 13C Control 0.78 0.03 5.4 4.87 6.41 0.68 7.42 avg. (SE) 0.82 6.10 (0.62) 4.41 (0.36) 2.53 (1.42) 1 2N Nitrogen 0.91 0.04 5.41 5.88 1.12 3.38 5.99 2 6N Nitrogen 0.53 0.04 3.22 7.69 0.6 1.29 6.97 3 9N Nitrogen 0.85 0.04 9.68 7.93 1.66 0.74 6.54 4 14N Nitrogen n/a 8.03 5.98 2.59 1.5 1.51 avg. (SE) 0.76 4.98 (2.87) 6.87 (0.55) 0.54 (0.89) 1 4P Phosphorus 0.89 0.03 4.56 1.37 14.49 4.13 8.12 2 8P Phosphorus 0.83 0.02 5.83 3.47 10.6 3.35 9.36 3 12P Phosphorus n/a 4.97 7.22 16.61 5.29 6.87 4 15P Phosphorus 0.51 0.03 1.74 3.74 16.23 2.41 11.28 avg. (SE) 0.74 4.28 (0.89) 3.95 (1.21) 14.48 (1.37) 1 3NP N + P 0.41 0.01 6.05 5.15 2.06 0.82 13.66 2 7NP N + P 0.59 0.01 14.85 5.08 2.67 1.11 11.5 3 11NP N + P 0.42 0.04 2.3 4.47 0.36 0.85 7.46 4 16NP N + P 0.41 0.03 2.93 4.29 1.52 1.43 9.47 avg. (SE) 0.46 3.92 (4.18) 4.75 (0.22) 1.65 (0.49)
116 Figure 4 1 Foliar 15 N values from black spruce trees were strongly correlated with %N across fertilization treatments ( 15 N = 7.72 + 4.09 %N, R 2 = 0.75, P < 0.001) but no t %P. Each point represents the mean of three or four trees from each of the 16 fertilized plots.
117 ! Figure 4 2 Black spruce and fungal response SE to five years of fertilization with nitrogen (N), phosphorus (P), both (NP), or none (C) Different letters indicate differences either relative to the control (Dunnett's ANOVA with blocking effect) in panels' a d, or Tukey's HSD with blocking effect panels' e f. (A) foliar N concentration of mature black spruce. (B) Black spruce needle (grey bars) and root (checkered bars) 15 N values. (C) Black spruce needle P concentration. (D) Organic soil content of the spec ific phospholipid fatty acid (PLFA) 18:2 6,9 regarded as a metric of fungal biomass. (E) Hyphal ingrowth measured in sand filled mesh bags incubated for the 2007 growing season. (F) Ectomycorrhizal sporocarps opportunistically collected from the plots du ring 2005 2008.
118 Figure 4 3 Response s of soil N 15 N values to five years of fertilization with ammonium nitrate (N), orthophosphate (P), both (N + P), or none (control) Double a sterisks indicates differences from control at # = 0.05, single asterisk at # = 0.10. The 15 N value of resin extractable N H 4 + was enriched in the N treatment relative to the control ( P = 0.07, Dunnett's test) and in the N treatment relative to the P treatments ( P < 0.1, Tukey's HSD). The 15 N value of resin extractable NO 3 was enriched in P relative to all trea tments ( P < 0.001, Tukey's HSD ) and the N+P treatment relative to the control ( P = 0.07, Dunnett's test ). Red arrows on right axis of figures indicate the average resin exchangeable [NO 3 ] for each treatment as a corollary to the spike in 15 N NO3 enrichment. Note that the scaling of NO 3 concentration was adjusted between treatments with and without N fertilization.
119 Figure 4 4. Model of the patte rns of N flux es across our treatment types as informed by mass balance mixing models. The Control and Phosphorus (P) treatments illustrate the high proportional flux of N through ECM hyphae (photo inset) to black spruce (red arrow), a high dependency on DON, and a lack of NO 3 use The N treatment in the middle illustrates a more complex pattern where NH 4 + and NO 3 fertilizer are seen to supplant reliance on DON uptake yet reliance on ECM derive d N remains unchanged. The N + P treatme nt on the right illustrates the most complex pattern of nutrient uptake with similar reliance upon N fertilizer but with a reduced reliance on ECM fungi
120 Figure 4 5 Correlations between black spruce proportional dependence on ECM derived N ( ) and other isotopic components in experimentally fertilized black spruce forest in central Alaska. (A) A n NH 4 + based enrichment factor ( = 15 N foliar NH4 ) R 2 = 0.43. (B) A DON based enrichment factor ( = 15 N foliar 15 N DON ) R 2 = 0.41. (C) Black spruce foliar 15 N R 2 = 0.43 (D) Salt extracted soil [DON], R 2 = 0.75. Open square = Control, blue circle = N, blue triangle = N+P, black diamond = P treatment.
121 APPENDIX A COLLECTOR BASED MISSCLASSIFICATIONS OF FUNGI A 2 Potential collector based misclassifications based on multivariate discriminant analysis of site normalized funga l isotope values ( 13 C, 15 N). Type = collector based classification as mycorrhizal (m) or saprotrophic (s), ECM P = percent probability of sporocarp being ectomycorrhizal or saprotrophic ( SAP P ), weak, ** = moderate, *** = strong support for assigned category. Author Species Site Type ECM P SAP P Koh zu et al. 1999 Russula sp. Asihu m 37 63 Laccaria sp. Asihu m 17 83 ** Trametes versicolor Asihu s 69 31 Agaricus subrutilescens Asihu s 100 0 *** Coprinus sp. Asihu s 84 16 ** Pholiota lenta Ashiu s 69 31 Pleurotus ostreatus Chiba s 96 4 *** Amanita abrupta Kyoto m 49 51 Amanita esculenta Kyoto m 37 63 Amanita pantherina Kyoto m 36 64 Boletus pseudocalopus Kyoto m 28 72 Boletus pseudocalopus Kyoto m 37 63 Boletaceae sp. Kyoto m 49.7 50.3 Cortinarius sp. Kyoto m 4 96 *** Entoloma clypeatu m Kyoto m 11 89 ** Lactarius chrysorrheus Kyoto m 43 57 Lactarius chrysorrheus Kyoto m 36 64 Lentinula edodes Kyoto s 71 29 Psathyrella sp. Kyoto s 55 45 Marasmius maximu s Kyoto s 97 3 *** Mycena pura Kyoto s 73 27 Mycena sp. Kyoto s 92 8 *** Tylopilus sp. Lambir m 6 94 *** Tylopilus sp. Lambir m 42 58 Ganoderma australe Lambir s 84 16 ** Microporus vernicipes Lambir s 91 9 *** Trametes versicolor Lambir s 58 42 Mycoleptodonoides aitchisonii Oodai s 60 40 Armillariella mel l e a Oodai s 65 35 Trametes sp. Shirahama s 67 32 Hobbie et al. 2001 Psathyrella sp. #2 Woods Creek s 80 20 ** Psathyrella sp. #1 Woods Creek s 53 47 Galerina heterocystis Woods Creek s 94 6 *** Henn & Chapela 2001 N/A California pine forest s 60 40 Taylor et al. 2003 Chalciporus piperatus Aheden m 40 60 Russula betularum Stadsskogen m 36 64 Russula vinosa Stadsskogen m 49.5 50.5
122 Gymn opilus junonius Stadsskogen s 56 44 Trudell et al. 2004 Cortinarius variosimilis Deer Park Rd m 2 98 *** Cystoderma amianthinum Deer Park Rd s 69 31 Cystoderma granulosum Deer Park Rd s 59 41 Pseudoplectania melaena Deer Park Rd s 62 38 Mycena clavicularis Hoh rainforest s 61 39 Pseudoplectania melaena Hoh rainforest s 64 36 Hart et al. 2006 Inocybe geophylla Lamar Haines m 39 61 Agaricus silvicola Lamar Haines s 65 35 Gymnopilus bellulus Lamar Haines s 94 6 *** Pholiota squarrosa Lamar Haines s 96 4 *** Pluteus lutescens Lamar Haines s 82 17 ** Hygrophorus camarophyllus Snowbowl m 39 61 Inocybe lacera Snowbowl m 19 81 ** Pholiota squarrosa Snowbowl s 91 9 *** Zeller et al. 2007 Lactarius chrysorrheus Breuil, France m 44 56 Leotia lubrica Breuil, France s 99 1 *** Amanita citrina Spruce plantation m 47 53 Clitopilus prunulus Spruce plantation s 62 38 Hypholoma fasciculare Spruce plantation s 66 34 Micromphale perforans Spruce plantation s 63 37 Mayor et al. this study Boletellus exiguus Guyana m 41 59 Cantherellus pleurotoides Guyana m 7 93 *** Cantherellus pleurotoides Guyana m 21 79 ** Cantherellus pleurotoides Guyana m 23 77 ** Russula sp. Guyana m 13 87 ** Tylopilus potamogeton var. irengensis Guyana m 22 78 ** Peren niporia inflexibilis Guyana s 57 43 Stipitochaete damaecornis Guyana s 58 42 Xylaria sp. Guyana s 71 29 Collybia aff. laccata Guyana s 68 32
123 APPENDIX B CLASSIFICATION OF FUNGI WITH UNKNOWN ECOLOGY B 2 Classification of fungi with unknown (unk) ecological roles based on multivariate discriminant analyses of site normalized fungal isotope values ( 13 C, 15 N). ECM P = percent probability of sporocarp being ectomycorrhizal or saprotrophic ( SAP P ), weak, ** = moderate, *** = strong support for assigned category. Descriptions of the sites can be found within the referenced articles detailed in the main text. Author Species Site Type ECM P SAP P Hobbie et al. 2001 Helvella lacunosa Woods Creek unk 89 11 ** Otidia aff. conci nna Woods Creek unk 94 6 *** Clavulina cf. cri stata Woods Creek unk 98 2 *** Ramaria Woods Creek unk 93 7 *** Helvella crispa Woods Creek unk 97 3 *** Otidea onotica Woods Creek unk 99 1 *** Helvella sp. Woods Creek unk 7 93 *** Ramaria sp. Woods Creek unk 100 0 *** Clavulina cristata Woods Creek unk 99 1 *** Clavulina rugosa Woods Creek unk 99 1 *** Trudell et al. 2004 Phylloporus rhodoxanthus Deer Park Rd unk 88 12 ** Tricholoma vaccinum Deer Park Rd unk 99.5 0.5 *** Bondarzewia mesenterica Hoh rainforest unk 36 64 Entoloma nitidum Hoh rainforest unk 81 19 ** Phylloporus rhodoxanthus Hoh rainforest unk 81 19 ** Hart et al. 2006 Lycoperdon perlatum Lamar Haines unk 1 99 *** Bovista spp. Snowbowl unk 47 53 Lycoperdon perlatum Snowbowl unk 7 93 *** Lycoperdon pyriforme Snowbowl unk 0 100 *** Clemmensen et al. 2006 Fayodia bisphaerigerella Tussock tundra unk 71 29 Mayor et al. this study Agaricus sp. Guyana unk 99 1 *** Coltriciella navispora Guyana unk 97 3 *** Coltriciella navispora Guyana unk 93 7 *** Coltriciella navispora Guyana unk 93 7 *** Coltriciella oblectabillis Guyana unk 99 1 *** Gymnopilus sp. Guyana unk 50.2 49.8 Tremellodendron ocreatum Guyana unk 91 9 ***
124 APPENDIX C ISOTOPE MASS BALANCE MIXING RESULTS C-3 Supplementary table of mass balance mixing model results on a plot -by-plot basis. See Methods for a definition of terms. !15N values were measured, green values are the average of all plots, red is the highest value recorded among all plots which was substituted to achieve mass balance where indicated. PLOT PARAMETERS NH4 NO3 T r 15 N fungi avg TKN 2 0.1 .23 .32 0.68 11.56 0.60 0.3 .27 .35 0.6 0.5 .31 .38 0.53 TKN 4 0.5 .46 .54 1 6.51 1.00 TKN 12 0.0 .70 .74 0.91 3.79 0.91 TKN 16 1 .19 .3 0.97 11 0.97 TKN 21 1 .2 .3 0.99 11 0.99 TKN 22 1 .3 .38 0.95 11 0.95 TKN 30 1 .42 .49 1 8.5 1.00 TKN 32 0.7 .45 .53 0.98 6.51 0.98 TKN 34 0.5 0.4 .54 .6 0.81 6.51 0.84 0.4 0.4 .54 .59 0.84 0.3 0.4 .53 .59 0.87 TKN 39 1 .34 .41 0.97 11 0.97 TKN 40 0.8 .52 .58 0.96 6.51 0.89 0.9 .54 .59 0.89 1 .55 .6 0.83 TKN 43 0.1 .18 .29 0.92 11 0.91 0.3 .19 .3 0.91 0.5 .21 .31 0.89 TKN 51 0.5 .24 .33 0.93 11 0.93 TKN 54 0.1 .35 .43 0.89 8.69 0.92 0.3 .34 .43 0.9 0.5 .34 .43 0.91 0.4 0.1 .33 .42 0.93 0.3 0.2 .32 .41 0.95 TKN 114 0.3 .15 .26 0.98 11 0.96 0.5 .18 .29 0.94 TKN 120 0.3 0.2 .47 .54 1 6.51 0.91 0.2 0.3 .51 .57 0.87 0.3 0.3 .51 .57 0.87 TKN 127 0.6 .54 .59 0.96 6.51 0.83 0.7 .57 .62 0.82 0.8 .59 .64 0.7 TKN 131 0.1 .23 .33 0.91 11 0.93 0.3 .23 .32 0.93
125 0.5 .22 .32 0.94 TKN 133 0.5 .13 .25 0.99 11 0.99 TKN 207 1 .08 .21 1 11 0.99 TKN 210 0.8 .56 .61 1 5.8 0.88 0.9 .58 .63 0.89 1 .6 .65 0.76 TKN 213 1 .32 .4 1 11 1.00 TKN 214 0.8 .26 .35 0.94 11 0.87 1 .34 .41 0.79 TKN 222 0.1 .24 .35 0.99 9.09 0.82 0.3 .25 .36 0.96 0.5 .27 .37 0.5 TKN 223 0.5 .43 .52 1 6.51 0.92 0.7 .5 .57 0.83 TKN 225 0.1 .28 .36 0.77 11 0.67 0.3 .29 .37 0.74 0.5 .31 .38 0.5 TKN 235 1 .32 .4 1 11 1.00 TKN 237 0.1 .5 .56 0.92 6.51 0.94 0.2 0.1 .5 .57 0.93 0.3 0.1 .49 .56 0.98 jrm2 0.3 .08 .21 0.93 11 0.89 0.5 .15 .26 0.84 jsn 0.1 .47 .54 0.82 6.51 0.65 0.3 .51 .58 0.65 0.5 .55 .61 0.47 DJ 0.1 .17 .29 0.93 9.79 0.89 0.3 .2 .31 0.89 0.5 .22 .33 0.86 Average 0.90 0.02
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151 BIOGRAPHICAL SKETCH Jordan Mayor grew up in northern Virginia and became interested in biology through Natural History and National Geographic magazines and during an advanced placement biology course in high school. These intere sts were supplemented through early explorations of the nearby woods with his mother and later alone in the neighboring Appalachian Mountains. He began his college career at George Mason University in 1994 at the age of 17 where he took a variety of forma tive courses ranging from Mushrooms, Molds, & Molecules taught by Albert Torzilli to Human Ecology from Mary Catherine Bateson. After soon exhausting the available biology courses he transferred to Virginia Polytechnic Institute & State University as a bi ology major. There he worked on undergraduate research projects related to the mycorrhization of pine trees under the late and great Dr. Orson K. Miller Jr. It is here he also had his first opportunities to conduct conservation biology field work chasing around red cockaded woodpeckers through chigger infested long leaf pine woods of North Carolina. After completing his B.S. in Biology in 1999, he moved to Northern California for more naturalizing. He worked a variety of positions throughout Northern Ca lifornia and Oregon mountains including as: a stream restoration intern to create salmon habitat; a wildlife biologist hooting for spotted owls late into the night; a Forest Service technician crawling about looking for rare mollusks, salamanders, and plan ts; and, as an independent subcontractor conducting rare plant and fungi surveys. After four years of sleeping under the stars, swimming in streams, tromping through high desserts, and drying gear in middle of nowhere motels, he returned to academia as a Master's student with Terry Henkel at Humboldt State University (HSU) in 2003. At HSU he conducted his research in tropical rainforests of Guyana focused on the influence of
152 ectomycorrhizal fungi on leaf litter decomposition. This work involved participa ting and leading four expeditions to the remote Pakaraima Mountains where he lived and worked among Patamona Amerindians under luxurious tarp and hammock accommodations. It is also here where he developed an inordinate fondness for dried beans. After two years he finished up and moved to FL to work on this PhD at University of Florida with Ted Schuur in 2005. After a brief tropical stint collecting in Guyana and assisting a postdoc in Colombia, he quickly realized the benefit that refrigeration and labor atory facilities could provide to the interpretability of his soil N extractions. He also began to believe that it is questions that should drive science, not the system or techniques. These beliefs, combined with logistical concerns, led him to work in Alaska, where the promise of laboratories and preexisting data overpowered his love of tropical forests. He does not regret that decision because an ecosystem ecologist should truly have a global perspective. Upon submission of this dissertation he will r eturn to tropical forest but with a question driven approach backed by ample methodological experience. He will conduct similar fundamental research based on understanding the underlying controls and utility of 15 N measureme nts throughout Panamanian rainforest thanks to a generous National Science Foundation International Research Fellowship. This work will be a natural extension of his PhD work and will permit him to address multiple competing hypotheses in a diverse, dynam ic, and N rich system.