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Soil nutrient status and fungal community structure of high and low phosphatase microsites in a mixed… Godin, Aaron Michael 2013

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	 ?Soil	 ?nutrient	 ?status	 ?and	 ?fungal	 ?community	 ?structure	 ?of	 ?high	 ?and	 ?low	 ?phosphatase	 ?microsites	 ?in	 ?a	 ?mixed	 ?Douglas-??fir	 ?paper	 ?birch	 ?stand	 ?	 ?by	 ?AARON	 ?MICHAEL	 ?GODIN	 ?B.Sc.,	 ?The	 ?University	 ?of	 ?Toronto,	 ?2010	 ?A	 ?THESIS	 ?SUBMITTED	 ?IN	 ?PARTIAL	 ?FULFILLMENT	 ?OF	 ?	 ?THE	 ?REQUIREMENTS	 ?FOR	 ?THE	 ?DEGREE	 ?OF	 ?MASTER	 ?OF	 ?SCIENCE	 ?in	 ?The	 ?College	 ?of	 ?Graduate	 ?Studies	 ?(Biology)	 ?THE	 ?UNIVERSITY	 ?OF	 ?BRITISH	 ?COLUMBIA	 ?(Okanagan)	 ?Sept	 ?2013	 ??	 ?Aaron	 ?Michael	 ?Godin	 ?2013	 ?  ii ABSTRACT	 ?Phosphorus (P) plays an important role in driving primary production in terrestrial ecosystems. However, the majority of P in soil is covalently bound to complex organic compounds and is largely inaccessible to plants. Soil fungi facilitate the release of mineral P from organic forms, through the release of extracellular phosphatase enzymes. To date, very little work has been done to identify fungal communities physically located with phosphatase activity in situ in the field. In the current study, I examined soil nutrient status and fungal communities associated with high and low phosphatase areas. I used an enzyme imprinting method to detect mm-scale phosphatase activity from soil profiles in a mixed Douglas fir and paper birch stand in British Columbia. Small (0.05 g) soil samples were removed from areas of high and low phosphatase activity at five root windows. Total extractable P (p=0.95), inorganic phosphate (p=0.87), and soluble organic P (p=0.20) were not different between areas of high and low phosphatase activity across all windows, suggesting that P availability alone was not important in driving phosphatase activity. However, percent total carbon (p=0.05) and percent total nitrogen (p=0.05) were higher in microsites with high phosphatase activity. This implies that higher levels of carbon and nitrogen, especially relative to P, stimulated phosphatase activity. Additions of carbon (C) and nitrogen (N) to randomly-selected microsites, to test this hypothesis, were inconclusive. I used pyrosequencing to characterize fungal communities from microsites differing in phosphatase activity. When examined as assemblages of operational taxonomic units (OTUs), fungal communities were not different (Bray Curtis, p=0.53; Jaccard p=0.52) between areas of high and low phosphatase activity  iii across all windows, though communities did differ among the five windows (Bray Curtis, p<0.01; Jaccard p<0.01). Furthermore, the number of sequences as OTUs grouped by trophic status differed between microsites in some windows. Specifically, the ratio of saprotrophic (SAP) to ectomycorrhizal (EM) fungi was higher in high than low phosphatase sites in windows with low EM fungal richness. The results of these experiments contribute to our understanding of fine-scale controls of P cycling in forest soils, as well as the relative importance of various spatial scales in structuring soil fungal communities.        iv PREFACE	 ?With	 ? guidance	 ? from	 ?my	 ? supervisor,	 ? Dr.	 ?Melanie	 ? Jones,	 ? as	 ?well	 ? as	 ?Dr.	 ?Matthew	 ?Whiteside,	 ? I	 ?was	 ? responsible	 ? for	 ? the	 ? experimental	 ? design,	 ? implementation,	 ? lab	 ?analysis,	 ?and	 ?statistical	 ?analysis	 ?of	 ?the	 ?study	 ?described	 ?in	 ?Chapters	 ?2	 ?and	 ?3.	 ?I	 ?was	 ?assisted	 ? in	 ? the	 ? field	 ?with	 ? imprinting,	 ? sample	 ? collection,	 ? and	 ?nutrient	 ? injections	 ?by	 ?research	 ?assistants.	 ?A	 ?version	 ?of	 ?Chapter	 ?2	 ?is	 ?in	 ?the	 ?final	 ?stage	 ?of	 ?preparation	 ?for	 ?submission,	 ?and	 ?is	 ?being	 ?prepared	 ?by	 ?Dr.	 ?Matthew	 ?Whiteside.	 ?With	 ?guidance	 ?from	 ?Dr.	 ?Jones,	 ?I	 ?was	 ?also	 ?responsible	 ?for	 ?the	 ?writing	 ?of	 ?this	 ?entire	 ?thesis.	 ?    v TABLE	 ?OF	 ?CONTENTS	 ?ABSTRACT	 ?..................................................................................................................................	 ?ii	 ?PREFACE	 ?....................................................................................................................................	 ?iv	 ?TABLE	 ?OF	 ?CONTENTS	 ?..............................................................................................................	 ?v	 ?LIST	 ?OF	 ?TABLES	 ?.....................................................................................................................	 ?viii	 ?LIST	 ?OF	 ?FIGURES	 ?.....................................................................................................................	 ?ix	 ?ACKNOWLEDGEMENTS	 ?.........................................................................................................	 ?xi	 ?1.	 ? GENERAL	 ?INTRODUCTION	 ?............................................................................................	 ?1	 ?1.1.	 ? PHOSPHORUS	 ?IN	 ?SOIL	 ?..........................................................................................................	 ?3	 ?1.2.	 ? PHOSPHATASE	 ?.......................................................................................................................	 ?5	 ?1.3.	 ? SOIL	 ?FUNGI	 ?..............................................................................................................................	 ?7	 ?1.4.	 ? MOLECULAR	 ?CHARACTERIZATION	 ?OF	 ?SOIL	 ?FUNGI	 ?....................................................	 ?9	 ?1.5.	 ? STUDY	 ?OBJECTIVES	 ?AND	 ?PREDICTIONS	 ?......................................................................	 ?11	 ?2.	 ? Nutrient	 ?status	 ?of	 ?high	 ?and	 ?low	 ?phosphatase	 ?microsites	 ?................................	 ?14	 ?2.1.	 ? SYNOPSIS	 ?...............................................................................................................................	 ?14	 ?2.2.	 ? METHODS	 ?..............................................................................................................................	 ?16	 ?2.2.1.	 ? Site	 ?Description	 ?and	 ?Study	 ?Design	 ?........................................................................	 ?16	 ?2.2.2.	 ? Soil	 ?Sampling	 ?................................................................................................................	 ?17	 ?2.2.3.	 ? Analysis	 ?of	 ?Soil	 ?Nutrients	 ?.........................................................................................	 ?19	 ?2.2.4.	 ? Soil	 ?Moisture	 ?and	 ?pH	 ?.................................................................................................	 ?19	 ?2.2.5.	 ? Nutrient	 ?Addition	 ?Experiment	 ?...............................................................................	 ?20	 ?2.2.6.	 ? Comparison	 ?of	 ?Imprinting	 ?Assays	 ?with	 ?Standard	 ?Methods	 ?..........................	 ?21	 ? vi 2.2.7.	 ? Data	 ?Analysis	 ?................................................................................................................	 ?22	 ?2.3.	 ? RESULTS	 ?................................................................................................................................	 ?23	 ?2.3.1.	 ? Soil	 ?Properties	 ?.............................................................................................................	 ?23	 ?2.3.2.	 ? Nutrient	 ?addition	 ?experiment	 ?................................................................................	 ?25	 ?2.3.3.	 ? Comparison	 ?of	 ?imprinting	 ?assays	 ?with	 ?standard	 ?methods	 ?...........................	 ?27	 ?2.4.	 ? DISCUSSION	 ?..........................................................................................................................	 ?27	 ?2.4.1.	 ? Nutrient	 ?status	 ?of	 ?soil	 ?microsites	 ?..........................................................................	 ?28	 ?2.4.2.	 ? Nutrient	 ?addition	 ?........................................................................................................	 ?30	 ?3.	 ? Fungal	 ?community	 ?structure	 ?of	 ?high	 ?and	 ?low	 ?phosphatase	 ?microsites	 ?......	 ?33	 ?3.1.	 ? SYNOPSIS	 ?...............................................................................................................................	 ?33	 ?3.2.	 ? METHODS	 ?..............................................................................................................................	 ?36	 ?3.2.1.	 ? Site	 ?description	 ?...........................................................................................................	 ?36	 ?3.2.2.	 ? Soil	 ?Sampling	 ?................................................................................................................	 ?36	 ?3.2.3.	 ? DNA	 ?extraction,	 ?PCR	 ?amplification,	 ?and	 ?pyrosequencing	 ?of	 ?fungal	 ?DNA	 ?from	 ?soil	 ?samples	 ?......................................................................................................................	 ?36	 ?3.2.4.	 ? Data	 ?analysis	 ?................................................................................................................	 ?38	 ?3.3.	 ? RESULTS	 ?................................................................................................................................	 ?39	 ?3.4.	 ? DISCUSSION	 ?..........................................................................................................................	 ?55	 ?3.4.1.	 ? Fungal	 ?communities	 ?and	 ?fine-??scale	 ?phosphatase	 ?activities	 ?.........................	 ?55	 ?3.4.2.	 ? Ectomycorrhizal	 ?and	 ?saprotrophic	 ?fungi	 ?............................................................	 ?58	 ?3.4.3.	 ? Differences	 ?in	 ?fungal	 ?communities	 ?between	 ?windows	 ?..................................	 ?61	 ?3.4.4.	 ? The	 ?fungal	 ?community	 ?detected	 ?by	 ?454	 ?pyrosequencing	 ?.............................	 ?62	 ?4.	 ? CONCLUSION	 ?...................................................................................................................	 ?64	 ?4.1.	 ? GENERAL	 ?DISCUSSION	 ?......................................................................................................	 ?64	 ? vii 4.2.	 ? STRENGTHS	 ?AND	 ?WEAKNESSES	 ?....................................................................................	 ?66	 ?4.3.	 ? FUTURE	 ?DIRECTIONS	 ?AND	 ?SIGNIFICANCE	 ?.................................................................	 ?68	 ?REFERENCES	 ?...........................................................................................................................	 ?71	 ?APPENDIX	 ?A:	 ?Soil	 ?profile	 ?.....................................................................................................	 ?85	 ? 	 ? 	 ? viii LIST	 ?OF	 ?TABLES	 ?Table	 ?3.1:	 ?The	 ?50	 ?most	 ?frequently	 ?detected	 ?OTUs,	 ?representing	 ?approximately	 ?50%	 ?of	 ? the	 ? total	 ? reads	 ?across	 ? the	 ? site,	 ? and	 ? their	 ?average	 ?number	 ?of	 ?reads	 ?found	 ?per	 ?microsite	 ?in	 ?each	 ?window	 ?(?SE).	 ?.............................................	 ?41	 ?Table	 ? 3.2:	 ? P-??values	 ? from	 ? nested	 ? ANOVAs	 ? (phosphatase	 ? status	 ? nested	 ? within	 ?window)	 ? using	 ? number	 ? of	 ? reads	 ? and	 ? occurrence	 ? of	 ? OTUs	 ? from	 ?ectomycorrhizal	 ? (EM)	 ? and	 ? saprotrophic	 ? (SAP)	 ? fungi,	 ? and	 ? EM:SAP	 ?from	 ?five	 ?root	 ?windows. ............................................................................... 46	 ?Table	 ?3.3:	 ?P-??values	 ?from	 ?PERMANOVAs	 ?comparing	 ?communities	 ?between	 ?high	 ?and	 ?low	 ?phosphatase	 ?areas	 ?from	 ?five	 ?root	 ?windows	 ?using	 ?Jaccard	 ?and	 ?Bray-??Curtis	 ? similarity	 ? indices.	 ? OTUs	 ? were	 ? ungrouped	 ? (top)	 ? and	 ?grouped	 ?(bottom)	 ?at	 ?the	 ?family	 ?level. .......................................................... 51	 ?Table	 ? 3.4:	 ? P-??values	 ? from	 ? ANOVAs	 ? using	 ? occurrence	 ? of	 ? OTUs	 ? and	 ? number	 ? of	 ?reads	 ?from	 ?ectomycorrhizal	 ?fungi	 ?(EM),	 ?saprotrophic	 ?fungi	 ?(SAP)	 ?and	 ?EM:SAP.	 ? One-??way	 ? ANOVAs	 ? were	 ? performed	 ? separately	 ? for	 ? each	 ?window/functional	 ?group	 ?combination. ..................................................... 52	 ?  	 ? ix LIST	 ?OF	 ?FIGURES	 ?Figure	 ? 2.1:	 ? A)	 ? Percent	 ? total	 ? carbon,	 ? B)	 ? percent	 ? total	 ? nitrogen,	 ? C)	 ? carbon	 ? to	 ?nitrogen	 ? ratio,	 ?D)	 ? total	 ? extractable	 ? phosphorus,	 ? E)	 ? soluble	 ? organic	 ?phosphorus,	 ?and	 ?F)	 ?inorganic	 ?phosphorus,	 ?from	 ?soils	 ?collected	 ?from	 ?four	 ?roots	 ?windows.	 ?P-??values	 ?based	 ?on	 ?paired	 ?t-??tests	 ?(n=4). ................. 24	 ?Figure	 ?2.2:	 ?A)	 ?Gravimetric	 ?soil	 ?moisture	 ?and	 ?B)	 ?soil	 ?solution	 ?pH	 ?from	 ?samples	 ?collected	 ?from	 ?four	 ?root	 ?windows.	 ?P-??values	 ?based	 ?on	 ?paired	 ?t-??tests	 ?(n=4). ........................................................................................................... 25	 ?Figure	 ?2.3:	 ?Average	 ?phosphatase	 ?intensity	 ?(0-??256)	 ?per	 ?treatment	 ?per	 ?root,	 ?with	 ?average	 ? intensity	 ? of	 ? dry	 ? controls	 ? subtracted	 ? separately	 ? for	 ? each	 ?window.	 ?Sampling	 ?was	 ?conducted	 ?five	 ?times	 ?over	 ?a	 ?432-??hour	 ?period	 ?following	 ? nutrient	 ? injections.	 ? P-??values	 ? based	 ? on	 ? nested	 ? ANOVA?s,	 ?with	 ?treatment	 ?nested	 ?within	 ?window,	 ?and	 ?conducted	 ?separately	 ?for	 ?each	 ?time	 ?(n=4). .......................................................................................... 26	 ?Figure	 ?2.4:	 ?Phosphatase	 ?activity	 ?of	 ?soils	 ?collected	 ?from	 ?five	 ?root	 ?windows	 ?from	 ?(a)	 ?June	 ?29,	 ?2012	 ?and	 ?(b)	 ?July	 ?7,	 ?2012.	 ?P-??values	 ?based	 ?on	 ?paired	 ?t-??tests	 ?(n=4). .................................................................................................. 27	 ?Figure	 ?3.1:	 ?Accumulation	 ?curves	 ?showing	 ?the	 ?number	 ?of	 ? fungal	 ?OTUs	 ?(defined	 ?as	 ? 97.5%	 ? sequence	 ? similarity)	 ? detected	 ? in	 ? each	 ?microsite	 ? at	 ? every	 ?window. ....................................................................................................... 43	 ?Figure	 ?3.2:	 ?Taxonomic	 ?distribution	 ?of	 ?OTUs	 ?at	 ?the	 ?family	 ?level,	 ?according	 ?to	 ?the	 ?top	 ?BLAST	 ?hits	 ?in	 ?the	 ?NCBI	 ?database.	 ?OTUs	 ?that	 ?were	 ?not	 ?classifiable	 ?at	 ?the	 ?family	 ?level	 ?were	 ?omitted. ............................................................... 44	 ?Figure	 ? 3.3:	 ? Non-??metric	 ? multi-??dimensional	 ? scaling	 ? with	 ? (A)	 ? Jaccard	 ? [stress	 ? =	 ?0.15]	 ?and	 ?(B)	 ?Bray-??Curtis	 ?[stress	 ?=	 ?0.17]	 ?similarity	 ?indices	 ?from	 ?high	 ?(green	 ? symbols)	 ? and	 ? low	 ? (blue	 ? symbols)	 ? phosphatase	 ? areas	 ? from	 ?Windows	 ?A	 ?through	 ?E. ................................................................................ 46	 ?Figure	 ?3.4:	 ?Bars	 ?(?SE)	 ?represent	 ?mean	 ?number	 ?of	 ?ectomycorrhizal	 ?(EM)	 ?fungal	 ?reads	 ? (A)	 ? and	 ? OTU	 ? occurrence	 ? (B)	 ? per	 ? microsample	 ? per	 ? window.	 ?Different	 ? letters	 ? indicate	 ? significant	 ? differences	 ? based	 ? on	 ? one-??way	 ?ANOVA	 ? followed	 ? by	 ? Tukey?s	 ? honest	 ? significance	 ? test	 ? (?	 ?=	 ? 0.05),	 ?corrected	 ?for	 ?multiple	 ?comparisons. ......................................................... 47	 ?Figure	 ? 3.5:	 ? Bars	 ? (?SE)	 ? represent	 ? mean	 ? number	 ? of	 ? saprotrophic	 ? (SAP)	 ? fungal	 ?reads	 ? (A)	 ? and	 ? OTU	 ? occurrence	 ? (B)	 ? per	 ? microsample	 ? per	 ? window.	 ?Different	 ? letters	 ? indicate	 ? significant	 ? differences	 ? based	 ? on	 ? one-??way	 ?ANOVA	 ? followed	 ? by	 ? Tukey?s	 ? honest	 ? significance	 ? test	 ? (?	 ?=	 ? 0.05),	 ?corrected	 ?for	 ?multiple	 ?comparisons. ......................................................... 48	 ? x Figure	 ? 3.6:	 ? Bars	 ? (?SE)	 ? represent	 ? ectomycorrhizal	 ? (EM)	 ? to	 ? saprotrophic	 ? (SAP)	 ?fungal	 ? ratios	 ? based	 ? on	 ? (A)	 ? number	 ? of	 ? fungal	 ? reads	 ? and	 ? (B)	 ? OTU	 ?occurrence	 ? averaged	 ? among	 ? microsamples	 ? per	 ? window.	 ? Different	 ?letters	 ? indicate	 ? significant	 ? differences	 ? based	 ? on	 ? one-??way	 ? ANOVA	 ?followed	 ? by	 ? Tukey?s	 ? honest	 ? significance	 ? test	 ? (?	 ?=	 ? 0.05),	 ? corrected	 ?for	 ?multiple	 ?comparisons. .......................................................................... 49	 ?Figure	 ?3.7:	 ?Boxplot	 ?of	 ?ectomycorrhizal	 ?(EM)	 ?to	 ?saprotrophic	 ?(SAP)	 ?fungal	 ?ratios	 ?based	 ? on	 ? number	 ? of	 ? fungal	 ? reads	 ? from	 ? high	 ? (H)	 ? and	 ? low	 ? (L)	 ?phosphatase	 ?areas	 ? from	 ? five	 ? (A-??E)	 ? root	 ?windows.	 ? (*)	 ? indicates	 ?p	 ?=	 ?0.05,	 ?while	 ?(?)	 ?indicates	 ?p	 ?=	 ?0.07	 ?between	 ?high	 ?and	 ?low	 ?phosphatase	 ?levels	 ?according	 ?to	 ?one-??way	 ?ANOVAs	 ?within	 ?the	 ?windows	 ?indicated. ... 53	 ?Figure	 ?3.8:	 ?Boxplot	 ?of	 ?number	 ?of	 ?ectomycorrhizal	 ?(EM)	 ?fungal	 ?reads	 ?from	 ?high	 ?(H)	 ? and	 ? low	 ? (L)	 ? phosphatase	 ? areas	 ? from	 ? five	 ? (A-??E)	 ? root	 ?windows.	 ?(*)	 ? indicates	 ? p	 ? =	 ? 0.05,	 ? between	 ? high	 ? and	 ? low	 ? phosphatase	 ? levels	 ?according	 ?to	 ?a	 ?one-??way	 ?ANOVA	 ?within	 ?the	 ?window	 ?indicated. .............. 54	 ?Figure	 ?3.9:	 ?Boxplot	 ?of	 ?number	 ?of	 ?saprotrophic	 ?(SAP)	 ?fungal	 ?reads	 ?from	 ?high	 ?(H)	 ?and	 ? low	 ? (L)	 ? phosphatase	 ? areas	 ? from	 ? five	 ? (A-??E)	 ? root	 ? windows.	 ? (?)	 ?indicates	 ? p	 ? =	 ? 0.07,	 ? between	 ? high	 ? and	 ? low	 ? phosphatase	 ? levels	 ?according	 ?to	 ?a	 ?one-??way	 ?ANOVA	 ?within	 ?the	 ?window	 ?indicated. .............. 54	 ?   !"#$%&'()*+!"#$%!&'#($%)!('#*!+$,-#+!./!///////////////////////////////////////////////////////////////////////////////////////!01! xi ACKNOWLEDGEMENTS	 ?I	 ? would	 ? like	 ? to	 ? thank	 ? the	 ? members	 ? of	 ? my	 ? committee	 ? for	 ? their	 ? insight	 ? and	 ?support,	 ?especially	 ?my	 ?supervisor,	 ?Professor	 ?Melanie	 ?Jones.	 ?I	 ? thank	 ?Valerie	 ?Ward	 ?and	 ?Sherrie	 ?Maxwell	 ? for	 ?assistance	 ?in	 ?the	 ? lab,	 ?and	 ?Denise	 ?Brooks,	 ? Travis	 ? Dickson,	 ? Carrie	 ? van	 ? Dorp,	 ? Taylor	 ? Holland,	 ? Lucy	 ? Liu,	 ? Logan	 ?Makaroff,	 ? Bailey	 ? Nicholson,	 ? Einav	 ? Shalev,	 ? Alicia	 ? Tymstra,	 ? Shayle	 ? Wiebe,	 ? and	 ?Matthew	 ?Whiteside	 ? for	 ? assistance	 ? in	 ? the	 ? field.	 ? I	 ? also	 ? thank	 ?Miranda	 ? Hart	 ? and	 ?Jason	 ? Pither	 ? for	 ? their	 ? assistance	 ?with	 ? statistics,	 ? as	 ?well	 ? as	 ? the	 ? countless	 ? other	 ?professors,	 ?post-??docs,	 ?and	 ?grad	 ?students	 ?who	 ?helped	 ?me	 ?out	 ?along	 ?the	 ?way.	 ?Lastly,	 ? I	 ? would	 ? like	 ? to	 ? thank	 ? my	 ? family	 ? and	 ? friends	 ? for	 ? their	 ? unconditional	 ?support. 1 1. GENERAL	 ?INTRODUCTION	 ?Fungi	 ? play	 ? an	 ? integral	 ? role	 ? in	 ? coniferous	 ? forest	 ? ecosystems.	 ? In	 ? addition	 ? to	 ?contributing	 ? to	 ? a	 ? substantial	 ? portion	 ? of	 ? soil	 ? biomass	 ? (Wallender	 ? et	 ? al.,	 ? 2001;	 ?Clemmensen	 ? et	 ? al.,	 ? 2013),	 ? they	 ? play	 ? an	 ? important	 ? role	 ? in	 ? nutrient	 ? cycling.	 ? The	 ?release	 ? of	 ? extracellular	 ? enzymes	 ? by	 ? fungi	 ? initiates	 ? the	 ? rate-??limiting	 ? step	 ? of	 ?decomposition:	 ? the	 ? depolymerization	 ? and	 ? subsequent	 ? mineralization	 ? of	 ? complex	 ?macromolecules	 ? into	 ?assimilable	 ?nutrients	 ? (Dighton	 ?and	 ?Boddy	 ?1989;	 ?Schimel	 ?and	 ?Bennett,	 ?2004).	 ?Phosphatase,	 ?an	 ?extracellular	 ?enzyme	 ?secreted	 ?by	 ? fungi,	 ? facilitates	 ?the	 ? release	 ? of	 ? inorganic	 ? phosphorus	 ? (P)	 ? from	 ? organic	 ? forms	 ? (Nygren	 ? &	 ? Rosling,	 ?2009;	 ?Courty	 ? et	 ? al.,	 ? 2010).	 ? In	 ? forest	 ? soils,	 ? a	 ? large	 ?portion	 ?of	 ?P	 ? in	 ? soil	 ? is	 ? covalently	 ?bound	 ? to	 ? complex	 ? organic	 ? compounds	 ? (Plassard	 ? and	 ? Dell,	 ? 2010)	 ? and	 ? is,	 ?consequently,	 ? inaccessible	 ? to	 ? plants.	 ? To	 ? date,	 ? very	 ? little	 ? work	 ? has	 ? been	 ? done	 ? in	 ?examining	 ?phosphatase	 ?activity	 ?and	 ?corresponding	 ?fungal	 ?communities	 ?in	 ?situ	 ?in	 ?the	 ?field.	 ? Furthermore,	 ? enzyme	 ? activities	 ? associated	 ? with	 ? fungal	 ? hyphae	 ? in	 ? situ	 ? have	 ?been	 ?largely	 ?uncharacterized	 ?due	 ?to	 ?the	 ?fragile	 ?nature	 ?of	 ?hyphae	 ?and	 ?the	 ?difficulty	 ?in	 ?measuring	 ? fine-??scale	 ? production	 ? of	 ? enzymes.	 ? Instead,	 ?most	 ? knowledge	 ? of	 ? enzyme	 ?excretion	 ?comes	 ?from	 ?lab	 ?studies	 ?on	 ?bulk	 ?soil,	 ?or	 ?from	 ?studies	 ?on	 ?root	 ?tips	 ?colonized	 ?by	 ? ectomycorrhizal	 ? (EM)	 ? fungi.	 ? Recently	 ? developed	 ? in	 ? situ	 ? enzyme	 ? assays	 ? have	 ?allowed	 ?for	 ?the	 ?detection	 ?of	 ?soil	 ?enzyme	 ?activities	 ?in	 ?soil	 ?at	 ?a	 ?fine	 ?scale,	 ?with	 ?minimal	 ?disturbance	 ? to	 ? microbial	 ? communities	 ? (Dong	 ? et	 ? al.,	 ? 2007;	 ? Wallenstein	 ? and	 ?Weintraub,	 ?2008)	 ? 2 In	 ? recent	 ? studies,	 ? Brooks	 ? (2010)	 ? and	 ? Brooks	 ? et	 ? al.	 ? (2013)	 ? used	 ? an	 ? enzyme	 ?imprinting	 ?method	 ? to	 ? detect	 ? fine	 ? scale	 ? (mm)	 ? phosphatase	 ? activity	 ? in	 ? organic	 ? and	 ?mineral	 ?soil	 ?horizons	 ?across	 ?different	 ?aged	 ?(4-??6yr,	 ?21-??30yr,	 ?60-??70yr,	 ?and	 ?90-??103yr)	 ?Douglas-??fir	 ?and	 ?paper	 ?birch	 ?stands.	 ?Soils	 ?were	 ?sampled	 ?at	 ?soil	 ?windows	 ?from	 ?areas	 ?of	 ?high	 ?and	 ?low	 ?phosphatase	 ?activity	 ?and	 ?their	 ?fungal	 ?communities	 ?compared	 ?using	 ?Terminal	 ?Restriction	 ?Fragment	 ?Length	 ?Polymorphism	 ?(TRFLP)	 ?signatures.	 ?In	 ?all	 ?but	 ?two	 ? forest	 ? stands,	 ? soil	 ?micro-??sites	 ?with	 ? high	 ? phosphatase	 ? activity	 ? exhibited	 ? lower	 ?species	 ? richness	 ? of	 ? EM	 ? fungi	 ? than	 ? micro-??sites	 ? with	 ? undetectable	 ? phosphatase	 ?activity	 ? (Brooks,	 ? 2010).	 ? Furthermore,	 ? the	 ? overall	 ? EM	 ? fungal	 ? fingerprints	 ? were	 ?different	 ?between	 ?high	 ?and	 ?low	 ?phosphatase	 ?microsites	 ?across	 ?the	 ?chronosequence.	 ?	 ?There	 ? were	 ? several	 ? limitations	 ? to	 ? this	 ? study,	 ? however.	 ? First,	 ? the	 ? TRFLP	 ? analysis	 ?could	 ?only	 ?be	 ?used	 ?to	 ?distinguish	 ?species	 ?richness,	 ?not	 ?relative	 ?abundance	 ?because	 ?each	 ?taxon	 ?produces	 ?only	 ?one	 ?fingerprint	 ?and	 ?TRFLP	 ?is	 ?not	 ?a	 ?quantitative	 ?technique.	 ?Furthermore,	 ? TRFLP	 ? signatures	 ? belonging	 ? to	 ? fungi	 ? other	 ? than	 ? EM	 ? fungi	 ? were	 ? not	 ?identified,	 ? because	 ? researchers	 ?had	 ?only	 ? a	 ?database	 ?of	 ?EM	 ? fungal	 ?DNA	 ? from	 ? these	 ?sites.	 ?Lastly,	 ?nothing	 ?was	 ?known	 ?about	 ? the	 ?chemical	 ?composition	 ?of	 ? the	 ?soil	 ? in	 ? the	 ?microsites	 ?with	 ?high	 ?and	 ?low	 ?phosphatase	 ?activities.	 ?Therefore,	 ?the	 ?overall	 ?structure	 ?and	 ? function	 ? of	 ? fungal	 ? communities	 ? in	 ? these	 ? micro-??sites	 ? remained	 ? largely	 ?uncharacterized.	 ? The	 ? overarching	 ? objective	 ? of	 ? my	 ? thesis	 ? was	 ? to	 ? characterize	 ?relevant	 ?aspects	 ?of	 ? the	 ?chemistry	 ?of	 ?high	 ?and	 ? low-??phosphatases	 ?microsites	 ?at	 ? root	 ?windows	 ? at	 ? one	 ? of	 ? the	 ? sites	 ? used	 ? by	 ? Brooks	 ? (2010)	 ? and	 ? to	 ? use	 ? next	 ? generation	 ?sequencing	 ?to	 ?describe	 ?the	 ?fungal	 ?communities	 ?present	 ?there.	 ?	 ? 3 1.1. PHOSPHORUS	 ?IN	 ?SOIL	 ?Most	 ?mineral	 ?nutrients	 ?required	 ?for	 ?plant	 ?growth	 ?are	 ?present	 ?in	 ?millimolar	 ?amounts	 ?in	 ? soil	 ? solution.	 ? However,	 ? inorganic	 ? P	 ? is	 ? present	 ? in	 ? only	 ? micromolar	 ? amounts	 ?(Gyeneshwar	 ? et	 ? al,	 ? 2002),	 ? ranging	 ? between	 ? 0.1	 ? and	 ? 10	 ? ?M	 ? (Hisinger,	 ? 2001).	 ?Orthophosphate,	 ? the	 ?main	 ? form	 ?absorbed	 ?by	 ?plant	 ? roots,	 ? constitutes	 ? less	 ? than	 ?one	 ?percent	 ? of	 ? total	 ? P	 ? in	 ? soil	 ? solution	 ? at	 ? a	 ? given	 ? time	 ? (Sylvia	 ? et	 ? al.,	 ? 2005).	 ? Next	 ? to	 ?nitrogen	 ?(N),	 ?P	 ?is	 ?one	 ?of	 ?the	 ?main	 ?growth-??limiting	 ?nutrients	 ?in	 ?northern	 ?coniferous	 ?forest	 ?soils	 ?(Akselsson	 ?et	 ?al.,	 ?2008;	 ?Quesnel	 ?and	 ?Cote,	 ?2009).	 ?	 ?The	 ?low	 ?availability	 ?of	 ?inorganic	 ?P	 ?is	 ?due	 ?mainly	 ?to	 ?its	 ?high	 ?reactivity	 ?in	 ?soil	 ?solution	 ?(Hisinger,	 ? 2001).	 ? 	 ? In	 ? alkaline	 ? soils,	 ? inorganic	 ? phosphate	 ? complexes	 ? with	 ? calcium	 ?compounds,	 ? whereas	 ? acidic	 ? soils	 ? favor	 ? the	 ? formation	 ? of	 ? aluminum	 ? and	 ? iron	 ?phosphate	 ? compounds,	 ? thus	 ? rendering	 ? phosphate	 ? insoluble	 ? (Hisinger,	 ? 2001;	 ?Gyeneshwar	 ? et	 ? al.,	 ? 2002).	 ? 	 ? Vitousek	 ? et	 ? al.	 ? (2010)	 ? summarized	 ? several	 ? other	 ?mechanisms	 ?by	 ?which	 ?P	 ?can	 ?become	 ?limited	 ?in	 ?terrestrial	 ?soils.	 ?Physical	 ?soil	 ?barriers	 ?may	 ?make	 ?portions	 ?of	 ?soil	 ? inaccessible	 ?to	 ?roots	 ?or	 ?mycorrhizal	 ? fungi.	 ?For	 ?example,	 ?the	 ? development	 ? of	 ? clay-??rich	 ? horizons	 ? may	 ? prevent	 ? root	 ? access	 ? to	 ? deeper	 ? soil	 ?horizons	 ?containing	 ?P-??rich	 ?patches.	 ?Secondly,	 ?soils	 ?developed	 ?over	 ?parent	 ?material	 ?with	 ?low	 ?phosphorus	 ?content	 ?will	 ?be	 ?limited	 ?in	 ?phosphorus,	 ?thus	 ?constraining	 ?total	 ?availability.	 ? Lastly,	 ? in	 ? soils	 ? where	 ? N	 ? is	 ? in	 ? abundance,	 ? high	 ? N:P	 ? ratios	 ? can	 ? cause	 ? P	 ?limitation.	 ? This	 ? may	 ? occur	 ? when	 ? parent	 ? material	 ? weathers	 ? slowly	 ? enough	 ? that	 ?phosphorus	 ?supply	 ?constrains	 ?plant	 ?growth	 ?All	 ?P	 ?in	 ?soil	 ?initially	 ?originates	 ?from	 ?mineral	 ?sources	 ?in	 ?the	 ?Earth?s	 ?crust,	 ?with	 ?apatite	 ? 4 (Ca10(PO4)6(OH,F,Cl)2)	 ?being	 ?the	 ?dominant	 ?contributor	 ?(Nygren	 ?and	 ?Rosling,	 ?2009).	 ?P	 ? is	 ? absorbed	 ? by	 ? roots	 ? and	 ? other	 ? soil	 ? organisms	 ? from	 ? soil	 ? solution	 ? as	 ? inorganic	 ?orthophosphate	 ? ions	 ? 	 ? (Raghothoma,	 ? 2005).	 ? Dissolved	 ? orthophosphate	 ? ions	 ? will	 ?associate	 ? with	 ? one,	 ? two,	 ? or	 ? three	 ? hydrogen	 ? ions	 ? (i.e.,	 ? HPO42-??,	 ? H2PO41-??,	 ? or	 ? H3PO4)	 ?depending	 ?on	 ?the	 ?pH	 ?of	 ?the	 ?soil.	 ?In	 ?most	 ?forest	 ?soils,	 ?H2PO4-??	 ?is	 ?the	 ?most	 ?common	 ?of	 ?these	 ? three	 ? forms	 ? (Fisher	 ?and	 ?Binkley,	 ?2000).	 ?After	 ?being	 ?absorbed	 ?by	 ?organisms,	 ?most	 ?P	 ? is	 ?assimilated	 ? into	 ?organic	 ?molecules,	 ?which	 ?are	 ?ultimately	 ?returned	 ?to	 ? the	 ?soil	 ? in	 ? litter.	 ? These	 ? must	 ? be	 ? re-??mineralized	 ? in	 ? order	 ? to	 ? be	 ? accessible	 ? to	 ? soil	 ?organisms.	 ?Organic	 ?P	 ?typically	 ?represents	 ?30	 ?to	 ?50	 ?percent	 ?of	 ?total	 ?P	 ?in	 ?most	 ?soils	 ?(Sylvia	 ?et	 ?al.,	 ?2005),	 ?although	 ?it	 ?may	 ?range	 ?from	 ?as	 ?low	 ?as	 ?20	 ?percent	 ?to	 ?as	 ?much	 ?as	 ?90	 ?percent	 ?in	 ?highly	 ? organic	 ? soils	 ? (Kogel-??Knaber,	 ? 2006).	 ? Organic	 ? forms	 ? of	 ? phosphate	 ? in	 ? soils	 ?include	 ? phosphomonoesters	 ? (i.e.	 ? inositol	 ? phosphates,	 ? sugar	 ? phosphates,	 ? and	 ?mononucleotides),	 ? phosphodiesters	 ? (i.e.	 ? DNA,	 ? RNA,	 ? and	 ? phospholipids)	 ? and	 ?phosphonates	 ?(containing	 ?C-??P	 ?bonds)	 ?(Kogel-??Knaber,	 ?2006).	 ?Inositol	 ?phosphates	 ?are	 ?the	 ?most	 ? abundant	 ? form	 ? of	 ? phosphomonoesters,	 ? contributing	 ? up	 ? to	 ? 50	 ? percent	 ? of	 ?organic	 ?phosphate	 ?in	 ?soil	 ?(Gyeneshwar	 ?et	 ?al.,	 ?2002).	 ?Inositol	 ?phosphates	 ?are	 ?a	 ?family	 ?of	 ?phosphoric	 ?esters	 ?of	 ?inositol	 ?in	 ?various	 ?states	 ?of	 ?phosphorylation,	 ?of	 ?which	 ?one	 ?to	 ?six	 ? orthophosphate	 ? ions	 ? are	 ? bound	 ? to	 ? inositol	 ? (Turner	 ? et	 ? al,	 ? 2002).	 ? 	 ? Phytate,	 ? a	 ?hexaphosphate	 ? ester	 ? of	 ? inositol,	 ? is	 ? the	 ?most	 ? abundant	 ? from	 ? of	 ? inositol	 ? phosphate	 ?and	 ?can	 ?contribute	 ?to	 ?a	 ?significant	 ?amount	 ?of	 ?total	 ?organic	 ?phosphate	 ?in	 ?soil.	 ?Inositol	 ?phosphates	 ? containing	 ? one	 ? to	 ? five	 ? phosphate	 ? groups	 ? are	 ? less	 ? common,	 ? and	 ? are	 ?thought	 ?to	 ?be	 ?degradation	 ?products	 ?of	 ?phytate.	 ?(Kogel-??Knaber,	 ?2006).	 ?Extracellular	 ? 5 enzymes	 ?excreted	 ?by	 ?a	 ?range	 ?of	 ?soil	 ?organisms	 ?mineralize	 ?these	 ?forms	 ?of	 ?organic	 ?P	 ?into	 ?absorbable	 ?forms	 ?of	 ?inorganic	 ?P.	 ?1.2. PHOSPHATASE	 ?Soil	 ?fungi,	 ?bacteria,	 ?and	 ?plant	 ?roots,	 ?exude	 ?extracellular	 ?acid	 ?phosphatases,	 ?including	 ?phosphomoneresterases	 ? and	 ? phosphodiesterases,	 ? which	 ? act	 ? to	 ? cleave	 ? phosphate-??ester	 ? bonds	 ? to	 ? release	 ? inorganic	 ? P	 ? (orthophosphate)	 ? from	 ? a	 ? range	 ? of	 ? substrates	 ?including	 ? inositol	 ? phosphate,	 ? phosphorylated	 ? sugars	 ? (Nygren	 ? and	 ? Rosling,	 ? 2009;	 ?Courty	 ? et	 ? al.,	 ? 2010),	 ? and	 ? organic	 ? materials	 ? present	 ? intracellularly	 ? or	 ? in	 ? the	 ? soil	 ?(Ragothama,	 ? 2005).	 ? Fungi	 ? and	 ? microbes	 ? can	 ? also	 ? produce	 ? intracellular	 ? alkaline	 ?phosphatases,	 ?which	 ?are	 ?similar	 ? to	 ?acid	 ?phosphatases	 ?but	 ?are	 ?active	 ?at	 ?higher	 ?pHs	 ?and	 ? produced	 ? in	 ? lower	 ? quantities	 ? (van	 ? Aarle	 ? and	 ? Plassard,	 ? 2010).	 ? The	 ? relative	 ?contribution	 ?of	 ?soil	 ?bacteria,	 ?fungi,	 ?and	 ?roots	 ?to	 ?P	 ?mineralization	 ?in	 ?soils	 ?is	 ?unclear.	 ?EM	 ? fungi	 ? significantly	 ? increase	 ? P	 ? uptake	 ? in	 ? host	 ? plants	 ? through	 ? the	 ? production	 ? of	 ?acid	 ? phosphomonoesterases	 ? and	 ? phosphodiesterases	 ? (Tibett	 ? et	 ? al.,	 ? 1998;	 ? Nygren	 ?and	 ?Rosling,	 ?2009;	 ?van	 ?Aarle	 ?and	 ?Plassard,	 ?2010).	 ?For	 ?example,	 ?Bending	 ?and	 ?Read	 ?(1995)	 ? colonized	 ? organic	 ?matter	 ? collected	 ? from	 ? the	 ? fermentation	 ? layer	 ? of	 ? a	 ? beech	 ?forest	 ? with	 ? the	 ? EM	 ? fungus	 ? Paxillus	 ? involutus,	 ? and	 ? found	 ? that	 ? colonized	 ? organic	 ?matter	 ?had	 ?significantly	 ?more	 ?phosphatase	 ?activity	 ?than	 ?uncolonized	 ?organic	 ?matter,	 ?when	 ?colonized	 ?for	 ?28-??50	 ?days.	 ?In	 ?a	 ?parallel	 ?study,	 ?organic	 ?matter	 ?patches	 ?colonized	 ?with	 ? the	 ? EM	 ? fungus	 ? Suillus	 ? bovinus	 ? had	 ? significantly	 ? reduced	 ? concentrations	 ? of	 ? P	 ?versus	 ? uncolonized	 ? organic	 ? matter	 ? patches	 ? (Bending	 ? and	 ? Read,	 ? 1995b).	 ? Non-??mycorrhizal	 ? fungi	 ? also	 ? produce	 ? extracellular	 ? phosphatase	 ? enzymes.	 ? For	 ? example,	 ?Colpaert	 ? and	 ? van	 ? Laere	 ? (1996)	 ? found	 ? increased	 ? phosphatase	 ? activities	 ? in	 ? beech	 ? 6 litter	 ?colonized	 ?with	 ?the	 ?decomposer	 ?fungus	 ?Lepista	 ?nuda.	 ?Phosphatase	 ? production	 ? in	 ? soil	 ? is	 ? influenced	 ? by	 ? a	 ? variety	 ? of	 ? biotic	 ? and	 ? abiotic	 ?factors.	 ?The	 ?extent	 ?to	 ?which	 ?extracellular	 ?phosphatase	 ? in	 ?activity	 ? in	 ?soil	 ? is	 ?affected	 ?by	 ?microbial	 ? community	 ? composition	 ? is	 ?not	 ?well	 ? understood.	 ? Some	 ? fungal	 ? species	 ?have	 ?been	 ?associated	 ?with	 ?higher	 ?activities	 ?than	 ?others	 ?in	 ?beech	 ?litter	 ?(Colpaert	 ?and	 ?van	 ? Laere	 ? (1996),	 ? and	 ? soil	 ? (Burke	 ? et	 ? al.,	 ? 2012),	 ? and	 ? high	 ? phosphatase	 ?microsites	 ?have	 ? been	 ? associated	 ? with	 ? a	 ? lower	 ? number	 ? of	 ? EMF	 ? taxa	 ? than	 ? low	 ? phosphatase	 ?microsites	 ?in	 ?forest	 ?soil	 ?(Brooks,	 ?2010).	 ?Furthermore,	 ?phosphatase	 ?activity	 ?has	 ?been	 ?associated	 ? with	 ? high	 ? bacterial	 ? biomass	 ? in	 ? litter	 ? (Criquet	 ? et	 ? al.,	 ? 2004)	 ? and	 ? soil	 ?(Sakurai	 ?et	 ?al	 ?2008).	 ?The	 ?pH	 ?optimum	 ?for	 ?acid	 ?phosphomonoesterase	 ?is	 ?between	 ?4	 ?and	 ?7,	 ?and	 ?is	 ?variable	 ?depending	 ?on	 ?the	 ?type	 ?of	 ?soil	 ?(Niemi	 ?and	 ?Vepsalainen,	 ?2005;	 ?Turner,	 ? 2010).	 ? Soil	 ? moisture	 ? has	 ? been	 ? positively	 ? correlated	 ? with	 ? phosphatase	 ?activity	 ? in	 ? oak	 ? litter	 ? (Criquet	 ? et	 ? al.,	 ? 2004)	 ? and	 ? soil	 ? (Huang	 ? et	 ? al.,	 ? 2011).	 ? Lastly,	 ?nutrient	 ?status	 ?can	 ?be	 ?important	 ?in	 ?controlling	 ?phosphatase	 ?activity.	 ?A	 ?recent	 ?meta-??analysis	 ? of	 ? 34	 ? separate	 ? studies	 ? found	 ? that	 ? N	 ? fertilization	 ? increased	 ? phosphatase	 ?activity,	 ? while	 ? P	 ? fertilization	 ? strongly	 ? suppressed	 ? activity	 ? (Marklein	 ? and	 ? Houlton,	 ?2012).	 ?There	 ?are	 ?four	 ?main	 ?ways	 ?by	 ?which	 ?enzyme	 ?production	 ?is	 ?regulated	 ?by	 ?nutrients	 ?in	 ?soil,	 ?as	 ?described	 ?by	 ?Giesseler	 ?et	 ?al.	 ?(2010).	 ?Substrate	 ?induction	 ?(i),	 ?occurs	 ?when	 ?the	 ?presence	 ?of	 ? substrate	 ? induces	 ? enzyme	 ?activity.	 ? End	 ?product	 ? repression	 ? (ii)	 ? occurs	 ?when	 ?high	 ?concentrations	 ?of	 ?available	 ?nutrients	 ?reduce	 ?the	 ?production	 ?of	 ?enzymes	 ?that	 ? break	 ? down	 ? more	 ? recalcitrant	 ? substrates	 ? (Schimel	 ? et	 ? al.,	 ? 1992;	 ? Allison	 ? and	 ? 7 Vitousek,	 ?2005).	 ?Conversely,	 ?resource	 ?limitation	 ?(iii)	 ?would	 ?be	 ?expected	 ?to	 ?stimulate	 ?activity.	 ? For	 ? example	 ? orthophosphate	 ? limitation	 ? has	 ? been	 ? shown	 ? to	 ? stimulate	 ?phosphatase	 ? activity	 ? in	 ? plant	 ? roots	 ? (Duff	 ? et	 ? al.,	 ? 1994,	 ? and	 ? references	 ? therein),	 ?bacteria	 ?(Lenburg	 ?and	 ?O?Shea	 ?1996),	 ?and	 ?EM	 ?fungi	 ?(van	 ?Aarle	 ?and	 ?Plassard,	 ?2010).	 ?Furthermore,	 ? the	 ?addition	 ?of	 ? some	 ?nutrients	 ?may	 ? increase	 ? the	 ?activity	 ?of	 ?enzymes	 ?that	 ? release	 ? other	 ? nutrients	 ? because	 ? the	 ? demand	 ? for	 ? the	 ? other	 ? nutrients	 ? has	 ?effectively	 ? been	 ? increased	 ? by	 ? the	 ? added	 ? resources.	 ? For	 ? example,	 ? Allison	 ? and	 ?Vitousek	 ?(2005)	 ?found	 ?that	 ?additions	 ?of	 ? labile	 ?carbon	 ?and	 ?nitrogen	 ?in	 ?the	 ?forms	 ?of	 ?sodium	 ?acetate	 ?and	 ?ammonium	 ?chloride	 ?increased	 ?acid	 ?phosphatase	 ?activity	 ?to	 ?25%	 ?higher	 ? than	 ? controls.	 ? Lastly,	 ? enzymes	 ?may	 ? be	 ? constitutively	 ? produced	 ? (iv)	 ? at	 ? low	 ?concentrations,	 ? allowing	 ?microbes	 ? to	 ? detect	 ? and	 ? respond	 ? to	 ? changes	 ? in	 ? substrate	 ?availability	 ?(Chrost,	 ?1991;	 ?Allison	 ?and	 ?Vitousek,	 ?2005).	 ?1.3. SOIL	 ?FUNGI	 ?Fungi	 ? are	 ?heterotrophic	 ? eukaryotic	 ?microorganisms	 ?present	 ? in	 ? soil	 ? as	 ? filamentous	 ?hyphae,	 ?spores,	 ?or	 ?single	 ?celled	 ?yeasts.	 ?Approximately	 ?99,000	 ?species	 ?of	 ?fungi	 ?have	 ?been	 ?described	 ?(Blackwell,	 ?2011),	 ?and	 ?estimates	 ?suggest	 ?1.5	 ?(Hawksworth	 ?1991)	 ?to	 ?5.1	 ? million	 ? (Blackwell,	 ? 2011)	 ? species	 ? may	 ? exist.	 ? Soil	 ? fungi	 ? living	 ? as	 ? filamentous	 ?hyphae	 ?acquire	 ?nourishment	 ?by	 ?extending	 ?their	 ?hyphae	 ?into	 ?surrounding	 ?substrate	 ?and	 ? absorbing	 ? nutrients.	 ? Fungi	 ? assimilate	 ? mineral	 ? nutrients	 ? from	 ? organic	 ? forms	 ?through	 ?production	 ?of	 ?extracellular	 ?enzymes	 ?(Nygren	 ?and	 ?Rosling,	 ?2009;	 ?van	 ?Aerle	 ?and	 ? Plassard,	 ? 2010).	 ? The	 ? ability	 ? to	 ? produce	 ? extracellular	 ? N-??	 ? and	 ? P-??mobilizing	 ?enzymes,	 ?such	 ?as	 ?phosphatases,	 ?chitinases,	 ?and	 ?proteases,	 ?is	 ?widespread	 ?across	 ?the	 ?fungal	 ? kingdom	 ? (Dighton,	 ? 2007).	 ? Some	 ? fungi	 ? can	 ? also	 ? access	 ? nutrients	 ? from	 ? 8 insoluble	 ? mineral	 ? sources	 ? through	 ? production	 ? of	 ? low	 ? molecular	 ? weight	 ? organic	 ?acids.	 ? For	 ? example,	 ? EM	 ? fungi	 ? have	 ? been	 ? shown	 ? to	 ? mobilize	 ? P	 ? from	 ? apatite	 ?(Wallander,	 ? 2000),	 ? and	 ?potassium	 ? from	 ?biotite	 ? (Wallander	 ? and	 ?Whickman,	 ? 1999).	 ?Furthermore,	 ?fungi	 ?are	 ?capable	 ?of	 ?parasitizing	 ?the	 ?hyphae	 ?of	 ?other	 ?fungi.	 ?Nutrients	 ?present	 ? in	 ? fungal	 ?mycelia	 ?may	 ?pose	 ?an	 ?attractive	 ?nutrient	 ? source	 ? for	 ? saprotrophic	 ?	 ?and	 ?mycorrhizal	 ? fungi	 ? (Lindahl	 ? et	 ? al.,	 ? 1999).	 ? Fungi	 ? play	 ? several	 ? unique	 ? ecological	 ?roles	 ? in	 ? soils	 ? including:	 ? saprotrophs	 ? (SAP),	 ? mutualists	 ? and	 ? symbionts	 ? of	 ?phototrophic	 ? organisms	 ? including	 ? lichens	 ? and	 ? mycorrhizal	 ? fungi,	 ? parasites	 ? and	 ?pathogens,	 ?and	 ?endophytes.	 ?For	 ?this	 ?paper,	 ?I	 ?will	 ?focus	 ?mainly	 ?on	 ?SAP	 ?and	 ?EM	 ?fungi	 ?because	 ?of	 ?their	 ?primary	 ?roles	 ?as	 ?decomposers	 ?(Rayner	 ?and	 ?Boddy,	 ?1988),	 ?and	 ?their	 ?substantial	 ?contribution	 ?to	 ?below	 ?ground	 ?biomass	 ?(Hogberg	 ?and	 ?Hogberg,	 ?2002)	 ?in	 ?forest	 ?soils.	 ?Saprotrophic	 ? fungi	 ? are	 ? free-??living	 ? fungi	 ? that	 ? receive	 ? their	 ? energy	 ? from	 ? decaying	 ?organic	 ?matter.	 ?Some	 ?species	 ?are	 ?capable	 ?of	 ?breaking	 ?down	 ?lignin	 ?(Dix	 ?and	 ?Webster	 ?1995),	 ?which	 ?represents	 ?10	 ?to	 ?40%	 ?of	 ?woody	 ?plant	 ?tissue	 ?(Aber	 ?and	 ?Melillo	 ?1991).	 ?Other	 ?species	 ?can	 ?also	 ?efficiently	 ?degrade	 ?cellulose	 ?(Valaskova	 ?and	 ?Baldrian,	 ?2006),	 ?the	 ?most	 ?abundant	 ?polysaccharide	 ?on	 ?earth	 ?and	 ?the	 ?main	 ?polymeric	 ?component	 ?of	 ?plant	 ?cell	 ?walls	 ?(Baldrian	 ?and	 ?Valaskova,	 ?2008).	 ?Saprotrophic	 ? fungi	 ?are	 ?considered	 ?to	 ? be	 ? the	 ? most	 ? efficient	 ? decomposers	 ? of	 ? these	 ? biopolymers	 ? (Baldrian,	 ? 2008)	 ? and	 ?thus,	 ? their	 ? ecological	 ? role	 ? in	 ? forest	 ? soils	 ? is	 ? extremely	 ? important.	 ? Ectomycorrhizal	 ?fungi	 ? form	 ?a	 ?symbiotic	 ? relationship	 ?with	 ?host	 ? tree	 ? roots,	 ? receiving	 ?photosynthates	 ?from	 ?the	 ?host	 ?and	 ?assisting	 ? in	 ? the	 ?uptake	 ?of	 ?otherwise	 ? inaccessible	 ?nutrients	 ? from	 ?soil.	 ?Furthermore,	 ?EM	 ?fungi	 ?can	 ? increase	 ?water	 ?absorption,	 ?disease	 ?resistance,	 ?and	 ? 9 significantly	 ?increase	 ?the	 ?surface	 ?area	 ?of	 ?roots	 ?through	 ?extramatrical	 ?hyphae,	 ?which	 ?extend	 ?from	 ?the	 ?mycorrhizal	 ?roots	 ?into	 ?the	 ?soil	 ?(Smith	 ?and	 ?Read,	 ?2008).	 ?Because	 ?EM	 ?fungi	 ?depend	 ?strongly	 ?on	 ? their	 ?hosts	 ? for	 ?C,	 ? they	 ?are	 ? less	 ?dependent	 ?on	 ?cellulolytic	 ?enzymes	 ? for	 ? carbohydrate	 ? acquisition	 ? (Dighton	 ? et	 ? al.,	 ? 2005).	 ? Both	 ? SAP	 ? and	 ?mycorrhizal	 ? fungi	 ? play	 ? an	 ? important	 ? role	 ? in	 ? the	 ? turnover	 ? of	 ? carbon	 ? and	 ? organic	 ?nutrients	 ? such	 ? as	 ? nitrogen	 ? and	 ? phosphorus	 ? in	 ? terrestrial	 ? soils	 ? (Smith	 ? and	 ? Read,	 ?2008;	 ?Taylor	 ?et	 ?al.,	 ?2010;	 ?Talbot	 ?et	 ?al.,	 ?2013),	 ?but	 ?EM	 ?fungi	 ?generally	 ? lack	 ?some	 ?of	 ?the	 ?oxidative	 ?enzymes	 ?present	 ? in	 ?the	 ?most	 ?effective	 ?SAP	 ?fungi	 ?(Martin	 ?et	 ?al.,	 ?2007;	 ?Floudas	 ?et	 ?al.,	 ?2012;	 ?Rineau	 ?et	 ?al.,	 ?2012).	 ?	 ?1.4. MOLECULAR	 ?CHARACTERIZATION	 ?OF	 ?SOIL	 ?FUNGI	 ?The	 ?internal	 ?transcribed	 ?spacer	 ?(ITS)	 ?region,	 ?located	 ?between	 ?the	 ?18S	 ?small	 ?sub	 ?unit	 ?and	 ? the	 ?28S	 ? large	 ? sub	 ?unit	 ?of	 ?nuclear	 ? ribosomal	 ?DNA,	 ? is	 ? the	 ?most	 ? commonly	 ?used	 ?genetic	 ? marker	 ? for	 ? molecular	 ? identification	 ? of	 ? fungi	 ? from	 ? environmental	 ? samples	 ?(White	 ? et	 ? al.,	 ? 1990;	 ? Gardes	 ? and	 ? Bruns,	 ? 1993;	 ? Rydberg	 ? et	 ? al.,	 ? 2009;	 ? Nilsson	 ? et	 ? al.,	 ?2010).	 ? The	 ? ITS1	 ? region	 ? is	 ? a	 ? suitable	 ? target	 ? for	 ? amplification	 ? because	 ? it	 ? has	 ? high	 ?variation	 ?at	 ? the	 ? species	 ? level,	 ? shows	 ?a	 ?high	 ? rate	 ?of	 ? evolution,	 ? and	 ?contains	 ? a	 ? large	 ?number	 ? of	 ? copies	 ? per	 ? cell	 ? (Bruns	 ? &	 ? Shefferson,	 ? 2004;	 ? Nilsson	 ? et	 ? al.,	 ? 2009).	 ?Traditional	 ? cloning,	 ? followed	 ? by	 ? Sanger	 ? sequencing	 ? of	 ? individual	 ? clones	 ? for	 ?enumerating	 ?and	 ?identifying	 ?fungi	 ?in	 ?communities	 ?suffer	 ?from	 ?a	 ?high	 ?cost	 ?and	 ?time	 ?investment	 ?and	 ?low	 ?throughput,	 ?which	 ?may	 ?limit	 ?the	 ?detection	 ?of	 ?some	 ?taxa	 ?leading	 ?to	 ?an	 ?underestimation	 ?of	 ?fungal	 ?biodiversity	 ?(Lim	 ?et	 ?al.,	 ?2010;	 ?Tedersoo	 ?et	 ?al.,	 ?2010).	 ?Pyrosequencing	 ? offers	 ? a	 ? faster	 ? and	 ? more	 ? affordable	 ? sequence	 ? platform	 ? that	 ? can	 ?produce	 ? hundreds	 ? of	 ? thousands	 ? of	 ? short	 ? sequences	 ? (or	 ? reads)	 ? (Tedersoo	 ? et	 ? al.,	 ? 10 2010),	 ?exceeding	 ?the	 ?capacity	 ?of	 ?traditional	 ?Sanger	 ?sequencing	 ?by	 ?several	 ?orders	 ?of	 ?magnitude	 ? (Margulies	 ? et	 ? al.,	 ? 2005).	 ? As	 ? a	 ? result,	 ? it	 ? is	 ? possible	 ? to	 ? examine	 ? a	 ? large	 ?number	 ? of	 ? individual	 ? samples	 ? simultaneously	 ? within	 ? a	 ? reasonable	 ? time	 ? and	 ? cost	 ?(Meyer	 ?et	 ?al.,	 ?2008;	 ?Jumpponen	 ?et	 ?al.,	 ?2010).	 ?	 ?	 ?With	 ?pyrosequencing,	 ?samples	 ?are	 ?tagged	 ?with	 ?an	 ?annealing	 ?sequence,	 ?which	 ?allows	 ?each	 ? individual	 ? sequence	 ? to	 ? be	 ? attached	 ? to	 ? a	 ? micro-??bead	 ? (Marguiles	 ? et	 ? al.,	 ? 2005;	 ?Mayer	 ? et	 ? al.,	 ? 2007;	 ?Hamaday	 ? et	 ? al.,	 ? 2008).	 ? In	 ? the	 ?next	 ? step,	 ? the	 ?micro-??beads,	 ? each	 ?with	 ? an	 ? individual	 ? sequence,	 ? are	 ? amplified	 ? in	 ? an	 ? emulsion	 ?PCR,	 ? so	 ? that	 ? each	 ?bead	 ?ends	 ?up	 ?with	 ?multiple	 ? copies	 ?of	 ? the	 ?original	 ? sequence.	 ?The	 ?beads	 ? are	 ? then	 ? spread	 ?onto	 ?a	 ?plate	 ?with	 ?approximately	 ?one	 ?million	 ?wells,	 ?such	 ?that	 ?each	 ?bead	 ?has	 ?its	 ?own	 ?well.	 ? The	 ? plate	 ? is	 ? then	 ? sequenced	 ? in	 ? a	 ? way	 ? that	 ? each	 ? nucleotide	 ? added	 ? to	 ? each	 ?sequence	 ?from	 ?each	 ?well	 ?can	 ?be	 ?recorded	 ?(Margulies	 ?et	 ?al.,	 ?2005;	 ?Meyer	 ?et	 ?al.,	 ?2008).	 ?In	 ? addition,	 ? each	 ?DNA	 ? sample	 ? extracted	 ? from	 ?soil	 ? can	 ?be	 ?barcoded	 ?with	 ? a	 ? specific	 ?sequence	 ? tag.	 ? By	 ? doing	 ? so,	 ? sequences	 ? from	 ? each	 ? sample	 ? can	 ? be	 ? distinguished,	 ?allowing	 ?extracts	 ?from	 ?many	 ?samples	 ?to	 ?be	 ?run	 ?in	 ?combination	 ?in	 ?the	 ?same	 ?section	 ?of	 ? a	 ? plate	 ? (Hamaday	 ? et	 ? al.,	 ? 2008).	 ? 	 ? In	 ? comparison	 ? to	 ? other	 ? next-??gen	 ? sequencing	 ?platforms,	 ? such	 ? as	 ? Illumina,	 ? pyrosequencing	 ? can	 ? achieve	 ? longer	 ? (approximately	 ?600bp	 ? vs	 ? 250bp)	 ? and	 ? higher	 ? quality	 ? sequences,	 ? which	 ? is	 ? useful	 ? for	 ? identifying	 ?organisms	 ?down	 ?to	 ?the	 ?genus	 ?level	 ?(Claesson	 ?et	 ?al.,	 ?2010).	 ?However,	 ?pyrosequencing	 ?produces	 ?fewer	 ?sequences	 ?than	 ?Illumina	 ?	 ?(approximately	 ?1	 ?000	 ?000	 ?vs.	 ?15	 ?000	 ?000	 ?to	 ?over	 ?60	 ?000	 ?000),	 ?and	 ?is	 ?more	 ?costly.	 ? 11 1.5. STUDY	 ?OBJECTIVES	 ?AND	 ?PREDICTIONS	 ?Using	 ?fine-??scale	 ?sampling	 ?of	 ?soils	 ?targeted	 ?by	 ?soil	 ?imprinting	 ?for	 ?enzyme	 ?activity	 ?in	 ?root	 ?windows	 ?across	 ?a	 ?chronosequence	 ?of	 ?mixed	 ?Douglas-??fir	 ?and	 ?paper	 ?birch	 ?stands,	 ?Brooks	 ? (2010)	 ? found	 ? that	 ? mm-??scale	 ? microsites	 ? with	 ? high	 ? phosphatase	 ? activity	 ?contained	 ? lower	 ? EM	 ? fungal	 ? species	 ? richness	 ? than	 ? microsites	 ? with	 ? undetectable	 ?phosphatase	 ? activity.	 ? Several	 ? hypotheses	 ? were	 ? developed	 ? to	 ? explain	 ? the	 ?observations	 ? (Brooks,	 ? 2010).	 ? All	 ? hypotheses	 ? assumed	 ? that	 ? microsites	 ? high	 ? in	 ?phosphatase	 ?represented	 ?nutrient-??rich	 ?patches,	 ?because	 ?soil	 ?phosphatase	 ?activities	 ?are	 ? often	 ? correlated	 ?with	 ?microbial	 ? biomass	 ? (Boerner	 ? et	 ? al.,	 ? 2006),	 ? although	 ? that	 ?relationship	 ?was	 ?not	 ?confirmed	 ?by	 ?Brooks	 ?(2010)	 ?for	 ?her	 ?sites.	 ?Brook?s	 ?hypotheses	 ?were:	 ?i) Saprotrophic	 ?fungi	 ?may	 ?exclude	 ?some	 ?EM	 ?fungi	 ?from	 ?nutrient-??rich	 ?microsites	 ?	 ? ii) Phosphatase	 ?activities	 ?are	 ?low	 ?where	 ?EM	 ?fungal	 ?biomass	 ?is	 ?high,	 ?because	 ?EM	 ?fungi	 ?select	 ?for	 ?soil	 ?bacteria	 ?with	 ?low	 ?phosphatase	 ?activities,	 ?as	 ?observed	 ?by	 ?Brooks	 ?et	 ?al.	 ?(2011).	 ?	 ?	 ? iii) Low	 ?EM	 ?fungal	 ?richness	 ?in	 ?microsites	 ?with	 ?high	 ?phosphatase	 ?activities	 ?is	 ?a	 ?result	 ?of	 ?priority	 ?effects,	 ?where	 ?the	 ?first	 ?EM	 ?fungal	 ?species	 ?to	 ?colonize	 ?a	 ?microsite	 ?dominates	 ?the	 ?location	 ?and	 ?excludes	 ?other	 ?fungal	 ?species	 ?from	 ?colonizing.	 ?	 ?In	 ? my	 ? thesis,	 ? some	 ? experiments	 ? are	 ? directed	 ? at	 ? hypothesis	 ? (i),	 ? as	 ? well	 ? as	 ? the	 ? 12 underlying	 ? assumption	 ? that	 ? higher	 ? phosphatase	 ? microsites	 ? are	 ? rich	 ? in	 ? organic	 ?matter.	 ?My	 ?study	 ?was	 ?conducted	 ?at	 ?one	 ?of	 ?the	 ?same	 ?sites	 ?used	 ?by	 ?Brooks	 ?(2010),	 ?but	 ?on	 ?five	 ?replicate	 ?windows	 ?per	 ?site,	 ?whereas	 ?Brooks	 ?imprinted	 ?only	 ?one	 ?window	 ?per	 ?site.	 ?	 ?This	 ?site	 ?was	 ?in	 ?one	 ?of	 ?the	 ?age	 ?classes	 ?(stem	 ?exclusion,	 ?61-??71	 ?years	 ?old)	 ?where	 ?Brooks	 ? (2010)	 ? detected	 ? differences	 ? in	 ? EM	 ? fungal	 ? richness	 ? between	 ? high	 ? and	 ? low	 ?phosphatase	 ?microsites.	 ?Specifically,	 ?the	 ?objectives	 ?for	 ?my	 ?thesis	 ?were:	 ?a) To	 ?compare	 ?soil	 ?nutrient	 ?status	 ?in	 ?high	 ?and	 ?low	 ?phosphatase	 ?microsites	 ?(Chapter	 ?2)	 ?	 ? b) To	 ?test	 ?whether	 ?the	 ?addition	 ?of	 ?C	 ?and	 ?N	 ?to	 ?soil	 ?profiles	 ?in	 ?situ	 ?influences	 ?phosphatase	 ?activity	 ?(Chapter	 ?2)	 ?	 ? c) To	 ?characterize	 ?fungal	 ?communities	 ?in	 ?high	 ?and	 ?low	 ?phosphatase	 ?microsites	 ?using	 ?pyrosequencing	 ?(Chapter	 ?3)	 ?	 ?Predictions:	 ?	 ?i) High	 ?phosphatase	 ?activities	 ?will	 ?be	 ?associated	 ?with	 ?microsites	 ?high	 ?in	 ?C	 ?and	 ?N,	 ?and	 ?low	 ?in	 ?inorganic	 ?P	 ?	 ? ii) Additions	 ?of	 ?C	 ?and	 ?N	 ?will	 ?stimulate	 ?phosphatase	 ?activity	 ?	 ? 13 iii) High	 ?phosphatase	 ?microsites	 ?will	 ?be	 ?associated	 ?with	 ?different	 ?fungal	 ?communities	 ?than	 ?low	 ?phosphatase	 ?microsites,	 ?which	 ?would	 ?confirm	 ?the	 ?results	 ?of	 ?Brooks	 ?	 ? iv) High	 ?phosphatase	 ?microsites	 ?will	 ?have	 ?lower	 ?richness	 ?of	 ?EM	 ?fungi	 ?than	 ?low	 ?phosphatase	 ?microsites,	 ?again	 ?confirming	 ?the	 ?results	 ?of	 ?Brooks.	 ?	 ? v) The	 ?richness	 ?and	 ?number	 ?of	 ?reads	 ?of	 ?SAP	 ?fungi	 ?will	 ?be	 ?greater	 ?in	 ?high	 ?than	 ?low	 ?phosphatase	 ?microsites.	 ?	 ? vi) The	 ?ratio	 ?of	 ?SAP	 ?to	 ?EM	 ?fungal	 ?sequences	 ?would	 ?be	 ?higher	 ?in	 ?high	 ?than	 ?low	 ?phosphatase	 ?microsites,	 ?which	 ?would	 ?be	 ?consistent	 ?with	 ?Brooks?	 ?hypothesis	 ?(i).	 ?	 ?     14 2. Nutrient	 ?status	 ?of	 ?high	 ?and	 ?low	 ?phosphatase	 ?microsites	 ?2.1. SYNOPSIS	 ?Phosphorus	 ? (P)	 ? plays	 ? an	 ? important	 ? role	 ? in	 ? driving	 ? primary	 ? productivity	 ? in	 ?terrestrial	 ? ecosystems.	 ? It	 ? is	 ? a	 ? structural	 ? building	 ? block	 ? of	 ? nucleic	 ? acids	 ? and	 ?phospholipids,	 ?and,	 ?as	 ?a	 ?component	 ?of	 ?ATP,	 ?is	 ?crucial	 ?to	 ?intracellular	 ?energy	 ?transfer	 ?in	 ?all	 ?organisms.	 ?P	 ? initially	 ?enters	 ? the	 ?soil	 ?solution	 ?as	 ?soluble	 ?orthophosphate	 ? ions	 ?(PO4-??3),	 ? after	 ? slow	 ? weathering	 ? of	 ? primary	 ? minerals	 ? (Filippelli,	 ? 2008).	 ? 	 ? However,	 ?orthophosphate	 ? ions	 ? are	 ? highly	 ? reactive,	 ? and	 ? tend	 ? to	 ? co-??precipitate	 ? with	 ? iron,	 ?calcium,	 ? and	 ? aluminum,	 ? or	 ? become	 ? adsorbed	 ? onto	 ? soil	 ? particle	 ? surfaces	 ?(Gyaneshwar	 ? et	 ? al.,	 ? 2002).	 ?The	 ? fraction	 ? remaining	 ? in	 ? solution	 ? is	 ? incorporated	 ? into	 ?organic	 ?matter	 ? after	 ? being	 ? taken	 ? up	 ? and	 ? assimilated	 ? by	 ? plants	 ? or	 ?microbes,	 ? or	 ? is	 ?removed	 ? from	 ? the	 ? system	 ? as	 ? runoff.	 ? Orthophosphate	 ? usually	 ? constitutes	 ? less	 ? than	 ?one	 ?percent	 ?of	 ?total	 ?phosphorus	 ?in	 ?soil	 ?at	 ?a	 ?given	 ?time	 ?(Sylvia	 ?et	 ?al.,	 ?2005),	 ?and	 ?thus	 ?is	 ? one	 ? of	 ? the	 ? least	 ? available	 ? plant	 ? nutrients	 ? found	 ? in	 ? the	 ? rhizosphere	 ? (Duff	 ? et	 ? al.,	 ?1994).	 ? Its	 ? availability	 ? in	 ? native	 ? soils	 ? is	 ? rarely	 ? adequate	 ? for	 ? optimal	 ? plant	 ? growth	 ?(Abel	 ?et	 ?al.,	 ?2001).	 ?Much	 ? of	 ? the	 ? P	 ? in	 ? soil,	 ? usually	 ? between	 ? 30-??50%	 ? of	 ? total	 ? P	 ? (Syliva	 ? et	 ? al.,	 ? 2005),	 ? is	 ?sequestered	 ? in	 ?organic	 ? forms	 ? (Kogel-??Knaber,	 ?2006;	 ?Richardson	 ?et	 ? al.,	 ? 2009).	 ?Plant	 ?roots,	 ? bacteria,	 ? and	 ? fungi	 ? produce	 ? a	 ? variety	 ? of	 ? extracellular	 ? phosphatase	 ? enzymes	 ?that	 ?mineralize	 ?organic	 ?P	 ?in	 ?soil,	 ?thereby	 ?releasing	 ?orthophosphate	 ?ions.	 ?These	 ?ions	 ?can	 ?then	 ?be	 ?taken	 ?up	 ?by	 ?plants	 ?or	 ?immobilized	 ?by	 ?microbes.	 ?Phosphatase	 ?enzymes	 ?are	 ? classified	 ? as	 ? either	 ? acid	 ? or	 ? alkaline	 ? based	 ? on	 ? whether	 ? their	 ? optimal	 ? pH	 ? for	 ? 15 catalysis	 ? is	 ? below	 ? or	 ? above	 ? pH	 ? 7	 ? (Vincent	 ? et	 ? al.,	 ? 1992).	 ? Bacteria,	 ? fungi,	 ? and	 ? plant	 ?roots	 ? are	 ? known	 ? to	 ? produce	 ? both	 ? acid	 ? and	 ? alkaline	 ? phosphatases	 ? (Tarafdar	 ? and	 ?Jungk,	 ? 1987;	 ?Duff	 ? et	 ? al.,	 ? 1994;	 ? Van	 ?Aarle	 ? and	 ? Plassard,	 ? 2010;	 ? Sakurai	 ? et	 ? al	 ? 2012).	 ?Alkaline	 ?phosphatases	 ?are	 ?generally	 ?rather	 ?substrate	 ?specific	 ?(Duff	 ?et	 ?al.,	 ?1994),	 ?and	 ?are	 ? produced	 ? in	 ? lower	 ? quantities	 ? (van	 ? Aarle	 ? and	 ? Plassard,	 ? 2010)	 ? than	 ? acid	 ?phosphatases.	 ? Since	 ? phosphomonoesters,	 ? such	 ? as	 ? phytate,	 ? comprise	 ? a	 ? significant	 ?portion	 ? of	 ? total	 ? organic	 ? P	 ? in	 ? soil	 ? (up	 ? to	 ? 70%)(Richardson	 ? et	 ? al.,	 ? 2009),	 ? acid	 ?phosphomonoesterases	 ?are	 ?likely	 ?playing	 ?a	 ?vital	 ?role	 ?in	 ?organic	 ?P	 ?mineralization	 ?in	 ?the	 ? rhizosphere,	 ? as	 ? well	 ? as	 ? mediating	 ? the	 ? overall	 ? availability	 ? of	 ? P	 ? to	 ? plants	 ? and	 ?microbes.	 ?	 ?A	 ? large	 ? challenge	 ? of	 ? studying	 ? soil	 ? enzyme	 ? dynamics	 ? is	 ? that	 ? many	 ? processes	 ? that	 ?regulate	 ?their	 ?activity	 ?occur	 ?at	 ?very	 ?fine	 ?mm	 ?to	 ?sub-??mm	 ?scales,	 ?making	 ?them	 ?difficult	 ?to	 ? measure.	 ? Schimel	 ? and	 ? Bennet	 ? (2004)	 ? suggested	 ? that	 ? the	 ? ability	 ? of	 ? plants	 ? to	 ?compete	 ?with	 ?microbes	 ?for	 ?available	 ?nitrogen	 ?depends	 ?on	 ?the	 ?existence	 ?soil	 ?micro-??sites,	 ?which	 ?are	 ?spatially	 ?distinct	 ?yet	 ?ubiquitous	 ?throughout	 ?the	 ?soil.	 ?Each	 ?micro-??site	 ?has	 ?unique	 ?features,	 ?such	 ?as	 ?the	 ?availability	 ?of	 ?labile	 ?and	 ?complex	 ?substrates,	 ?which	 ?control	 ? rates	 ? of	 ? mobilization	 ? and	 ? assimilation	 ? in	 ? that	 ? site.	 ? Such	 ? micro-??scale	 ?characteristics	 ? are	 ? important	 ? in	 ? regulating	 ? macro-??scale	 ? processes	 ? of	 ? ecosystem	 ?functioning	 ? (Giesseler	 ? et	 ? al.,	 ? 2010).	 ? Furthermore,	 ? traditional	 ? lab	 ? enzyme	 ? assays	 ?suffer	 ?from	 ?several	 ?limitations	 ?(reviewed	 ?by	 ?Wallenstein	 ?and	 ?Weintraub,	 ?2008),	 ?and	 ?may	 ?not	 ? reflect	 ? actual	 ? enzyme	 ? activities	 ? occurring	 ? in	 ?situ.	 ? A	 ? recently	 ? developed	 ? in	 ?situ	 ?enzyme	 ?assay	 ?using	 ?imprinting	 ?of	 ?soil	 ?profiles	 ?at	 ?root	 ?windows	 ?installed	 ?in	 ?the	 ?field	 ? has	 ? allowed	 ? for	 ? the	 ? detection	 ? of	 ? mm-??scale	 ? activities	 ? of	 ? several	 ? extracellular	 ? 16 enzymes	 ? with	 ? minimal	 ? disturbance	 ? to	 ? soil	 ? microbial	 ? communities	 ? (Dong	 ? et	 ? al.,	 ?2007).	 ?	 ?The	 ? study	 ? reported	 ? here	 ? used	 ? the	 ? Dong	 ? et	 ? al.	 ? (2007)	 ? method	 ? to	 ? study	 ? the	 ?distribution	 ?of	 ?acid	 ?phosphomonoesterase	 ?activities	 ?at	 ?mm	 ?scales	 ?on	 ?soil	 ?profiles	 ?of	 ?a	 ?mixed	 ?Douglas	 ?fir	 ?and	 ?paper	 ?birch	 ?stand	 ?in	 ?British	 ?Columbia.	 ?	 ?My	 ?overall	 ?objective	 ?was	 ?to	 ?better	 ?understand	 ?the	 ?chemical	 ?and	 ?microbial	 ?features	 ?associated	 ?with	 ?high	 ?activities	 ?at	 ?fine	 ?scales	 ?in	 ?situ.	 ?Specifically,	 ?I	 ?aimed	 ?to	 ?(i)	 ?compare	 ?soil	 ?nutrient	 ?status	 ?in	 ? high	 ? and	 ? low	 ? phosphatase	 ? micro-??sites;	 ? and	 ? (ii)	 ? test	 ? whether	 ? the	 ? addition	 ? of	 ?nutrients	 ?to	 ?soil	 ?profiles	 ?in	 ?situ	 ?influenced	 ?phosphatase	 ?activity.	 ?I	 ?hypothesized	 ?that	 ?high	 ?activities	 ?would	 ?be	 ?associated	 ?with	 ?microsites	 ?high	 ?in	 ?carbon	 ?(C)	 ?and	 ?nitrogen	 ?(N),	 ?and	 ?low	 ?in	 ?inorganic	 ?P.	 ?	 ?2.2. METHODS	 ?2.2.1. Site	 ?Description	 ?and	 ?Study	 ?Design	 ?The	 ?study	 ?took	 ?place	 ?in	 ?a	 ?mixed	 ?Douglas-??fir	 ?(Pseudotsuga	 ?menziesii	 ?(Mirb.)	 ?Franco),	 ?paper	 ?birch	 ? (Betula	 ?papyrifera	 ?Marsh)	 ?stand	 ? located	 ? in	 ? the	 ?moist,	 ?warm	 ?variant	 ?of	 ?the	 ? Interior	 ? Cedar-??Hemlock	 ? biogeoclimactic	 ? zone	 ? of	 ? southern	 ? interior	 ? British	 ?Columbia	 ? (Lloyd	 ? et	 ? al.,	 ? 1990;	 ? Twieg	 ? et	 ? al.,	 ? 2007;	 ? Twieg	 ? et	 ? al.,	 ? 2009).	 ? The	 ? stand	 ?regenerated	 ?naturally	 ? after	 ? a	 ?wildfire	 ? in	 ? approximately	 ? 1945	 ? (Twieg	 ? et	 ? al.,	 ? 2007).	 ?The	 ?canopy	 ?consisted	 ?of	 ?approximately	 ?38%	 ?Douglas-??fir,	 ?43%	 ?paper	 ?birch,	 ?and	 ?4%	 ?Western	 ? redcedar	 ? (Thuja	 ? plicata	 ? (Donn	 ? ex	 ? D.	 ? Don)	 ? Spach)	 ? (Twieg	 ? et	 ? al.,	 ? 2009).	 ?Annual	 ?average	 ?air	 ?temperature	 ?and	 ?precipitation	 ?for	 ?this	 ?region	 ?are	 ?7.5	 ??C	 ?and	 ?656	 ? 17 mm,	 ? respectively	 ? (Twieg	 ? et	 ? al.,	 ? 2009).	 ? 	 ? Soils	 ? are	 ? Podzols	 ? or	 ? Brunisols	 ?with	 ? loamy	 ?texture	 ?and	 ?moder	 ?humus	 ?form	 ?(Tweig	 ?et	 ?al.,	 ?2007).	 ?Five	 ? root	 ? windows	 ? were	 ? installed	 ? during	 ? the	 ? summers	 ? of	 ? 2004	 ? and	 ? 2005.	 ? The	 ?windows	 ?consisted	 ?of	 ?transparent	 ?acrylic	 ?panels	 ?(77	 ?x	 ?56	 ?x	 ?0.6	 ?cm),	 ?each	 ?with	 ?a	 ?trap	 ?door	 ?(30	 ?x	 ?30	 ?cm)	 ?in	 ?the	 ?center.	 ?The	 ?root	 ?windows	 ?were	 ?dug	 ?vertically	 ?into	 ?the	 ?soil	 ?midway	 ?between	 ?birch	 ?and	 ?Douglas	 ? fir	 ? trees.	 ?They	 ?were	 ? then	 ?anchored	 ? into	 ?place	 ?with	 ? 1.27	 ? cm-??diameter	 ? iron	 ? rods.	 ? After	 ? installation,	 ? gaps	 ? between	 ? the	 ? soil	 ? profile	 ?and	 ? the	 ?windows	 ?were	 ? filled	 ?with	 ? retained	 ? soil,	 ?maintaining	 ? the	 ? soil	 ? horizons.	 ? To	 ?protect	 ? the	 ?windows,	 ? plywood	 ? panels	 ?were	 ? placed	 ? in	 ? against	 ? them,	 ? and	 ? the	 ? holes	 ?were	 ? back-??filled	 ?with	 ? soil.	 ? Each	 ? root	 ?window	 ?was	 ? between	 ? 5-??15	 ?m	 ? from	 ? the	 ? next	 ?closest	 ?window.	 ?2.2.2. Soil	 ?Sampling	 ?For	 ? approximately	 ? 3	 ?weeks	 ? from	 ? June	 ? 14	 ? to	 ? July	 ? 5	 ? 2011,	 ? fine-??scale	 ? soil	 ? sampling	 ?took	 ? place	 ? from	 ? the	 ? soil	 ? profile	 ? accessed	 ? through	 ? the	 ? trap	 ? door.	 ? Four	 ? sets	 ? of	 ? soil	 ?samples	 ?were	 ? collected	 ? for	 ? separate	 ? analysis	 ? of	 ?C	 ? and	 ?N,	 ?P,	 ? pH,	 ? and	 ? soil	 ?moisture.	 ?The	 ?sampling	 ?was	 ?targeted	 ?to	 ?areas	 ?differing	 ?in	 ?phosphatase	 ?activity,	 ?as	 ?determined	 ?by	 ? the	 ? imprinting	 ? technique	 ?of	 ?Dong	 ?et	 ?al.	 ? (2007)	 ?and	 ? Jones	 ?et	 ?al.	 ? (2011).	 ? In	 ?brief,	 ?acid	 ? phosphomonoesterase-??reactive	 ? imprinting	 ? sheets	 ? were	 ? created	 ? by	 ? soaking	 ?chromatography	 ?paper	 ?(Whatman,	 ?20	 ?x	 ?20	 ?cm,	 ?Cat	 ?No.	 ?3001-??861)	 ?for	 ?1	 ?min	 ?in	 ?a	 ?1:10	 ?(v/v)	 ?mixture	 ?of	 ?freshly	 ?prepared	 ?50	 ?mM	 ??-??naphthyl	 ?phosphate	 ?(Sigma	 ?N7255)	 ?and	 ?10	 ?mM	 ? Fast	 ? Red	 ? TR	 ? (Sigma	 ? F2768),	 ? both	 ? in	 ? 50	 ?mM	 ? pH	 ? 5.6	 ? citrate	 ? buffer	 ? (Fisher	 ?S279).	 ?Phosphomonoesterase	 ?hydrolyzes	 ??-??naphthyl	 ?phosphate,	 ? releasing	 ?napthol,	 ? 18 which	 ?reacts	 ?with	 ?the	 ?Fast	 ?Red	 ?TR	 ?(a	 ?diazonium	 ?salt)	 ?to	 ?form	 ?a	 ?stable	 ?red	 ?precipitate	 ?on	 ?the	 ? imprinting	 ?sheet	 ?(Dong	 ?et	 ?al.,	 ?2007).	 ?The	 ?sheets	 ?were	 ?air	 ?dried	 ?for	 ?3	 ?hours,	 ?wrapped	 ?in	 ?aluminum	 ?foil,	 ?and	 ?stored	 ?in	 ?a	 ?sealed	 ?plastic	 ?bag	 ?at	 ?4	 ??C	 ?for	 ?up	 ?to	 ?7	 ?days	 ?until	 ? use	 ? in	 ? the	 ? field.	 ? A	 ? sheet	 ? of	 ? ?	 ? inch	 ? thick	 ? foam	 ? was	 ? cut	 ? to	 ? the	 ? exact	 ? inner	 ?dimensions	 ?of	 ? the	 ? trap	 ?door	 ? (30	 ?x	 ?30	 ?cm).	 ?Then	 ?a	 ?20	 ?x	 ?20	 ?cm	 ?hole	 ?was	 ?cut	 ? in	 ? the	 ?center	 ?of	 ?the	 ?foam	 ?board	 ?for	 ?use	 ?as	 ?a	 ?border.	 ?	 ?A	 ?30	 ?x	 ?30	 ?cm	 ?sheet	 ?of	 ?Mylar?	 ?plastic	 ?was	 ? taped	 ? to	 ? the	 ? foam	 ? border.	 ? The	 ? border-??Mylar?	 ? combination	 ? was	 ? then	 ? taped	 ?inside	 ? the	 ? trap	 ? door	 ? hole,	 ? with	 ? the	 ? Mylar?	 ? facing	 ? the	 ? soil	 ? profile	 ? for	 ? use	 ? as	 ? an	 ?alignment	 ?and	 ? sampling	 ?guide.	 ?Areas	 ? in	 ? the	 ? soil	 ?profile	 ? that	 ? could	 ?not	 ?be	 ? sampled	 ?(i.e.	 ?rocks	 ?and	 ?roots)	 ?or	 ?where	 ?the	 ?soil	 ?did	 ?not	 ?have	 ?good	 ?contact	 ?with	 ?the	 ?Mylar?	 ?(i.e.	 ?holes,	 ?burrows	 ?and	 ?LFH	 ?layer)	 ?were	 ?marked	 ?onto	 ?the	 ?Mylar?	 ?with	 ?a	 ?felt	 ?tip	 ?pen.	 ?These	 ? layers	 ?were	 ? avoided	 ? during	 ? sampling.	 ? The	 ?Mylar?	 ?was	 ? then	 ? removed	 ? from	 ?the	 ? foam	 ? border	 ? and	 ? taped	 ? onto	 ? the	 ? inside	 ? of	 ? the	 ? trap	 ? door	 ? window.	 ? A	 ? treated	 ?imprinting	 ?sheet	 ?was	 ?taped	 ?onto	 ?the	 ?Mylar?,	 ?and	 ?was	 ?applied	 ?directly	 ?to	 ?the	 ?intact	 ?soil	 ?profile	 ?by	 ?closing	 ?the	 ?trap	 ?door.	 ?The	 ?imprint	 ?was	 ?incubated	 ?in	 ?place	 ?for	 ?an	 ?hour,	 ?with	 ?even	 ?pressure	 ?applied	 ?to	 ?the	 ?trap	 ?door	 ?to	 ?ensure	 ?contact.	 ?	 ?After	 ? incubation,	 ? the	 ?Mylar?	 ? sheet,	 ?with	 ? the	 ? imprint	 ? attached,	 ?was	 ? removed	 ? from	 ?the	 ?door	 ?and	 ?rinsed	 ?briefly	 ?with	 ?sterile	 ?dH2O.	 ?Ten	 ?of	 ?the	 ?darkest	 ?red	 ?areas	 ?of	 ?at	 ?least	 ?5	 ? mm	 ? diameter,	 ? which	 ? were	 ? visible	 ? on	 ? the	 ? back-??side	 ? of	 ? the	 ? imprint	 ? through	 ? the	 ?Mylar?,	 ?were	 ?marked	 ?onto	 ?the	 ?Mylar?	 ?with	 ?a	 ?permanent	 ?marker.	 ?These	 ?areas	 ?were	 ?considered	 ? to	 ? be	 ? high	 ? phosphatase	 ? microsites.	 ? Ten	 ? of	 ? the	 ? largest	 ? areas	 ? with	 ? no	 ?colour	 ?change,	 ?but	 ?with	 ?good	 ?soil	 ?contact,	 ?were	 ?considered	 ?to	 ?be	 ? low	 ?phosphatase	 ?microsites.	 ?The	 ?centers	 ?of	 ?these	 ?areas	 ?were	 ?marked	 ?in	 ?another	 ?colour	 ?on	 ?the	 ?Mylar?	 ? 19 sheet,	 ?which	 ?was	 ?then	 ?removed	 ?from	 ?the	 ?imprint.	 ?A	 ?knife	 ?was	 ?used	 ?to	 ?cut	 ?5	 ?x	 ?5	 ?mm	 ?holes	 ?in	 ?the	 ?Mylar?	 ?in	 ?the	 ?marked	 ?areas	 ?of	 ?high	 ?and	 ?low	 ?phosphatase	 ?activity.	 ?The	 ?Mylar?	 ? sheet	 ?was	 ? then	 ? attached	 ? to	 ? the	 ? foam	 ? border	 ? and	 ? replaced	 ? inside	 ? the	 ? trap	 ?door	 ? hole.	 ? The	 ? foam	 ?border	 ? ensured	 ? that	 ? the	 ?Mylar?	 ? sheet	 ?was	 ? replaced	 ? into	 ? the	 ?window	 ? against	 ? the	 ? soil	 ? profile	 ? exactly	 ? in	 ? the	 ? exact	 ? location	 ? as	 ? previously.	 ? A	 ? soil	 ?sample	 ? of	 ? approximately	 ? 0.05	 ? g	 ? was	 ? collected	 ? from	 ? each	 ? microsite	 ? using	 ? sterile	 ?forceps,	 ?placed	 ?into	 ?a	 ?microcentrifuge	 ?tube,	 ?and	 ?stored	 ?on	 ?ice	 ?during	 ?transport	 ?back	 ?to	 ? the	 ? lab.	 ?This	 ?procedure	 ?was	 ? repeated	 ? for	 ? all	 ? five	 ?windows	 ?over	 ? the	 ? course	 ?of	 ? a	 ?day.	 ?2.2.3. Analysis	 ?of	 ?Soil	 ?Nutrients	 ?From	 ?each	 ?of	 ?the	 ?five	 ?root	 ?windows,	 ?on	 ?each	 ?collection	 ?date,	 ? the	 ?ten	 ?samples	 ?from	 ?high	 ? phosphatase	 ? areas	 ? were	 ? pooled,	 ? and,	 ? separately,	 ? the	 ? ten	 ? samples	 ? from	 ? low	 ?phosphatase	 ?areas	 ?were	 ?pooled.	 ?Samples	 ?were	 ?stored	 ?at	 ?4	 ??C,	 ?dried,	 ?and	 ?shipped	 ?on	 ?ice	 ? to	 ? the	 ? Canadian	 ? Forest	 ? Service	 ? Pacific	 ? Forestry	 ? Centre	 ? in	 ? Victoria,	 ? British	 ?Columbia.	 ? Percent	 ? total	 ? C	 ? and	 ? N	 ? (combustion)	 ? was	 ? determined	 ? for	 ? one	 ? set	 ? of	 ?samples.	 ?Total	 ?extractable	 ?P	 ? (Inductively	 ?coupled	 ?plasma	 ?mass	 ?spectrometry	 ?(ICP-??MS)	 ?after	 ?standard	 ?Bray-??P1	 ?extraction),	 ?and	 ?inorganic	 ?phosphate	 ?(colorimetric	 ?after	 ?standard	 ? Bray-??P1	 ? extraction)(Kalra	 ? and	 ? Maynard,	 ? 1991)	 ? were	 ? determined	 ? on	 ?another	 ? set	 ? of	 ? samples.	 ? Soluble	 ? organic	 ? P	 ?was	 ? estimated	 ? by	 ? subtracting	 ? inorganic	 ?phosphate	 ?values	 ?from	 ?total	 ?extractable	 ?P.	 ?	 ?2.2.4. Soil	 ?Moisture	 ?and	 ?pH	 ?After	 ? pooling	 ? as	 ? described	 ? above,	 ? gravimetric	 ? soil	 ? moisture	 ? was	 ? calculated	 ? for	 ? a	 ? 20 third	 ?set	 ?of	 ?samples	 ?by	 ?weighing	 ?soils	 ?before	 ?and	 ?after	 ?oven	 ?drying	 ?at	 ?105	 ??C	 ?for	 ?3	 ?days.	 ? Soil	 ? pH	 ? was	 ? measured	 ? on	 ? a	 ? fourth	 ? set	 ? of	 ? samples	 ? using	 ? a	 ? glass	 ? electrode	 ?(Beckman	 ?511052),	 ? in	 ? a	 ?1:2	 ? ratio	 ?of	 ? soil:deionized	 ?water,	 ? after	 ? several	 ?minutes	 ?of	 ?equilibration.	 ?2.2.5. Nutrient	 ?Addition	 ?Experiment	 ?To	 ? test	 ? for	 ? the	 ? effects	 ? of	 ? nutrient	 ? limitation	 ? on	 ? phosphatase	 ? activity,	 ? I	 ? added	 ?relatively	 ? labile	 ? forms	 ?of	 ?C	 ?and	 ?N	 ?to	 ?randomly-??selected	 ?microplots	 ?on	 ?soil	 ?profiles,	 ?and	 ? analyzed	 ? phosphatase	 ? activities	 ? by	 ? imprinting.	 ? I	 ? selected	 ? sodium	 ? acetate	 ? and	 ?ammonium	 ?chloride	 ?as	 ?labile	 ?forms	 ?of	 ?C	 ?and	 ?N,	 ?based	 ?on	 ?the	 ?results	 ?of	 ?Allison	 ?and	 ?Vitousk,	 ? (2005),	 ? who	 ? found	 ? that	 ? these	 ?molecules	 ? stimulated	 ? phosphatase	 ? activity	 ?when	 ?applied	 ?to	 ?bulk	 ?soils.	 ?The	 ?following	 ?five	 ?treatments	 ?were	 ?applied	 ?to	 ?four	 ?1	 ?cm2	 ?microplots	 ? each	 ? on	 ? soil	 ? profiles	 ? accessed	 ? via	 ? the	 ? root	 ? windows:	 ? 417	 ?mM	 ? sodium	 ?acetate,	 ? 417	 ? mM	 ? ammonium	 ? chloride,	 ? 417	 ? mM	 ? sodium	 ? acetate	 ? plus	 ? 417	 ? mM	 ?ammonium	 ? chloride,	 ? reverse	 ? osmosis	 ? water,	 ? and	 ? a	 ? no-??addition,	 ? dry	 ? control	 ?treatment.	 ? Nutrient	 ? solutions	 ? were	 ? buffered	 ? to	 ? pH	 ? 5.2,	 ? with	 ? sodium	 ? hydroxide	 ?buffer,	 ?to	 ?mimic	 ?the	 ?soil	 ?pH	 ?at	 ?the	 ?site.	 ?The	 ?volume	 ?of	 ?nutrient	 ?solution	 ?added	 ?was	 ?equal	 ?to	 ?the	 ?amount	 ?of	 ?liquid	 ?needed	 ?to	 ?saturate	 ?approximately	 ?1	 ?cm3	 ?of	 ?the	 ?native	 ?soil,	 ? as	 ? determined	 ? in	 ? a	 ? preliminary	 ? experiment.	 ? Five	 ? Mylar?	 ? sheets	 ? with	 ? 1	 ? cm2	 ?grids	 ?were	 ? created	 ? in	 ? the	 ? lab	 ? to	 ? guide	 ? nutrient	 ? additions.	 ? The	 ?Mylar?	 ? grids	 ?were	 ?sized	 ?to	 ?fit	 ?the	 ?foam	 ?borders	 ?(30	 ?x	 ?30	 ?cm).	 ?	 ?On	 ? June	 ? 3rd,	 ? 2012,	 ? nutrient	 ? additions	 ? were	 ? carried	 ? out	 ? in	 ? the	 ? field.	 ? Soil	 ? profile	 ?features	 ?were	 ?drawn	 ?onto	 ?the	 ?Mylar?	 ?grid	 ?supported	 ?by	 ?a	 ?foam	 ?border,	 ?as	 ?described	 ? 21 above.	 ?The	 ?grid	 ?was	 ? then	 ?removed	 ? from	 ?the	 ?soil	 ?profile,	 ?and	 ? four	 ?grid	 ? locations	 ? in	 ?the	 ?mineral	 ?soil	 ?were	 ?chosen	 ?using	 ?a	 ?random	 ?number	 ?generator	 ?for	 ?each	 ?of	 ?the	 ?five	 ?treatments.	 ?A	 ?knife	 ?was	 ?used	 ?to	 ?cut	 ?20	 ?1	 ?x	 ?1	 ?cm	 ?holes	 ?in	 ?the	 ?Mylar?	 ?in	 ?the	 ?selected	 ?grid	 ?locations.	 ?The	 ?foam-??supported	 ?Mylar?	 ?was	 ?then	 ?returned	 ?to	 ?the	 ?trap	 ?door	 ?hole	 ?and	 ? used	 ? as	 ? a	 ? template	 ? for	 ? nutrient	 ? additions.	 ? Using	 ? sterile	 ? micropipette	 ? tips,	 ?nutrient	 ?solutions	 ?were	 ?added	 ?to	 ? the	 ?upper	 ?portions	 ?of	 ?each	 ?1	 ?x	 ?1	 ?cm	 ?vertical	 ?soil	 ?surface	 ?accessible	 ?through	 ?the	 ?holes	 ?in	 ?the	 ?Mylar?.	 ?	 ?Soil	 ? imprinting	 ? took	 ?place	 ?1,	 ? 2,	 ? 4,	 ? 10,	 ? and	 ?18	 ?days	 ? after	 ?nutrient	 ? inputs,	 ? using	 ? the	 ?imprinting	 ?procedure	 ?described	 ?above.	 ?This	 ?procedure	 ?was	 ?carried	 ?out	 ? for	 ?all	 ? five	 ?root	 ?windows	 ?on	 ?the	 ?same	 ?day.	 ?Imprints	 ?were	 ?rinsed	 ?with	 ?sterile	 ?dH2O,	 ?wrapped	 ?in	 ?aluminum	 ? foil,	 ? and	 ? returned	 ? to	 ? the	 ? lab.	 ?The	 ? imprints	 ?were	 ? then	 ? rinsed	 ?once	 ?more	 ?with	 ? sterile	 ? dH2O	 ? and	 ? air-??dried.	 ? 	 ? After	 ? drying,	 ? the	 ? imprints	 ?were	 ? scanned	 ? (Canon	 ?CanoScan	 ?9000F)	 ?with	 ? the	 ?Mylar?	 ?grid	 ? sheet	 ? overlayed,	 ? and	 ? the	 ? intensity	 ? of	 ? each	 ?addition-??patch	 ? was	 ? measured	 ? using	 ? ImageJ64	 ?(http://rsbweb.nih.gov/ij/index.html).	 ? Scanned	 ? images	 ? were	 ? inverted	 ? into	 ? RGB	 ?scale,	 ?and	 ?the	 ?contrast	 ?was	 ?increased	 ?by	 ?five	 ?units.	 ?Then	 ?each	 ?sheet	 ?was	 ?calibrated	 ?to	 ?a	 ?150x150	 ?dark	 ?area	 ?on	 ?the	 ?imprint	 ?by	 ?adjusting	 ?the	 ?contrast	 ?so	 ?that	 ?the	 ?intensity	 ?of	 ?the	 ?dark	 ?area	 ?was	 ?0.	 ?A	 ?150x150	 ?box	 ?was	 ?used	 ?to	 ?measure	 ?the	 ?mean	 ?intensity	 ?of	 ?each	 ? patch	 ? using	 ? the	 ? histogram	 ? function	 ? of	 ? ImageJ64.	 ? Any	 ? spots	 ?with	 ?wrinkles	 ? or	 ?discoloration	 ?from	 ?soil	 ?were	 ?not	 ?included	 ?in	 ?the	 ?analysis.	 ?	 ?2.2.6. Comparison	 ?of	 ?Imprinting	 ?Assays	 ?with	 ?Standard	 ?Methods	 ?In	 ? June-??July	 ? 2012,	 ? two	 ? groups	 ? of	 ? soil	 ? samples	 ? were	 ? collected	 ? for	 ? two	 ? separate	 ? 22 fluorimetric	 ?assays	 ?of	 ?acid	 ?phosphatase	 ?(EC	 ?3.2.1.31).	 ?Samples	 ?were	 ?stored	 ?for	 ?one	 ?day	 ?at	 ?4?C	 ?before	 ?analysis	 ?as	 ?per	 ?the	 ?recommendations	 ?of	 ?Lorenz	 ?and	 ?Dick	 ?(2011).	 ?Substrates	 ? were	 ? analyzed	 ? on	 ? 96-??well	 ? plates.	 ? The	 ? assays	 ? were	 ? performed	 ? as	 ?described	 ?by	 ?Sinsabaugh	 ?et	 ?al.	 ? (2003).	 ?Fluorescence	 ?was	 ?measured	 ?on	 ?a	 ?FLUOstar	 ?Galaxy	 ? (BMG	 ? Lab	 ? Technologies,	 ? Ortenburg,	 ? Germany)	 ? microplate	 ? reader,	 ? with	 ?excitation	 ? set	 ? to	 ? 360/40	 ? nm	 ? and	 ? emission	 ? at	 ? 460/40	 ? nm.	 ? Activities	 ? were	 ? later	 ?calculated	 ?as	 ?nmol	 ?of	 ?substrate	 ?converted	 ?per	 ?hour	 ?per	 ?gram	 ?of	 ?soil	 ?using	 ?a	 ?10	 ??M	 ?4-??methylumbelliferone	 ?standard.	 ?2.2.7. Data	 ?Analysis	 ?All	 ? statistical	 ? analyses	 ? were	 ? carried	 ? out	 ? using	 ? RStudio	 ? version	 ? (0.96.316),	 ? with	 ?significance	 ? for	 ? all	 ? tests	 ? set	 ? to	 ? ?=0.05	 ? unless	 ? stated	 ? otherwise.	 ? I	 ? used	 ? boxplots	 ? to	 ?visually	 ?check	 ?for	 ?homogeneity	 ?of	 ?variance	 ?and	 ?normality.	 ?Boxplots	 ?were	 ?not	 ?grossly	 ?asymmetrical	 ? or	 ? different	 ? in	 ? size	 ? and	 ? thus,	 ? no	 ? transformations	 ? to	 ? the	 ? data	 ? were	 ?needed.	 ?Data	 ?points	 ?from	 ?one	 ?of	 ?the	 ?windows	 ?were	 ?consistent	 ?outliers,	 ?so	 ?I	 ?removed	 ?that	 ? window	 ? from	 ? all	 ? analyses.	 ? Otherwise,	 ? the	 ? assumptions	 ? of	 ? homogeneity	 ? of	 ?variance	 ?and	 ?normality	 ?were	 ?met.	 ?I	 ?used	 ?paired	 ?t-??tests	 ?to	 ?test	 ?for	 ?differences	 ?among	 ?high	 ? and	 ? low	 ? phosphatase	 ? areas	 ? for	 ? soil	 ? nutrient	 ? variables,	 ? soil	 ?moisture,	 ? pH	 ? and	 ?assayed	 ?enzyme	 ?activities	 ?from	 ?samples	 ?from	 ?high	 ?and	 ?low	 ?phosphatase	 ?microsites	 ?as	 ? directed	 ? by	 ? the	 ? imprinting	 ?method.	 ? 	 ? For	 ? these	 ? analyses,	 ? N=4,	 ? with	 ? the	 ? pooled	 ?samples	 ?per	 ?root	 ?window	 ?serving	 ?as	 ?replicates.	 ?For	 ? the	 ? nutrient	 ? addition	 ? experiment,	 ? I	 ? subtracted	 ? the	 ? average	 ? dry,	 ? non-??addition	 ?treatment	 ?per	 ?window	 ?(N=4	 ?microplots)	 ?from	 ?intensity	 ?readings	 ?of	 ?all	 ?of	 ?the	 ?other	 ? 23 treatments	 ?in	 ?that	 ?window.	 ?This	 ?procedure	 ?was	 ?repeated	 ?for	 ?all	 ?four	 ?windows	 ?over	 ?all	 ?dates.	 ?After	 ?I	 ?checked	 ?for	 ?homogeneity	 ?of	 ?variance	 ?and	 ?normality,	 ?I	 ?used	 ?a	 ?nested	 ?analysis	 ?of	 ? variance	 ? (ANOVA)	 ? to	 ? test	 ? for	 ?differences	 ?between	 ? the	 ? treatments,	 ?with	 ?treatment	 ?nested	 ?within	 ?window.	 ?This	 ?analysis	 ?was	 ?carried	 ?out	 ?separately	 ?for	 ?each	 ?sampling	 ?date.	 ?2.3. RESULTS	 ?2.3.1. Soil	 ?Properties	 ?Soil	 ?samples	 ?that	 ?differed	 ?in	 ? imprintable	 ?phosphatase	 ?activity,	 ?and	 ?sampled	 ?at	 ?mm	 ?scales,	 ? differed	 ? in	 ? several	 ? chemical	 ? and	 ? physical	 ? properties.	 ? Specifically,	 ? percent	 ?total	 ? C	 ? (p=0.05)	 ? and	 ? percent	 ? total	 ? N	 ? (p=0.05)	 ? were	 ? higher	 ? in	 ? high	 ? phosphatase	 ?microsites	 ? across	 ? four	 ? root	 ? windows	 ? (Figure	 ? 2.1).	 ? The	 ? C:N	 ? ratio	 ? was	 ? marginally	 ?higher	 ? in	 ? high	 ? phosphatase	 ?microsites	 ? (p=0.06;	 ? Figure	 ? 2.1).	 ? Surprisingly,	 ? levels	 ? of	 ?extractable	 ? P	 ? were	 ? not	 ? different	 ? between	 ? high	 ? and	 ? low	 ? phosphatase	 ? microsites,	 ?regardless	 ?of	 ?the	 ?form	 ?of	 ?P	 ?measured	 ?(organic,	 ?p=0.2;	 ?inorganic,	 ?p=0.9;	 ?total	 ?p=1.0;	 ?Figure	 ?2.1).	 ?Likewise,	 ?gravimetric	 ? soil	 ?moisture	 ? (p=0.3)	 ?and	 ?pH	 ?(p=0.3)	 ?were	 ?both	 ?similar	 ?between	 ?the	 ?two	 ?types	 ?of	 ?microsites	 ?(Figure	 ?2.2).	 ?	 ?	 ?	 ? 	 ? 24  Figure	 ?2.1:	 ?A)	 ?Percent	 ?total	 ?carbon,	 ?B)	 ?percent	 ?total	 ?nitrogen,	 ?C)	 ?carbon	 ?to	 ?nitrogen	 ?ratio,	 ? D)	 ? total	 ? extractable	 ? phosphorus,	 ? E)	 ? soluble	 ? organic	 ? phosphorus,	 ? and	 ? F)	 ?inorganic	 ?phosphorus,	 ?from	 ?soils	 ?collected	 ?from	 ?microsites	 ?differing	 ?in	 ?imprintable	 ?phosphomonoesterase	 ?activities	 ? in	 ? four	 ?root	 ?windows.	 ?P-??values	 ?based	 ?on	 ?paired	 ?t-??tests	 ?(n=4).	 ? A)	 ? 	 ? 	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ? 	 ?	 ?	 ?	 ?p=0.05	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ? B)	 ? 	 ? 	 ?	 ?	 ?	 ?	 ?	 ? 	 ?	 ?	 ?	 ?p=0.05	 ?E)	 ? 	 ? 	 ? 	 ?	 ?	 ?	 ?p=0.34	 ?D)	 ? 	 ? 	 ? 	 ?	 ?	 ?	 ?	 ?	 ?	 ?p=0.6	 ?C)	 ? 	 ? 	 ? 	 ?	 ?	 ?	 ?p=0.06	 ?F)	 ? 	 ? 	 ? 	 ?	 ?	 ?	 ?p=0.46	 ? 25  Figure	 ? 2.2:	 ? A)	 ? Gravimetric	 ? soil	 ? moisture	 ? and	 ? B)	 ? soil	 ? solution	 ? pH	 ? from	 ? samples	 ?collected	 ? from	 ?microsites	 ? differing	 ? in	 ? imprintable	 ? phosphomonoesterase	 ? activities	 ?in	 ?four	 ?root	 ?windows.	 ?P-??values	 ?based	 ?on	 ?paired	 ?t-??tests	 ?(n=4).	 ? 2.3.2. Nutrient	 ?addition	 ?experiment	 ?Given	 ? the	 ? higher	 ? levels	 ? of	 ? C	 ? and	 ? N	 ? in	 ? microsites	 ? with	 ? higher	 ? imprintable	 ?phosphatase	 ? activities,	 ? I	 ? ran	 ? a	 ? manipulative	 ? experiment	 ? to	 ? distinguish	 ? among	 ?several	 ? possible	 ? reasons	 ? for	 ? this	 ? association.	 ?When	 ? C	 ? and	 ? N	 ?were	 ? added	 ? in	 ? fairly	 ?simple	 ? forms	 ?(sodium	 ?acetate	 ?and	 ?ammonium	 ?chloride,	 ? respectively),	 ? they	 ?did	 ?not	 ?significantly	 ?affect	 ?imprintable	 ?phosphatase	 ?activities	 ?(Figure	 ?2.3).	 ?This	 ?was	 ?true	 ?for	 ?all	 ?five	 ?measurements	 ?between	 ?24	 ?hours	 ?and	 ?18	 ?days,	 ?and	 ?for	 ?C	 ?and	 ?N	 ?added	 ?alone	 ?or	 ? together.	 ? However,	 ? after	 ? 48	 ? hours,	 ? the	 ? treatments	 ? were	 ? marginally	 ? different	 ?(p=0.06),	 ?with	 ?mean	 ?activity	 ? in	 ?the	 ?C	 ?treatment	 ?being	 ?slightly	 ?higher	 ?than	 ? in	 ?other	 ?treatments	 ?(Figure	 ?2.3).	 ?  A)	 ? 	 ? 	 ? 	 ?	 ?	 ?	 ?p=0.3	 ? B)	 ? 	 ? 	 ? 	 ?	 ?	 ?	 ?p=0.3	 ? 26 	 ?Figure	 ? 2.3:	 ? Average	 ? phosphatase	 ? intensity	 ? (0-??256)	 ? per	 ? treatment	 ? per	 ? root,	 ? with	 ?average	 ?intensity	 ?of	 ?dry	 ?control	 ?microplots	 ?subtracted	 ?separately	 ?for	 ?each	 ?window.	 ?Sampling	 ?was	 ?conducted	 ? five	 ? times	 ?over	 ?a	 ?432-??hour	 ?period	 ? following	 ? injections	 ?of	 ?sodium	 ? acetate	 ? (C),	 ? sodium	 ? acetate	 ? and	 ? ammonium	 ? chloride	 ? (CN),	 ? ammonium	 ?chloride	 ?(N),	 ?or	 ?sterile	 ?deionized	 ?water	 ?(W).	 ?P-??values	 ?based	 ?on	 ?nested	 ?ANOVAs,	 ?with	 ?treatment	 ?nested	 ?within	 ?window,	 ?and	 ?conducted	 ?separately	 ?for	 ?each	 ?time	 ?(n=4).	 ?24hrs	 ? 	 ? 	 ? 	 ?	 ?	 ?	 ?p=0.20	 ? 48hrs	 ? 	 ? 	 ? 	 ?	 ?	 ?	 ?p=0.06	 ?96hrs	 ? 	 ? 	 ? 	 ?	 ?	 ?	 ?p=0.80	 ? 240hrs	 ? 	 ? 	 ?	 ?	 ?	 ?p=0.36	 ?432hrs	 ? 	 ? 	 ?	 ?	 ?	 ?p=0.52	 ? 27 2.3.3. Comparison	 ?of	 ?imprinting	 ?assays	 ?with	 ?standard	 ?methods	 ?The	 ? imprinting	 ? technique	 ? is	 ? a	 ? fairly	 ? new	 ? technique	 ? for	 ? measuring	 ? phosphatase	 ?activities	 ?and	 ?has	 ?not	 ?been	 ?compared	 ?directly	 ?with	 ?standard	 ?lab	 ?assays.	 ?I	 ?measured	 ?phosphatase	 ?activities	 ?of	 ?soils	 ?sampled	 ?on	 ?two	 ?dates	 ?from	 ?high	 ?and	 ?low	 ?phosphatase	 ?microsites	 ?using	 ?standard	 ?lab	 ?assays.	 ?	 ?Phosphatase	 ?activities	 ?measured	 ?in	 ?the	 ?lab	 ?did	 ?not	 ?differ	 ?between	 ?high	 ?and	 ?low	 ?phosphatase	 ?microsites	 ?from	 ?samples	 ?acquired	 ?on	 ?June	 ? 29,	 ? 2012	 ? (Figure	 ? 2.4A),	 ? but	 ? were	 ? higher	 ? in	 ? high	 ? phosphatase	 ? microsites	 ?(p=0.02)	 ?from	 ?samples	 ?acquired	 ?on	 ?July	 ?7,	 ?2012	 ?(Figure	 ?2.4B).	 ? Figure	 ?2.4:	 ?Phosphatase	 ?activity,	 ?as	 ?measured	 ?by	 ?standard	 ?fluorogenic	 ?lab	 ?assays,	 ?of	 ?soils	 ? collected	 ? from	 ? microsites	 ? differing	 ? in	 ? imprintable	 ? phosphomonoesterase	 ?activities	 ?in	 ?five	 ?root	 ?windows	 ?from	 ?(a)	 ?June	 ?29,	 ?2012	 ?and	 ?(b)	 ?July	 ?7,	 ?2012.	 ?P-??values	 ?based	 ?on	 ?paired	 ?t-??tests	 ?(n=4).	 ? 2.4. DISCUSSION	 ?The	 ?release	 ?of	 ?extracellular	 ?enzymes	 ?by	 ?soil	 ?microbes	 ?is	 ?crucial	 ?to	 ?the	 ?degradation	 ?of	 ?large	 ? insoluble	 ? organic	 ? molecules	 ? into	 ? soluble	 ? monomers.	 ? This	 ? depolymerization	 ?and	 ?solubilization	 ?is	 ?important,	 ?as	 ?it	 ?regulates	 ?the	 ?availability	 ?of	 ?nutrients	 ?from	 ?litter	 ?A)	 ? 	 ? 	 ?	 ?	 ?	 ?	 ? 	 ?	 ?	 ?	 ?p=0.50	 ? B)	 ? 	 ? 	 ?	 ?	 ?	 ?	 ? 	 ?	 ?	 ?	 ?p=0.02	 ? 28 and	 ?organic	 ?matter	 ?into	 ?bioavailable	 ?forms	 ?that	 ?can	 ?be	 ?assimilated	 ?by	 ?microbes	 ?and	 ?plants	 ?(Schimel	 ?and	 ?Bennet,	 ?2004).	 ?	 ?Much	 ?of	 ?our	 ?current	 ?understanding	 ?of	 ?microbial	 ?enzyme	 ? dynamics	 ? in	 ? soil	 ? is	 ? derived	 ? from	 ? traditional	 ? lab-??based	 ? assays	 ? (eg.	 ?Sinsabaugh	 ?et	 ?al.,	 ?2008),	 ?which	 ?measure	 ?potential	 ?enzyme	 ?activities	 ?under	 ?optimal	 ?conditions	 ? (pH,	 ? temperature,	 ? unlimited	 ? substrate	 ? availability).	 ?While	 ? these	 ? assays	 ?continue	 ?to	 ?provide	 ?insight	 ?into	 ?the	 ?roles	 ?of	 ?enzymes	 ?in	 ?ecosystem	 ?functioning,	 ?they	 ?are	 ?not	 ? indicative	 ? of	 ? actual	 ? enzyme	 ?activities	 ? under	 ?natural	 ? soil	 ?conditions	 ? in	 ?situ.	 ?Actual	 ? enzyme	 ? activities	 ? are	 ? regulated	 ? by	 ? complex	 ? soil	 ? chemical	 ? and	 ? physical	 ?interactions,	 ? which	 ? can	 ? vary	 ? across	 ? different	 ? soil	 ? microclimates	 ? (Schimel	 ? and	 ?Bennet,	 ? 2004;	 ? Castillo-??Monroy	 ? et	 ? al.,	 ? 2010;	 ? Giesseler	 ? et	 ? al.,	 ? 2010).	 ? Using	 ? the	 ? root	 ?window	 ?approach	 ?allowed	 ?me	 ?to	 ?estimate	 ?more	 ?realistic	 ?enzyme	 ?activities	 ?in	 ?situ,	 ?in	 ?the	 ? field,	 ? and	 ? allowed	 ?me	 ? to	 ?detect	 ? fine	 ?mm-??scale	 ?differences	 ? in	 ? enzyme	 ?activities	 ?and	 ? associate	 ? them	 ?with	 ? various	 ? soil	 ? chemical	 ? and	 ?microclimate	 ? characteristics.	 ? I	 ?found	 ?that	 ?microsites	 ?high	 ?in	 ?imprintable	 ?phosphatase	 ?activities	 ?had	 ?higher	 ?total	 ?C	 ?and	 ?total	 ?N	 ?than	 ?low	 ?phosphatase	 ?microsites.	 ?In	 ?spite	 ?of	 ?this,	 ?additions	 ?of	 ?soluble	 ?N	 ?or	 ? C	 ? to	 ? randomly	 ? selected	 ? microsites	 ? did	 ? not	 ? result	 ? in	 ? increased	 ? phosphatase	 ?activities.	 ?2.4.1. Nutrient	 ?status	 ?of	 ?soil	 ?microsites	 ?Several	 ? studies	 ? have	 ? shown	 ? that	 ? substrate	 ? addition	 ? can	 ? induce	 ? enzyme	 ? activity	 ? in	 ?soils.	 ?For	 ?example,	 ?chitinase	 ?can	 ?be	 ?induced	 ?by	 ?the	 ?presence	 ?of	 ?chitin	 ?(Smucker	 ?and	 ?Kim,	 ?1987),	 ?whereas	 ?microbial	 ?proteases	 ?can	 ?be	 ?induced	 ?by	 ?the	 ?presence	 ?of	 ?proteins	 ?in	 ?liquid	 ?media	 ?(Kalisz,	 ?1988;	 ?Haab	 ?et	 ?al.,1990).	 ?There	 ?was	 ?no	 ?evidence	 ?for	 ?substrate	 ?induction	 ?in	 ?this	 ?system,	 ?as	 ?levels	 ?of	 ?soluble	 ?organic	 ?P	 ?were	 ?similar	 ?across	 ?high	 ?and	 ? 29 low	 ?phosphatase	 ?microsites.	 ?	 ?Another	 ? type	 ? of	 ? enzyme	 ? regulation	 ? observed	 ? in	 ? soil	 ? is	 ? end	 ? product	 ? repression	 ?(Giesseler	 ?et	 ?al.,	 ?2010).	 ?Although,	 ? fertilization	 ?with	 ? inorganic	 ?P	 ?has	 ?been	 ?shown	 ?to	 ?decrease	 ?phosphatase	 ?activity	 ? in	 ?a	 ? range	 ?of	 ? soils	 ? (Marklein	 ?and	 ?Houlton,	 ?2012),	 ? in	 ?this	 ? system,	 ? levels	 ? of	 ? soluble	 ? inorganic	 ? P	 ? were	 ? similar	 ? between	 ? high	 ? and	 ? low	 ?phosphatase	 ?microsites.	 ?When	 ?taken	 ?alone,	 ?these	 ?results	 ?do	 ?not	 ?provide	 ?evidence	 ?of	 ?end	 ?product	 ?repression	 ?in	 ?my	 ?system.	 ?There	 ?are	 ?several	 ?reasons	 ?why	 ?high	 ?C	 ?and	 ?N	 ?would	 ?be	 ?expected	 ?in	 ?microsites	 ?with	 ?high	 ? phosphatase	 ? activities.	 ? First,	 ? past	 ? studies	 ? have	 ? shown	 ? that	 ? phosphatase	 ?activities	 ? can	 ? be	 ? positively	 ? correlated	 ? with	 ? soil	 ? microbial	 ? biomass	 ? (Keeler	 ? et	 ? al.,	 ?2009;	 ? Brocket	 ? et	 ? al.,	 ? 2012),	 ? as	 ? well	 ? as	 ? bacterial	 ? and	 ? fungal	 ? biomass	 ? individually	 ?(Brocket	 ?et	 ?al.,	 ?2012).	 ?In	 ?this	 ?study,	 ?the	 ?high	 ?total	 ?carbon	 ?and	 ?total	 ?nitrogen	 ?found	 ?in	 ?high	 ?phosphatase	 ?microsites	 ?could	 ?simply	 ?indicate	 ?high	 ?microbial	 ?biomass	 ?in	 ?those	 ?microsites.	 ? Furthermore,	 ? enzyme	 ? production	 ? is	 ? energetically	 ? and	 ? nutritionally	 ?expensive,	 ?requiring	 ?a	 ?large	 ?investment	 ?of	 ?both	 ?carbon	 ?and	 ?nitrogen	 ?on	 ?the	 ?part	 ?of	 ?the	 ?microbe	 ?(Schimel	 ?and	 ?Weintraub,	 ?2003).	 ?Therefore,	 ?even	 ?with	 ?no	 ?difference	 ? in	 ?microbial	 ? biomass,	 ? one	 ?would	 ? expect	 ?microsites	 ?higher	 ? in	 ?C	 ? and	 ?N	 ? to	 ?have	 ?higher	 ?activities	 ?of	 ?extracellular	 ?enzymes.	 ?	 ?Finally,	 ?nutrient	 ?limitation	 ?can	 ?induce	 ?microbes	 ?to	 ? increase	 ? the	 ? production	 ? of	 ? enzymes	 ? to	 ? acquire	 ? the	 ? limiting	 ? nutrient	 ? (Sims	 ? and	 ?Wander,	 ? 2002;	 ? Allison	 ? and	 ?Vitousek,	 ? 2005).	 ? Though	 ? not	 ?mutually	 ? exclusive,	 ? there	 ?are	 ? two	 ? means	 ? by	 ? which	 ? a	 ? nutrient	 ? can	 ? be	 ? limiting:	 ? either	 ? the	 ? nutrient	 ? has	 ? low	 ?availability,	 ?or	 ?demand	 ? for	 ? the	 ?nutrient	 ? is	 ? increased	 ?by	 ?a	 ?high	 ?availability	 ?of	 ?other	 ? 30 resources.	 ? For	 ? example,	 ? chitinase	 ? activity	 ? has	 ? been	 ? shown	 ? to	 ? increase	 ? with	 ?decreasing	 ? availability	 ? of	 ? nitrogen	 ? along	 ? a	 ? soil	 ? chronosequence	 ? (Olander	 ? and	 ?Vitousek,	 ? 2000),	 ? and	 ? fertilization	 ? with	 ? nitrogen	 ? has	 ? been	 ? shown	 ? to	 ? increase	 ?phosphatase	 ?activity	 ?(Markelin	 ?and	 ?Houlton,	 ?2012).	 ?Furthermore,	 ?N	 ?and	 ?S	 ?limitation	 ?has	 ? induced	 ? protease	 ? synthesis	 ? in	 ? sand	 ? culture	 ? (Sims	 ? and	 ? Wander,	 ? 2002).	 ? The	 ?higher	 ? total	 ? C	 ? and	 ? N	 ? in	 ? high	 ? phosphatase	 ? microsites,	 ? with	 ? no	 ? difference	 ? in	 ?extractable	 ?phosphorus	 ? compared	 ? to	 ? low	 ?phosphatase	 ? sites,	 ?may	 ? indicate	 ? that	 ? the	 ?high	 ?phosphatase	 ?microsites	 ?were	 ?areas	 ?where	 ?phosphorus	 ?was	 ?limiting,	 ?relative	 ?to	 ?carbon	 ?and	 ?nitrogen,	 ?and	 ?where	 ?microbes	 ?were	 ?stimulated	 ?to	 ?increase	 ?production	 ?of	 ?phosphorus-??acquiring	 ?enzymes.	 ?In	 ? this	 ? study,	 ? it	 ? is	 ? difficult	 ? to	 ? distinguish	 ? among	 ? these	 ? three	 ? explanations	 ? for	 ? the	 ?relationship	 ? between	 ? soil	 ? C,	 ? soil	 ? N	 ? and	 ? phosphatase	 ? activities.	 ? I	 ? do	 ? not	 ? have	 ?microbial	 ? biomass	 ? data,	 ? although	 ? other	 ? studies	 ? in	 ? our	 ? lab	 ? using	 ? root-??window	 ?targeted	 ? sampling	 ? found	 ? no	 ? relationship	 ? between	 ? microbial	 ? biomass	 ? and	 ?phosphatase	 ?activity	 ? (Lidher,	 ?unpublished).	 ? In	 ?order	 ? to	 ? try	 ? to	 ?distinguish	 ?between	 ?high	 ?C	 ? and	 ?N	 ? representing	 ?high	 ?microbial	 ? biomass	 ? versus	 ? a	 ? nutrient-??rich	 ?patch	 ?of	 ?organic	 ? matter	 ? that	 ? might	 ? trigger	 ? excretion	 ? of	 ? nutrient-??acquiring	 ? enzymes,	 ? I	 ?conducted	 ?the	 ?nutrient-??addition	 ?experiment.	 ?	 ?2.4.2. Nutrient	 ?addition	 ?If	 ?high	 ?availability	 ?of	 ?soil	 ?C	 ?and	 ?N	 ?in	 ?soil	 ?microsites	 ?was	 ?driving	 ?phosphatase,	 ?either	 ?by	 ? providing	 ? energy	 ? sources	 ? and	 ? C	 ? and	 ? N	 ? skeletons	 ? for	 ? enzyme	 ? synthesis,	 ? or	 ? by	 ?creating	 ? a	 ? relative	 ? nutrient	 ? limitation	 ? by	 ? P,	 ? I	 ? expected	 ? to	 ? stimulate	 ? microbial	 ? 31 communities	 ?to	 ?produce	 ?phosphatase	 ?in	 ?microsites	 ?where	 ?I	 ?added	 ?soluble	 ?C	 ?and	 ?N.	 ?I	 ?was	 ?surprised,	 ?therefore,	 ?to	 ?find	 ?that	 ?additions	 ?of	 ?labile	 ?carbon	 ?and	 ?nitrogen,	 ?either	 ?alone	 ? or	 ? in	 ? combination,	 ? did	 ? not	 ? stimulate	 ? a	 ? detectable	 ? increase	 ? in	 ? phosphatase	 ?activity	 ? over	 ? control	 ? treatments.	 ? There	 ? could	 ? be	 ? several	 ? reasons	 ?why	 ? I	 ? could	 ? not	 ?stimulate	 ?activity.	 ?First,	 ?with	 ?added	 ?C	 ?and	 ?N,	 ?P	 ?demand	 ?could	 ?have	 ?been	 ?met	 ?through	 ?small	 ? amounts	 ? of	 ? constitutive	 ? production	 ? of	 ? phosphatase,	 ? making	 ? additional	 ?phosphatase	 ?production	 ?unnecessary.	 ?Second,	 ?enzyme	 ?synthesis	 ?is	 ?often	 ?induced	 ?by	 ?a	 ?combination	 ?of	 ?resource	 ? limitation	 ?and	 ?substrate	 ? induction.	 ?For	 ?example,	 ?Allison	 ?and	 ? Vitousek	 ? (2005)	 ? were	 ? able	 ? to	 ? stimulate	 ??-??glucosidase	 ? (a	 ? glucose	 ? acquiring	 ?enzyme)	 ? activity	 ? with	 ? addition	 ? of	 ? a	 ? combination	 ? of	 ? ammonium,	 ? phosphate	 ? and	 ?cellulose;	 ? glycine	 ? aminopeptidase	 ? (a	 ? nitrogen	 ? acquiring	 ? enzyme)	 ? activity	 ? was	 ?stimulated	 ? with	 ? additions	 ? of	 ? a	 ? mixture	 ? of	 ? acetate,	 ? phosphate,	 ? and	 ? collagen.	 ?Moreover,	 ?if	 ?P	 ?supply	 ?was	 ?adequate,	 ?it	 ?is	 ?possible	 ?that	 ?carbon	 ?and	 ?nitrogen	 ?additions	 ?were	 ?allocated	 ?towards	 ?the	 ?production	 ?of	 ?microbial	 ?biomass	 ?instead	 ?of	 ?phosphatase.	 ?For	 ?example,	 ?Allison	 ?and	 ?Vitousek	 ?(2005)	 ?added	 ?acetate	 ?and	 ?phosphate	 ?to	 ?soil,	 ?and	 ?found	 ? significant	 ? increases	 ? in	 ? microbial	 ? biomass	 ? carbon	 ? and	 ? microbial	 ? biomass	 ?phosphorus,	 ?but	 ?not	 ?glycine	 ?aminopeptidase	 ?or	 ?available	 ?nitrogen.	 ?Lastly,	 ?there	 ?was	 ?rain	 ? on	 ? the	 ? day	 ? of	 ? injections,	 ? as	 ?well	 ? as	 ? the	 ? first	 ? day	 ? of	 ? sampling.	 ? This	 ?may	 ? have	 ?caused	 ? changes	 ? in	 ? detectable	 ? phosphatase	 ? activity	 ? on	 ? the	 ? imprints.	 ? Since	 ? this	 ?investigation,	 ? our	 ? lab	 ? has	 ? performed	 ? a	 ? follow-??up	 ? study	 ? (Lidher,	 ? unpublished)	 ? to	 ?determine	 ? if	 ? other	 ? forms	 ? of	 ? carbon	 ? and	 ? nitrogen	 ? could	 ? stimulate	 ? imprintable	 ?phosphatase	 ? activity	 ? using	 ? the	 ? same	 ? root	 ? window	 ? approach	 ? used	 ? in	 ? this	 ? study.	 ?Lidher	 ? found	 ? that	 ? additions	 ? of	 ? ammonium	 ? chloride	 ? and	 ? sodium	 ? acetate,	 ? collagen,	 ? 32 and	 ?chitin	 ?stimulated	 ?activity	 ?over	 ?the	 ?water	 ?control.	 ?These	 ?results	 ?suggest	 ?that	 ?soil	 ?phosphatase	 ?activity	 ?may	 ?be	 ?driven	 ?by	 ?the	 ?availability	 ?of	 ?C	 ?and	 ?N	 ?in	 ?my	 ?sites.	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ? 33 3. Fungal	 ?community	 ?structure	 ?of	 ?high	 ?and	 ?low	 ?phosphatase	 ?microsites	 ?3.1. SYNOPSIS	 ?Fungi	 ? are	 ? a	 ? crucial	 ? component	 ? of	 ? forest	 ? ecosystems.	 ? They	 ? act	 ? as	 ? root-??associated	 ?mediators	 ? of	 ? below	 ? ground	 ? nutrient	 ? transport	 ? (Jones	 ? et	 ? al.	 ? 2009),	 ? respiration	 ?(Hogberg	 ?and	 ?Hogberg,	 ?2002),	 ?carbon	 ?sequestration	 ?(Clemmensen	 ?et	 ?al.	 ?2013),	 ?and	 ?are	 ? important	 ? decomposers	 ? of	 ? organic	 ? nutrients.	 ? The	 ? initial	 ? rate-??limiting	 ? step	 ? of	 ?decomposition	 ? is	 ? the	 ? depolymerization	 ? and	 ? solubilization	 ? of	 ? organic	 ? matter	 ? into	 ?smaller	 ?assimilatable	 ?molecules	 ?(Schimel	 ?and	 ?Bennett	 ?2003),	 ?which	 ?is	 ?facilitated	 ?by	 ?the	 ?release	 ?of	 ?extracellular	 ?hydrolytic	 ?enzymes	 ?by	 ? fungi	 ?and	 ?other	 ?microbes	 ? in	 ? the	 ?soil	 ? (Sinsabaugh,	 ? 1994;	 ? Burns	 ? and	 ? Dick,	 ? 2002).	 ? Phosphatase,	 ? an	 ? extracellular	 ?enzyme	 ? secreted	 ? by	 ? fungi	 ? and	 ? other	 ? soil	 ? organisms,	 ? breaks	 ? down	 ? phosphorus-??containing	 ?organic	 ?molecules	 ? to	 ?release	 ? inorganic	 ?phosphate	 ?(Juma	 ?and	 ?Tabatabai,	 ?1978).	 ? This	 ? process	 ? is	 ? important	 ? for	 ? plant	 ? communities,	 ? as	 ? inorganic	 ? phosphate	 ?levels	 ? are	 ? rarely	 ? adequate	 ? for	 ?plant	 ? growth	 ? (Abel	 ? et	 ? al.,	 ? 2002).	 ?To	 ?date,	 ? very	 ? little	 ?work	 ? has	 ? been	 ? done	 ? examining	 ? fungal	 ? communities	 ? with	 ? respect	 ? to	 ? their	 ? role	 ? in	 ?enzyme	 ?production	 ?in	 ?soils.	 ?Although	 ? fungi	 ? play	 ? an	 ? important	 ? role	 ? in	 ? forest	 ? ecosystem	 ? functioning,	 ? the	 ? spatial	 ?distribution	 ?of	 ?below-??ground	 ? fungal	 ?communities	 ?has	 ?only	 ?recently	 ?been	 ?explicitly	 ?studied,	 ? in	 ? large	 ?part	 ?due	 ?to	 ? the	 ?advent	 ?of	 ?molecular	 ? techniques	 ?(i.e.	 ?Rosling	 ?et	 ?al.,	 ?2003,	 ?Lindahl	 ?et	 ?al.,	 ?2007;	 ?Pickles	 ?et	 ?al.,	 ?2010;	 ?Baldrian	 ?et	 ?al.,	 ?2012;	 ?Clemmensen	 ?et	 ?al.,	 ? 2013).	 ? These	 ? studies	 ? have	 ? demonstrated	 ? that	 ? fungi	 ? are	 ? indeed	 ? not	 ? randomly	 ?distributed,	 ?but	 ?exhibit	 ?some	 ?degree	 ?of	 ?spatial	 ?patterning	 ?over	 ?a	 ?range	 ?of	 ?scales.	 ?At	 ? 34 larger	 ? regional	 ? scales	 ? (hundreds	 ? to	 ? thousands	 ?of	 ?km),	 ? fungal	 ?distribution	 ?patterns	 ?are	 ?driven	 ?largely	 ?by	 ?temperature	 ?and	 ?precipitation	 ?regimes	 ?(Tedersoo	 ?et	 ?al.,	 ?2012),	 ?dispersal	 ? limitation	 ?by	 ?geographic	 ?barriers	 ? (i.e.	 ?oceans	 ?and	 ?mountains)(Sato	 ?et	 ?al.,	 ?2011),	 ?host	 ?distribution	 ? (Sato	 ?et	 ? al.,	 ? 2011),	 ? altitude	 ? (Bahram	 ?et	 ? al.,	 ? 2011)	 ?and	 ? soil	 ?nutrient	 ?regimes	 ?(Jarvis	 ?et	 ?al.,	 ?2013).	 ?	 ?At	 ? local	 ?and	 ?stand	 ?scales	 ?(<100	 ?km),	 ? fungal	 ?communities	 ?are	 ?structured	 ? in	 ?part	 ?by	 ?nutrient	 ?gradients.	 ?For	 ?example,	 ?Kranabetter	 ?et	 ?al.	 ?(2009)	 ?found	 ?significant	 ?changes	 ?in	 ? EM	 ? fungal	 ? communities	 ? along	 ? a	 ? gradient	 ? of	 ? increasing	 ? soil	 ? nitrogen	 ? and	 ?phosphorus.	 ? Dispersal	 ? limitation	 ? can	 ? also	 ? play	 ? a	 ? role	 ? at	 ? this	 ? scale.	 ? For	 ? example,	 ?	 ?studies	 ? have	 ? shown	 ? reduced	 ? EM	 ? fungal	 ? spore	 ? dispersal	 ? at	 ? distances	 ? greater	 ? than	 ?100m	 ? (Peay	 ? et	 ? al.,	 ? 2010;	 ? Peay	 ? et	 ? al.,	 ? 2012).	 ? Interspecific	 ? interactions	 ? also	 ? play	 ? an	 ?important	 ?role	 ?in	 ?structuring	 ?fungal	 ?communities	 ?at	 ?this	 ?scale.	 ?Pickles	 ?et	 ?al.	 ?(2010)	 ?examined	 ? the	 ? horizontal	 ? distribution	 ? of	 ? ectomycorrhizas	 ? in	 ? a	 ? Scots	 ? Pine	 ? (Pinus	 ?sylvestris	 ? L.)	 ? forest,	 ? and	 ? found	 ? that	 ? most	 ? species	 ? displayed	 ? patchy	 ? distributions.	 ?Furthermore,	 ? several	 ? species	 ?were	 ? autocorrelated,	 ?while	 ?others	 ?were	 ?never	 ? found	 ?together	 ?over	 ?a	 ?two-??year	 ?period.	 ?	 ?Interspecific	 ?interactions	 ?are	 ?also	 ?very	 ?important	 ?in	 ?structuring	 ?fungal	 ?communities	 ?at	 ? fine	 ? scales	 ? (mm	 ? to	 ? cm)(Agerer	 ? et	 ? al.,	 ? 2002;	 ? Koide	 ? et	 ? al.,	 ? 2005).	 ? For	 ? example,	 ?Agerer	 ?et	 ?al.	 ?(2002)	 ?found	 ?that	 ?at	 ?the	 ?cm	 ?scale,	 ?there	 ?were	 ?several	 ?pairs	 ?of	 ?EM	 ?fungal	 ?species	 ? that	 ? were	 ? never	 ? found	 ? together,	 ? suggesting	 ? competitive	 ? exclusion.	 ? Other	 ?drivers	 ?of	 ?fungal	 ?communities	 ?structure	 ?at	 ?fine	 ?scales	 ?include	 ?spatial	 ?heterogeneity	 ?in	 ? various	 ? soil	 ? properties	 ? including	 ? pH	 ? and	 ? soil	 ? moisture	 ? (Morris	 ? and	 ? Boerner,	 ? 35 1999),	 ? litter	 ? chemistry	 ? (Baxter	 ? and	 ? Dighton	 ? 2005)	 ? and	 ? soil	 ? nutrient	 ? availability	 ?(Harvey	 ?et	 ?al.,	 ?1987;	 ?Lindahl	 ?et	 ?al	 ?2001).	 ?Most	 ?studies	 ?examining	 ?fine	 ?scale	 ?EM	 ?fungal	 ?community	 ?structure	 ?have	 ?focused	 ?on	 ?root	 ?tips	 ?because	 ?the	 ?fragile	 ?nature	 ?of	 ?fungal	 ?hyphae	 ?makes	 ?them	 ?difficult	 ?to	 ?study	 ?in	 ?soil.	 ? In	 ? a	 ? chronosequence	 ? of	 ? mixed	 ? paper	 ? birch	 ? and	 ? Douglas-??fir	 ? stands,	 ? Brooks	 ?(2010)	 ? used	 ? a	 ? fine-??scale	 ? imprinting	 ? approach	 ? combined	 ? with	 ? terminal-??restriction	 ?fragment	 ? length	 ? polymorphism	 ? (T-??RFLP)	 ? analysis	 ? to	 ? characterize	 ? EM	 ? fungal	 ?communities	 ?from	 ?soils	 ?associated	 ?with	 ?high	 ?and	 ?low	 ?phosphatase	 ?microsites	 ?(mm-??scale)	 ?in	 ?situ.	 ?She	 ?found	 ?that	 ?high	 ?phosphatase	 ?microsites	 ?contained	 ?a	 ?lower	 ?number	 ?of	 ? EM	 ? fungal	 ? species	 ? compared	 ? to	 ? low	 ?phosphatase	 ?microsites	 ? in	 ? some	 ? forest	 ? age	 ?classes.	 ? One	 ? hypothesis	 ? proposed	 ? to	 ? explain	 ? this	 ? observation,	 ? was	 ? that	 ? EM	 ? fungi	 ?were	 ?competitively	 ?excluded	 ?from	 ?high	 ?phosphatase	 ?microsites	 ?by	 ?SAP	 ?fungi,	 ?which	 ?can	 ?be	 ?more	 ?competitive	 ?when	 ?given	 ?a	 ?large	 ?supply	 ?of	 ?carbon	 ?(Lindahl	 ?et	 ?a.,	 ?2001).	 ?	 ?Given	 ?the	 ?results	 ?described	 ?in	 ?Chapter	 ?2	 ?of	 ?this	 ?thesis,	 ?which	 ?demonstrate	 ?that	 ?high	 ?phosphatase	 ?microsites	 ?are	 ?enriched	 ?in	 ?carbon	 ?and	 ?nitrogen,	 ?this	 ?hypothesis	 ?merits	 ?further	 ?investigation.	 ?In	 ? the	 ? study	 ? described	 ? in	 ? this	 ? chapter,	 ? I	 ? tested	 ? whether	 ? fine-??scale	 ? hotspots	 ? of	 ?phosphatase	 ? activity	 ? were	 ? associated	 ? with	 ? different	 ? fungal	 ? communities	 ? than	 ? the	 ?rest	 ? of	 ? the	 ? soil	 ? profile.	 ? I	 ? used	 ? an	 ? enzyme	 ? imprinting	 ? method	 ? (Dong	 ? et	 ? al.,	 ? 2007;	 ?Brooks,	 ?2010)	 ?to	 ?guide	 ?immediate	 ?mm-??scale	 ?sampling	 ?from	 ?soil	 ?profiles	 ?in	 ?the	 ?field,	 ?and	 ? characterized	 ? the	 ? corresponding	 ? fungal	 ? communities	 ? using	 ? pyrosequencing.	 ? I	 ?hypothesized	 ? that	 ?high	 ?phosphatase	 ?microsites	 ?would	 ?be	 ?associated	 ?with	 ?different	 ? 36 fungal	 ? communities	 ? than	 ? low	 ? phosphatase	 ? microsites.	 ? I	 ? also	 ? predicted	 ? that	 ? the	 ?richness	 ?and	 ?relative	 ?abundance	 ?of	 ?SAP	 ?fungi	 ?would	 ?be	 ?greater	 ?in	 ?high	 ?phosphatase	 ?microsites,	 ?especially	 ?relative	 ?to	 ?EM	 ?fungi. 3.2. METHODS	 ?3.2.1. Site	 ?description	 ?	 ?This	 ?study	 ?was	 ?conducted	 ?at	 ?the	 ?same	 ?root	 ?windows	 ?and	 ?site	 ?described	 ?in	 ?Chapter	 ?2.	 ?3.2.2. Soil	 ?Sampling	 ?Soil	 ? sampling	 ? was	 ? conducted	 ? in	 ? June	 ? 2011,	 ? in	 ? the	 ? same	 ? manner	 ? as	 ? described	 ? in	 ?Chapter	 ?2.	 ?No	 ?nutrient	 ?additions	 ?were	 ?made	 ?prior	 ? to	 ?sampling.	 ? 	 ?Soil	 ?was	 ?collected	 ?from	 ? three	 ? to	 ? five	 ?areas	 ?of	 ?high	 ?and	 ? low	 ?phosphatase	 ?activity,	 ? as	 ? indicated	 ?by	 ? soil	 ?imprinting,	 ? from	 ?each	 ?root	 ?window.	 ?Soils	 ?were	 ?sampled	 ?from	 ?Windows	 ?A,	 ?B	 ?and	 ?C	 ?on	 ?June	 ?3rd	 ?and	 ?from	 ?Windows	 ?D	 ?and	 ?E	 ?on	 ?June	 ?4th.	 ?	 ?	 ?3.2.3. DNA	 ?extraction,	 ? PCR	 ?amplification,	 ? and	 ?pyrosequencing	 ?of	 ? fungal	 ?DNA	 ?from	 ?soil	 ?samples	 ?DNA	 ? was	 ? extracted	 ? individually	 ? from	 ? each	 ? 0.25	 ? g	 ? soil	 ? sample	 ? using	 ? the	 ? MoBio	 ?PowerSoil	 ?DNA	 ?isolation	 ?kit	 ?(MoBio	 ?Laboratories	 ?Inc.,	 ?Carlsbad	 ?CA,	 ?U.S.A)	 ?following	 ?the	 ? manufacturer?s	 ? protocol.	 ? Samples	 ? were	 ? prepared	 ? for	 ? pyrosequencing	 ? by	 ?performing	 ? a	 ? nested	 ? PCR,	 ? using	 ? the	 ? fungal-??specific	 ? primers	 ? ITS1-??F	 ? [5?-??CTTGGTCATTTAGAGGAAGTAA-??3?]	 ? (Gardes	 ? and	 ? Bruns,	 ? 1993)	 ? and	 ? ITS4	 ? [5?-??TCCTCCGCTTATTGATATGC-??3?]	 ? (White	 ? et	 ? al.,	 ? 1990)	 ? in	 ? the	 ? first	 ? step,	 ? and	 ? fusion	 ?primers	 ?of	 ? ITS1F	 ?combined	 ?with	 ? ITS2	 ?[5?-??GCTGCGTTCTTCATCGATGC-??3?]	 ? (White	 ?et	 ? 37 al.,	 ?1990)	 ? in	 ?the	 ?nested	 ?step.	 ?A	 ?unique	 ?fusion	 ?primer	 ?was	 ?used	 ?for	 ?each	 ?sample,	 ?so	 ?that	 ? samples	 ? could	 ? be	 ? resolved	 ? after	 ? sequencing.	 ? Fusion	 ?primers	 ?were	 ? created	 ? by	 ?adding	 ?a	 ?unique	 ?10	 ?base-??pair	 ?Multiplex	 ?Identifier	 ?(MID)-??containing	 ?adapter	 ?to	 ?ITS1F	 ?for	 ? each	 ? sample.	 ? DNA	 ? from	 ? each	 ? soil	 ? sample	 ? was	 ? amplified	 ? in	 ? three	 ? separate	 ?replicates	 ?to	 ?account	 ?for	 ?heterogeneous	 ?amplification.	 ?Thermocycler	 ?conditions	 ?for	 ?both	 ?steps	 ?of	 ?the	 ?nested	 ?PCR	 ?were	 ?as	 ?follows:	 ?95?C	 ?for	 ?10	 ?minutes,	 ?followed	 ?by	 ?34	 ?cycles	 ?of	 ?94?C	 ?for	 ?60	 ?seconds,	 ?and	 ?a	 ?final	 ?extension	 ?step	 ?of	 ?72?C	 ?for	 ?7	 ?minutes	 ?before	 ?storage	 ? at	 ? 4?C.	 ? Triplicate	 ? samples	 ? were	 ? combined,	 ? and	 ? DNA	 ? was	 ? cleaned	 ? and	 ?normalized	 ?using	 ?the	 ?Invitrogen	 ?SequalPrep	 ?Normalization	 ?kit	 ?(Invitrogen,	 ?Carlsbad,	 ?CA,	 ?USA)	 ?following	 ?the	 ?manufacturer?s	 ?protocol.	 ?Pyrosequencing	 ?was	 ?performed	 ?at	 ?the	 ?Vancouver	 ? Prostate	 ? Centre	 ? Laboratory	 ? for	 ?Advanced	 ?Genome	 ? analysis	 ? using	 ? a	 ?half	 ?GS	 ?FLX+	 ?Titanium	 ?plate	 ?(Roche	 ?Applied	 ?Biosystems).	 ?The	 ? sequence	 ? data	 ? were	 ? analyzed	 ? using	 ? the	 ? QIIME	 ? (Quantitative	 ? Insights	 ? Into	 ?Microbial	 ? Ecology)	 ? pipeline	 ? (Caporaso	 ? et	 ? al.,	 ? 2010).	 ? Initial	 ? filtering	 ? and	 ? trimming	 ?were	 ?done	 ?using	 ?the	 ?split_libraries.py	 ?command.	 ?Sequences	 ?were	 ?removed	 ?from	 ?the	 ?data	 ?set	 ?if	 ?they	 ?were	 ?shorter	 ?than	 ?100	 ?bp	 ?in	 ?length,	 ?had	 ?any	 ?ambiguous	 ?bases,	 ?had	 ?more	 ?than	 ?8	 ?homopolymers,	 ?had	 ?a	 ?mean	 ?quality	 ?score	 ?below	 ?25,	 ?or	 ?had	 ?any	 ?barcode	 ?errors.	 ?Filtered	 ?sequences	 ?were	 ?then	 ?processed	 ?using	 ?the	 ?ITS	 ?extractor	 ?(Nilsson	 ?et	 ?al.,	 ? 2010),	 ? which	 ? extracts	 ? the	 ? variable	 ? ITS1	 ? region	 ? from	 ? the	 ? sequence.	 ? Sequences	 ?were	 ? clustered	 ? into	 ? operational	 ? taxonomic	 ? units	 ? (OTU)	 ? with	 ? a	 ? 97.5%	 ? similarity	 ?threshold,	 ?using	 ?uclust	 ?(Edgar,	 ?2010)	 ?in	 ?the	 ?pick_otus.py	 ?command.	 ?Taxonomy	 ?was	 ?assigned	 ?by	 ?searching	 ?representative	 ?OTUs	 ?against	 ? the	 ?UNITE	 ?and	 ?NCBI	 ?databases	 ?with	 ? the	 ?assign_taxonomy.py	 ?script.	 ?OTUs	 ?with	 ? fewer	 ? than	 ? three	 ?sequences	 ?across	 ? 38 all	 ? samples	 ? were	 ? removed	 ? using	 ? the	 ? filter_otu_table.py	 ? command.	 ? To	 ? account	 ? for	 ?heterogeneity	 ? in	 ? sampling	 ? effort,	 ? data	 ? sets	 ? were	 ? rarefied	 ? per	 ? sample	 ? using	 ? the	 ?single_rarefaction.py	 ?command.	 ?All	 ?OTUs	 ?were	 ?then	 ?placed	 ?into	 ?ecological	 ?functional	 ?groups:	 ? ectomycorrhizal	 ? fungi,	 ? saprotrophs,	 ? pathogens,	 ? endophytes,	 ? yeasts,	 ?mixed	 ?fungi,	 ?arbuscular	 ?mycorrhizal	 ?fungi,	 ?and	 ?unknown,	 ?by	 ?cross-??referencing	 ?with	 ?Lawry	 ?and	 ?Diederich	 ? (2003),	 ?Cannon	 ?and	 ?Kirk	 ? (2007),	 ?Rinaldi	 ? et	 ? al.	 ? (2008),	 ?Hibbet	 ? et	 ? al.	 ?(2000),	 ?and	 ?Tedersoo	 ?et	 ?al.	 ?(2010).	 ?	 ?3.2.4. Data	 ?analysis	 ?All	 ? multivariate	 ? analyses	 ? were	 ? carried	 ? out	 ? using	 ? PRIMER6	 ? (version	 ? 6.1.12)	 ? and	 ?PERMANOVA+	 ?(version	 ?1.02;	 ?Primer-??E,	 ? Ivybridge,	 ?UK).	 ?A	 ? Jaccard	 ?similarity	 ?matrix	 ?was	 ? calculated	 ? based	 ? on	 ? sequence	 ? presence/absence	 ? data,	 ? and	 ? a	 ? Bray-??Curtis	 ?similarity	 ? matrix	 ? was	 ? calculated	 ? based	 ? on	 ? square	 ? root-??transformed	 ? sequence	 ?abundance	 ?data.	 ?Similarities	 ?among	 ?fungal	 ?communities	 ?in	 ?each	 ?micro-??sample	 ?were	 ?displayed	 ? using	 ? non-??metric	 ? multi-??dimensional	 ? scaling	 ? (NMDS).	 ? To	 ? test	 ? for	 ?differences	 ? in	 ?community	 ?structure	 ?between	 ?high	 ?and	 ? low	 ?phosphatase	 ?microsites	 ?within	 ?each	 ?window,	 ?and	 ?with	 ?phosphatase	 ?activity	 ?nested	 ?within	 ?window	 ?across	 ?all	 ?five	 ?windows,	 ?I	 ?used	 ?permutational	 ?multivariate	 ?analysis	 ?of	 ?variance	 ?(PERMANOVA)	 ?with	 ? 9999	 ? unrestricted	 ? permutations,	 ? and	 ? significance	 ? for	 ? all	 ? tests	 ? set	 ? to	 ? ?=0.05,	 ?unless	 ? stated	 ? otherwise.	 ? This	 ? procedure	 ? was	 ? repeated	 ?with	 ? OTUs	 ? grouped	 ? at	 ? the	 ?family	 ?level.	 ?Species	 ?accumulation	 ?curves	 ?were	 ?created	 ?using	 ?the	 ?alpha_rarefaction.py	 ?command.	 ?I	 ? used	 ? analysis	 ? of	 ? variance	 ? (ANOVA)	 ? and	 ? Tukey?s	 ? honest	 ? significance	 ? test,	 ? as	 ? 39 implemented	 ? in	 ? RStudio	 ? (version	 ? 0.96.316),	 ? to	 ? test	 ? for	 ? differences	 ? in	 ?presence/absence	 ? and	 ? abundance	 ? of	 ? OTUs	 ? belonging	 ? to	 ? designated	 ? ecological	 ?functional	 ?groups,	 ?as	 ?well	 ?as	 ?EM:SAP	 ?between	 ?high	 ?and	 ?low	 ?phosphatase	 ?microsites.	 ?This	 ?analysis	 ?was	 ?conducted	 ?for	 ?each	 ?window	 ?separately,	 ?and	 ?for	 ?all	 ?windows	 ?with	 ?phosphatase	 ?activity	 ?nested	 ?within	 ?window.	 ?I	 ?used	 ?rarefied	 ?read	 ?abundance	 ?instead	 ?of	 ?relative	 ?abundance	 ?when	 ?comparing	 ?abundance	 ?between	 ?samples,	 ?as	 ?all	 ?samples	 ?were	 ?rarefied	 ?to	 ?800	 ?reads.	 ?Significance	 ?for	 ?all	 ?tests	 ?was	 ?set	 ?to	 ??=0.05	 ?unless	 ?stated	 ?otherwise.	 ?Lastly,	 ?indicator	 ?species	 ?analysis	 ?was	 ?carried	 ?out	 ?in	 ?RStudio	 ?(version	 ?0.96.316)	 ?with	 ?the	 ?multipatt	 ?function	 ?of	 ?the	 ??idicspecies?	 ?package	 ?(De	 ?C?aceres	 ?and	 ?Legendre,	 ?2009)	 ?using	 ?the	 ?OTU	 ?relative	 ?abundances	 ?grouped	 ?to	 ?the	 ?family	 ?level.	 ?The	 ?indicator	 ?species	 ?analysis	 ?indicates	 ?the	 ?taxa	 ?that	 ?tend	 ?to	 ?be	 ?found	 ?in	 ?a	 ?sample	 ?group	 ?or	 ?combination	 ?of	 ?sample	 ? groups	 ? versus	 ? others.	 ? I	 ? applied	 ? the	 ? Holm	 ? correction	 ? for	 ? multiple	 ?comparisons	 ?(Holm,	 ?1979)	 ?using	 ?the	 ?p.adjust	 ?function.	 ?Significance	 ?for	 ?all	 ?tests	 ?was	 ?set	 ?to	 ??=0.05.	 ?	 ?3.3. RESULTS	 ?After	 ?quality	 ?filtering	 ?and	 ?target	 ?extraction,	 ?I	 ?analyzed	 ?a	 ?total	 ?of	 ?64,495	 ?fungal	 ?ITS1	 ?reads,	 ? with	 ? an	 ? average	 ? length	 ? of	 ? 313.6	 ? bp.	 ? The	 ? total	 ? number	 ? of	 ? reads	 ? per	 ?micro-??sample	 ? ranged	 ? from	 ? 858	 ? to	 ? 2648,	 ? with	 ? an	 ? average	 ? per	 ? sample	 ? per	 ? window	 ? of	 ?1547.67	 ? to	 ?1775.11.	 ?The	 ?number	 ?of	 ? reads	 ?per	 ?OTU	 ? ranged	 ? from	 ?3	 ? to	 ?4219.	 ?There	 ?were	 ?6470	 ?reads	 ?removed	 ?for	 ?quality	 ?control,	 ?including	 ?2898	 ?reads	 ?less	 ?than	 ?100bp,	 ? 40 1492	 ?with	 ?ambiguous	 ?bases,	 ?and	 ?1265	 ?with	 ?homopolymer	 ?runs	 ?greater	 ?than	 ?8.	 ?Also,	 ?3733	 ?reads	 ?were	 ?removed	 ?that	 ?had	 ?fewer	 ?than	 ?three	 ?sequences	 ?across	 ?all	 ?samples.	 ?	 ?Sequence	 ?clustering	 ?yielded	 ?1103	 ?OTUs.	 ?The	 ?number	 ?of	 ?OTUs	 ?per	 ?soil	 ?microsample	 ?ranged	 ? from	 ? 90	 ? to	 ? 155,	 ? averaging	 ? 116	 ? to	 ? 138	 ? per	 ? sample	 ? per	 ?window.	 ? The	 ?most	 ?frequently	 ? encountered	 ? OTUs	 ?were	 ? an	 ?Oidiodendron	 ? (OTU1),	 ? a	 ?Wilcoxina	 ? (OTU2),	 ?and	 ? a	 ? member	 ? of	 ? the	 ? Pyronemataceae	 ? (OTU3)	 ? (Table	 ? 3.1).	 ? Many	 ? frequently	 ?encountered	 ?OTUs	 ?were	 ?present	 ? in	 ? only	 ? one	 ?or	 ? two	 ?windows,	 ? such	 ? as	 ? Inocybe	 ?sp.	 ?(OTU5),	 ? two	 ?uncultured	 ?Tomentella	 ? (OTU19	 ?and	 ?OTU20),	 ?an	 ?uncultured	 ?Piloderma	 ?(OTU29),	 ?and	 ?an	 ?uncultured	 ?Wilcoxina	 ?(OTU30).	 ?	 ?OTU	 ?accumulation	 ?curves	 ?were	 ?similar	 ?for	 ?all	 ?windows	 ?(Figure	 ?3.1).	 ?In	 ?spite	 ?of	 ?the	 ?small	 ?sizes	 ?of	 ?the	 ?soil	 ?samples,	 ?none	 ?of	 ?the	 ?curves	 ?reached	 ?an	 ?asymptote,	 ?suggesting	 ?that	 ?many	 ?fungal	 ?taxa	 ?remain	 ?undetected.	 ?I	 ?was	 ?able	 ?to	 ?group	 ?approximately	 ?66%	 ?of	 ?the	 ?OTUs	 ?into	 ?50	 ?families.	 ?The	 ?remaining	 ?34%	 ?could	 ?only	 ?be	 ?labeled	 ?with	 ?a	 ?higher	 ?taxonomic	 ?level	 ?and,	 ?therefore,	 ?were	 ?not	 ?included	 ?in	 ?the	 ?family-??level	 ?or	 ?functional-??group	 ?analyses.	 ?The	 ?families	 ?with	 ?the	 ?largest	 ?numbers	 ?of	 ?reads	 ?were	 ?Cortinariaceae	 ?(22.12%),	 ? Myxotrichaceae	 ? (16.83%),	 ? Pyronemataceae	 ? (16.34%),	 ? Thelephoraceae	 ?(10.63),	 ?and	 ?Atheliaceae	 ?(7.51%)(Figure	 ?3.2).	 ? I	 ? further	 ?grouped	 ?OTUs	 ? identified	 ?to	 ?the	 ? family	 ? level	 ? into	 ? functional	 ? groups.	 ? Sequences	 ? of	 ? EM	 ? fungi	 ? dominated	 ? the	 ?samples	 ? (80.66%	 ? of	 ? classified	 ? sequences,	 ? 290	 ? OTUs),	 ? followed	 ? by	 ? SAP	 ? fungi	 ?(11.02%,	 ? 100	 ? OTUs),	 ? and	 ? pathogenic	 ? fungi	 ? (5.48%,	 ? 35	 ? OTUs).	 ? Endophytic	 ? fungi,	 ?arbuscular	 ?mycorrhizal	 ?fungi,	 ?lichen	 ?and	 ?fungi	 ?that	 ?were	 ?part	 ?of	 ?multiple	 ?functional	 ?groups	 ? represented	 ? less	 ? than	 ? 2%	 ? of	 ? the	 ? sequences	 ? each. 41 Table	 ?3.1:	 ?The	 ?50	 ?most	 ?frequently	 ?detected	 ?OTUs,	 ?representing	 ?approximately	 ?50%	 ?of	 ?the	 ?total	 ?reads	 ?across	 ?the	 ?site,	 ?and	 ?the	 ?average	 ?number	 ?of	 ?reads	 ?found	 ?per	 ?microsite	 ?in	 ?each	 ?window	 ?(?SE).	 ?OTU	 ?I.D.	 ? Closest	 ?NCBI	 ?match	 ? Identity,	 ?length	 ?(%)	 ? No.	 ?of	 ?reads	 ? Accession	 ?number	 ?(closest	 ?match)	 ? A	 ? B	 ? C	 ? D	 ? E	 ?OTU1	 ? Uncultured	 ?Oidiodendron	 ? 164/165	 ?(99)	 ? 4219	 ? HQ022064	 ? 53.1?8.4	 ? 86.7?21.4	 ? 80?16.7	 ? 81?18.2	 ? 219.3?68.6	 ?OTU2	 ? Uncultured	 ?Wilcoxina	 ? 188/188	 ?(100)	 ? 2478	 ? GU452514	 ? 0.7?0.3	 ? 121.2?26.7	 ? 72.8?26.6	 ? 98.5?29.3	 ? 22.4?8.0	 ?OTU3	 ? Uncultured	 ?Pyronemataceae	 ? 184/191	 ?(96)	 ? 1597	 ? JX030921	 ? 92.7?27.1	 ? 26.8?3.6	 ? 2.9?0.8	 ? 27.5?13.2	 ? 50.2?31.5	 ?OTU4	 ? Uncultured	 ?fungus	 ?clone	 ? 186/186	 ?(100)	 ? 1497	 ? GU366685	 ? 0.1?0.1	 ? 88.7?18.5	 ? 44.4?21.1	 ? 55.5?7.9	 ? 10.8?6.6	 ?OTU5	 ? Inocybe	 ?sp.	 ? 262/263	 ?(99)	 ? 1480	 ? HQ604098	 ? 0	 ? 0	 ? 185?52.9	 ? 0	 ? 0	 ?OTU6	 ? Uncultured	 ?Agaricomycetes	 ? 176/176	 ?(100)	 ? 1166	 ? FJ553582	 ? 12.6?2.7	 ? 15.9?4.5	 ? 74.9?15.8	 ? 10.2?2.8	 ? 30.8?8.6	 ?OTU7	 ? Uncultured	 ?Wilcoxina	 ? 187/188	 ?(99)	 ? 1051	 ? GU452514	 ? 129.5?23.1	 ? 0.1?0.1	 ? 0.7?0.6	 ? 0.5?0.2	 ? 0.5?0.3	 ?OTU8	 ? Uncultured	 ?Cryptosporiopsis	 ? 181/182	 ?(99)	 ? 1047	 ? FJ440880	 ? 5.4?1.9	 ? 69.3?60.8	 ? 4.7?1.3	 ? 13.6?1.7	 ? 36.5?12.5	 ?OTU9	 ? Mortierella	 ?sp.	 ? 156/156	 ?(100)	 ? 934	 ? HQ446020	 ? 29.0?12.1	 ? 54.1?15.8	 ? 6.2?2.8	 ? 35?18.5	 ? 1?0.6	 ?OTU10	 ? Uncultured	 ?Cortinarius	 ? 161/162	 ?(99)	 ? 887	 ? DQ481733	 ? 0.1?0.1	 ? 53?30.6	 ? 0.1?0.1	 ? 0.1?0.1	 ? 51.1?51.1	 ?OTU11	 ? Inocybe	 ?sp.	 ? 259/259	 ?(100)	 ? 864	 ? HQ604298	 ? 0	 ? 2?0.7	 ? 88.1?17.4	 ? 23.5?9.1	 ? 0.2?0.1	 ?OTU12	 ? Uncultured	 ?fungus	 ? 174/176	 ?(99)	 ? 850	 ? GU366690	 ? 3.9?1.4	 ? 19.7?2.5	 ? 11.4?3.0	 ? 9.5?2.4	 ? 57?11.5	 ?OTU13	 ? Amphinema	 ?sp.	 ? 167/170	 ?(98)	 ? 742	 ? EU292238	 ? 32.8?11.6	 ? 54.1?9.2	 ? 0	 ? 4?2.5	 ? 2.5?1.1	 ?OTU14	 ? Inocybe	 ?sp.	 ? 258/259	 ?(99)	 ? 646	 ? HQ604298	 ? 0	 ? 1?0.7	 ? 68.8?15.7	 ? 14.6?6.8	 ? 0	 ?OTU15	 ? Uncultured	 ?fungus	 ?	 ? 175/75	 ?(100)	 ? 608	 ? AY097049	 ? 4.6?1.1	 ? 24.2?5.3	 ? 9.8?2.8	 ? 8.3?2.5	 ? 27.6?13.4	 ?OTU16	 ? Piloderma	 ?sp.	 ?	 ? 181/185	 ?(96)	 ? 586	 ? AY097039	 ? 0.1?0.1	 ? 0.1?0.1	 ? 72.9?29.0	 ? 0.2?0.2	 ? 0	 ?OTU17	 ? Penicillium	 ?sp.	 ?	 ? 173/173	 ?(100)	 ? 573	 ? AF033457	 ? 7.8?1.8	 ? 2.3?2.3	 ? 1.5?0.8	 ? 22.2?5.0	 ? 38.4?11.1	 ?OTU18	 ? Penicillium	 ?sp.	 ? 183/183	 ?(100)	 ? 565	 ? GU092965	 ? 0.6?0.4	 ? 2.1?1.2	 ? 5.6?2.1	 ? 13.3?3.1	 ? 46.4?17.7	 ?OTU19	 ? Uncultured	 ?Tomentella	 ? 199/201	 ?(99)	 ? 537	 ? GU220708	 ? 0	 ? 0.1?0.1	 ? 67?23.5	 ? 0	 ? 0	 ?OTU20	 ? Uncultured	 ?Tomentella	 ?	 ? 194/200	 ?(97)	 ? 526	 ? EF655697	 ? 65.7?18.7	 ? 0	 ? 0	 ? 0	 ? 0	 ?OTU21	 ? Uncultured	 ?Myxotrichaceae	 ?	 ? 168/168	 ?(100)	 ? 509	 ? FJ475547	 ? 12.7?3.6	 ? 19?5.0	 ? 12.8?6.6	 ? 11.5?5.1	 ? 9.3?4.2	 ?OTU22	 ? Uncultured	 ?Russula	 ? 198/200	 ?(99)	 ? 507	 ? EU711738	 ? 61.2?21.7	 ? 0	 ? 0	 ? 1?1.0	 ? 1.2?1.2	 ?OTU23	 ? Uncultured	 ?Venturia	 ? 159/159	 ?(100)	 ? 498	 ? FJ553706	 ? 0.2?0.2	 ? 0.1?0.1	 ? 0.2?0.2	 ? 0.3?0.2	 ? 54.5?44.3	 ?OTU24	 ? Cryptococcus	 ?sp.	 ? 163/163	 ?(100)	 ? 480	 ? JQ666590	 ? 44.8?32.8	 ? 10.6?3.3	 ? 0.2?0.2	 ? 4.3?3.0	 ? 0.8?0.6	 ? 42 OTU25	 ? Uncultured	 ?Pezizomycotina	 ? 160/160	 ?(100)	 ? 464	 ? GU083093	 ? 42.5?10.8	 ? 6?1.9	 ? 1.6?0.6	 ? 3.3?1.4	 ? 4.7?4.0	 ?OTU26	 ? Uncultured	 ?fungus	 ? 214/214	 ?(100)	 ? 442	 ? FN377841	 ? 4.2?1.8	 ? 30.3?15.1	 ? 6?3.5	 ? 1.7?0.8	 ? 12?4.5	 ?OTU27	 ? Uncultured	 ?mycorrhizal	 ?fungus	 ? 162/165	 ?(98)	 ? 437	 ? AB669637	 ? 0.1?0.1	 ? 54.4?11.9	 ? 0	 ? 0	 ? 0.1?0.1	 ?OTU28	 ? Uncultured	 ?Oidiodendron	 ? 535/535	 ?(100)	 ? 434	 ? HQ022064	 ? 2.2?0.5	 ? 3.9?1.1	 ? 8.9?2.1	 ? 16.5?3.9	 ? 23.9?5.2	 ?OTU29	 ? Uncultured	 ?Piloderma	 ? 190/191	 ?(99)	 ? 412	 ? HM488497	 ? 0	 ? 0	 ? 0	 ? 68.7?33.4	 ? 0	 ?OTU30	 ? Uncultured	 ?Wilcoxina	 ? 187/188	 ?(99)	 ? 407	 ? GU452514	 ? 0	 ? 50.8?17.6	 ? 0	 ? 0.2?0.2	 ? 0	 ?OTU31	 ? Uncultured	 ?fungus	 ? 175/175	 ?(100)	 ? 396	 ? HQ125510	 ? 17.2?7.1	 ? 11?2.2	 ? 4.1?2.1	 ? 1.7?1.5	 ? 14.1?7.5	 ?OTU32	 ? Tomentella	 ?sp.	 ? 194/199	 ?(97)	 ? 387	 ? HQ215812	 ? 48.4?13.2	 ? 0	 ? 0	 ? 0	 ? 0	 ?OTU33	 ? Uncultured	 ?fungus	 ? 211/214	 ?(99)	 ? 375	 ? AY702788	 ? 46.3?24.2	 ? 0	 ? 0.5?0.5	 ? 0	 ? 0	 ?OTU34	 ? Russula	 ?sp.	 ? 186/189	 ?(98)	 ? 371	 ? JQ975978	 ? 46.1?24.1	 ? 0	 ? 0.1?0.1	 ? 0.2?0.2	 ? 0	 ?OTU35	 ? Uncultured	 ?Basidiomycota	 ? 175/176	 ?(99)	 ? 356	 ? JX032353	 ? 13.8?5.5	 ? 1.9?0.7	 ? 3.4?1/0	 ? 7.7?3.6	 ? 17.4?10.5	 ?OTU36	 ? Uncultured	 ?Sistotrema	 ? 170/173	 ?(98)	 ? 354	 ? FJ196900	 ? 0	 ? 0	 ? 0	 ? 31?20.9	 ? 18.7?5.1	 ?OTU37	 ? Russula	 ?sp.	 ? 201/201	 ?(100)	 ? 349	 ? EF218808	 ? 43.6?18.4	 ? 0	 ? 0	 ? 0	 ? 0	 ?OTU38	 ? Uncultured	 ?Helotiales	 ? 156/158	 ?(99)	 ? 342	 ? DQ182441	 ? 0.1?0.1	 ? 41.5?27.6	 ? 0	 ? 0	 ? 1?1.0	 ?OTU39	 ? Uncultured	 ?Ascomycota	 ?clone	 ? 161/161	 ?(100)	 ? 338	 ? FJ553219	 ? 4.9?2.5	 ? 16.3?12.0	 ? 2.4?0.9	 ? 2.3?1.7	 ? 15.1?6.0	 ?OTU40	 ? Uncultured	 ?Basidiomycota	 ? 173/174	 ?(99)	 ? 331	 ? JQ666662	 ? 13.4?2.6	 ? 2.1?0.5	 ? 0.9?0.4	 ? 0.3?0.2	 ? 22?12.6	 ?OTU41	 ? Beauveria	 ?sp.	 ? 158/161	 ?(98)	 ? 327	 ? JN379794	 ? 0.3?0.2	 ? 0	 ? 40.4?38.7	 ? 0	 ? 0.2?0.2	 ?OTU42	 ? Uncultured	 ?Inocybe	 ? 277/282	 ?(98)	 ? 323	 ? FJ554164	 ? 0.1?0.1	 ? 0	 ? 11.3?8.9	 ? 0	 ? 25.8?6.1	 ?OTU43	 ? Inocybe	 ?sp.	 ? 268/269	 ?(99)	 ? 325	 ? HQ604165	 ? 0	 ? 40.6?10.7	 ? 0	 ? 0	 ? 0	 ?OTU44	 ? Uncultured	 ?fungus	 ? 164/166	 ?(99)	 ? 325	 ? EF434129	 ? 10?4.6	 ? 0.4?0.2	 ? 28.1?27	 ? 2.8?1.9	 ? 0	 ?OTU45	 ? Uncultured	 ?fungus	 ? 157/158	 ?(99)	 ? 310	 ? GQ159942	 ? 29?7.1	 ? 4.4?1.1	 ? 2.1?1.1	 ? 1.2?0.8	 ? 2.1?1.9	 ?OTU46	 ? Uncultured	 ?Oidiodendron	 ? 167/167	 ?(100)	 ? 302	 ? FJ553884	 ? 4.3?1.6	 ? 5.3?1.3	 ? 9.6?2.8	 ? 10.2?2.7	 ? 9.7?3.2	 ?OTU47	 ? Geopora	 ?cooperi	 ? 205/206	 ?(99)	 ? 290	 ? JN558642	 ? 0.3?0.2	 ? 0.3?0.2	 ? 35.8?10.7	 ? 0	 ? 0	 ?OTU48	 ? Uncultured	 ?Amphinema	 ? 168/170	 ?(99)	 ? 289	 ? EU292238	 ? 33.5?10.9	 ? 2?0.9	 ? 0.1?0.1	 ? 0.3?0.3	 ? 0.2?0.1	 ?OTU49	 ? Uncultured	 ?Pseudotomentella	 ? 215/216	 ?(99)	 ? 270	 ? FJ554425	 ? 0	 ? 0	 ? 0	 ? 31.7?10.6	 ? 8.9?2.7	 ?OTU50	 ? Uncultured	 ?fungus	 ? 183/183	 ?(100)	 ? 269	 ? FJ552894	 ? 0.1?0.1	 ? 0.3?0.2	 ? 1.1?0.5	 ? 3.3?1.4	 ? 26.3?15.4	 ?  43  Figure	 ? 3.1:	 ?Accumulation	 ? curves	 ? showing	 ? the	 ?number	 ?of	 ? fungal	 ?OTUs	 ? (defined	 ?as	 ?97.5%	 ?sequence	 ?similarity)	 ?detected	 ?in	 ?each	 ?microsite	 ?at	 ?every	 ?window.	 ?	 ?	 ?!"#!"$!"%!"&!"'!!"'#!"'$!"'%!"'&!"#!" '#!" ##!" (#!" $#!" )#!" %#!" *#!" &#!" +#!" '!(!" ''+!"!"#$%&'()'*+,-'./'0123'-4#45.&4/6'!"#$%&'()'-%7"%89%-',"-"."/"0" 44  Figure	 ?3.2:	 ?Taxonomic	 ?distribution	 ?of	 ?OTUs	 ?at	 ?the	 ?family	 ?level,	 ?according	 ?to	 ?the	 ?top	 ?BLAST	 ?hits	 ?in	 ?the	 ?NCBI	 ?database.	 ?OTUs	 ?that	 ?were	 ?not	 ?classifiable	 ?at	 ?the	 ?family	 ?level	 ?were	 ?omitted.	 ?	 ?Interestingly,	 ? fungal	 ? communities	 ? differed	 ? among	 ? windows,	 ? even	 ? though	 ? the	 ?windows	 ?were	 ?only	 ?separated	 ?by	 ?5-??15	 ?m	 ?in	 ?a	 ?stand	 ?selected	 ?for	 ?its	 ?uniformity.	 ?This	 ?was	 ?true	 ?for	 ?both	 ?analyses	 ?based	 ?on	 ?occurrence	 ?(PERMANOVA,	 ?p<0.01;	 ?Figure	 ?3.3A)	 ?and	 ? relative	 ? abundance	 ? (p<0.01;	 ? Figure	 ? 3.3B)	 ? of	 ? OTUs.	 ? In	 ? both	 ? ordinations,	 ? the	 ?communities	 ? in	 ?Windows	 ?A	 ?and	 ?B	 ?separated	 ? from	 ?the	 ?others	 ?along	 ?Axis	 ?1	 ?and	 ? the	 ?community	 ?in	 ?Window	 ?E	 ?separated	 ?from	 ?the	 ?others	 ?along	 ?Axis	 ?2.	 ?	 ?The	 ?distribution	 ?of	 ?functional	 ?groups	 ?also	 ?varied	 ?across	 ?windows	 ?(Table	 ?3.2,	 ?Figure	 ?3.4,	 ?Figure	 ?3.5,	 ?Figure	 ?3.6).	 ?Window	 ?C	 ?had	 ?higher	 ?rarefied	 ?abundance	 ?of	 ?EM	 ?fungal	 ? 45 reads	 ? 	 ?(Figure	 ?3.4A)	 ?and	 ?OTU	 ?occurrence-??based	 ?EM:SAP	 ?ratio	 ?than	 ?other	 ?windows	 ?(Figure	 ?3.6).	 ?This	 ?was	 ?the	 ?only	 ?window	 ?with	 ?visible	 ?EM	 ?roots	 ?and	 ?EM	 ?hyphae	 ?across	 ?the	 ?window	 ?(Figure	 ?A.1).	 ?There	 ?were	 ?a	 ?greater	 ?number	 ?of	 ?EM	 ?fungal	 ?OTUs	 ?detected	 ?in	 ? Windows	 ? C-??E	 ? than	 ? in	 ? Windows	 ? A	 ? and	 ? B	 ? (Figure	 ? 3.4B).	 ? Window	 ? E	 ? had	 ? higher	 ?rarefied	 ? abundance	 ? of	 ? SAP	 ? fungal	 ? reads	 ? than	 ?Window	 ? A,	 ? as	 ?well	 ? as	 ? a	 ? higher	 ? SAP	 ?richness	 ? than	 ? Window	 ? C	 ? (Figure	 ? 3.5). The	 ? following	 ? families	 ? were	 ? detected	 ? as	 ?indicator	 ? taxa	 ? for	 ? individual	 ? windows	 ? or	 ? groups	 ? of	 ? windows	 ? (p	 ? =	 ? 0.041	 ? after	 ?adjusting	 ?for	 ?multiple	 ?comparisons):	 ?Ceratobasidiaceae	 ?for	 ?window	 ?B,	 ?Clavulinaceae	 ?and	 ?Vibrisseaceae	 ?for	 ?window	 ?D,	 ?Russulaceae	 ?for	 ?windows	 ?A	 ?and	 ?D,	 ?Sistotremataceae	 ?for	 ?windows	 ?D	 ?and	 ?E,	 ?and	 ?Cortinariaceae	 ?for	 ?all	 ?windows	 ?except	 ?window	 ?A.	 ?	 ?	 ?	 ?A)	 ? 46  Figure	 ? 3.3:	 ?Non-??metric	 ?multi-??dimensional	 ? scaling	 ?with	 ? (A)	 ? Jaccard	 ? [stress	 ?=	 ?0.15]	 ?and	 ?(B)	 ?Bray-??Curtis	 ?[stress	 ?=	 ?0.17]	 ?similarity	 ?indices	 ?from	 ?high	 ?(green	 ?symbols)	 ?and	 ?low	 ?(blue	 ?symbols)	 ?phosphatase	 ?areas	 ?from	 ?Windows	 ?A	 ?through	 ?E.	 ? Table	 ? 3.2:	 ? P-??values	 ? from	 ? nested	 ? ANOVAs	 ? (phosphatase	 ? status	 ? nested	 ? within	 ?window)	 ?using	 ?number	 ?of	 ?reads	 ?and	 ?occurrence	 ?of	 ?OTUs	 ?from	 ?ectomycorrhizal	 ?(EM)	 ?and	 ?saprotrophic	 ?(SAP)	 ?fungi,	 ?and	 ?EM:SAP	 ?from	 ?five	 ?root	 ?windows.	 ?      !! "#$%&'($)*!+,(#-!./0!.%%#,,1$%1!2#341,!(5!,1)67!8'$6(9! !"# $%&%%'# $%&%%'#8'$6(9:;<(7-<)&)71=! !"# %&()# %&'(#8'$6(9! *+,# %&%-# %&%.#8'$6(9:;<(7-<)&)71=! *+,# %&/(# %&00#8'$6(9! !"1*+,# %&%%.# %&%'#8'$6(9:;<(7-<)&)71=! !"1*+,# %&2(# %&%.#B)	 ? 47  Figure	 ? 3.4:	 ? Bars	 ? (?SE)	 ? represent	 ? mean	 ? number	 ? of	 ? ectomycorrhizal	 ? (EM)	 ? fungal	 ?reads	 ? (A)	 ? and	 ?OTU	 ?occurrence	 ? (B)	 ?per	 ?microsample	 ?per	 ?window.	 ?Different	 ? letters	 ?indicate	 ? significant	 ? differences	 ? based	 ? on	 ? one-??way	 ? ANOVA	 ? followed	 ? by	 ? Tukey?s	 ?honest	 ?significance	 ?test	 ?(?	 ?=	 ?0.05),	 ?corrected	 ?for	 ?multiple	 ?comparisons.	 ? 	 ?P<0.001	 ?	 ?P<0.001	 ? 48  Figure	 ?3.5:	 ?Bars	 ?(?SE)	 ?represent	 ?mean	 ?number	 ?of	 ?saprotrophic	 ?(SAP)	 ?fungal	 ?reads	 ?(A)	 ?and	 ?OTU	 ?occurrence	 ?(B)	 ?per	 ?microsample	 ?per	 ?window.	 ?Different	 ?letters	 ?indicate	 ?significant	 ? differences	 ? based	 ? on	 ? one-??way	 ? ANOVA	 ? followed	 ? by	 ? Tukey?s	 ? honest	 ?significance	 ?test	 ?(?	 ?=	 ?0.05),	 ?corrected	 ?for	 ?multiple	 ?comparisons.	 ? 	 ?P=0.03	 ?	 ?P=0.04	 ? 49  Figure	 ? 3.6:	 ? Bars	 ? (?SE)	 ? represent	 ? ectomycorrhizal	 ? (EM)	 ? to	 ? saprotrophic	 ? (SAP)	 ?fungal	 ?ratios	 ?based	 ?on	 ?(A)	 ?number	 ?of	 ?fungal	 ?reads	 ?and	 ?(B)	 ?OTU	 ?occurrence	 ?averaged	 ?among	 ?microsamples	 ?per	 ?window.	 ?Different	 ? letters	 ? indicate	 ? significant	 ?differences	 ?based	 ?on	 ?one-??way	 ?ANOVA	 ? followed	 ?by	 ?Tukey?s	 ? honest	 ? significance	 ? test	 ? (?	 ?=	 ? 0.05)	 ?corrected	 ?for	 ?multiple	 ?comparisons.	 ? 	 ?P=0.01	 ?	 ?P=0.003	 ? 50 Fungal	 ? communities	 ? in	 ?high	 ? and	 ? low	 ?phosphatase	 ?microsites	 ? in	 ? the	 ? same	 ?window	 ?did	 ? not	 ? differ	 ? overall	 ? (Table	 ? 3.3),	 ? regardless	 ? of	 ?whether	 ? OTUs	 ?were	 ? grouped	 ? into	 ?families,	 ? or	 ? left	 ? as	 ? individual	 ? OTUs.	 ? However,	 ? there	 ? was	 ? an	 ? indication	 ? that	 ? the	 ?distribution	 ?of	 ?functional	 ?groups,	 ?specifically	 ?EM	 ?and	 ?SAP	 ?fungi,	 ?differed	 ?(Table	 ?3.2).	 ?The	 ?relationship	 ?between	 ?these	 ?two	 ?functional	 ?groups	 ?varied	 ?by	 ?window,	 ?however:	 ?the	 ?ratio	 ?of	 ?EM	 ?to	 ?SAP	 ?sequences	 ?was	 ?lower	 ?in	 ?Window	 ?A	 ?(p=0.05),	 ?and	 ?tended	 ?to	 ?be	 ?lower	 ? in	 ? Window	 ? B	 ? (p=0.07),	 ? in	 ? the	 ? high	 ? phosphatase	 ? compared	 ? to	 ? the	 ? low	 ?phosphatase	 ?microsites	 ?(Table	 ?3.4,	 ?Figure	 ?3.7).	 ?No	 ?other	 ?differences	 ?in	 ?abundance	 ?of	 ?functional	 ?groups	 ?with	 ?phosphatase	 ?activity	 ?were	 ?observed	 ?across	 ?windows	 ?(Table	 ?3.2),	 ? but	 ? some	 ?were	 ? seen	 ? in	 ? individual	 ?windows.	 ? Specifically,	 ? in	 ? high	 ?phosphatase	 ?compared	 ?to	 ?low	 ?phosphatase	 ?areas,	 ?the	 ?relative	 ?abundance	 ?of	 ?EM	 ?fungi	 ?was	 ?lower	 ?in	 ?window	 ?B	 ?(p=0.05;	 ?Figure	 ?3.8)	 ?and	 ?relative	 ?abundance	 ?of	 ?SAP	 ?fungi	 ?tended	 ?to	 ?be	 ?lower	 ?in	 ?window	 ?D	 ?(p=0.07;	 ?Figure	 ?3.9).	 ?There	 ?were	 ?no	 ?differences	 ?in	 ?the	 ?number	 ?of	 ?EM	 ?or	 ?SAP	 ?fungal	 ?OTUs	 ?(taxon	 ?richness	 ?per	 ?functional	 ?group)	 ?between	 ?high	 ?and	 ?low	 ?phosphatase	 ?areas	 ?in	 ?any	 ?window	 ?(Table	 ?3.4).	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ? 51 Table	 ? 3.3:	 ? P-??values	 ? from	 ? PERMANOVAs	 ? comparing	 ? fungal	 ? communities	 ? between	 ?high	 ? and	 ? low	 ? phosphatase	 ? areas	 ? from	 ? five	 ? root	 ?windows	 ? using	 ? Jaccard	 ? and	 ? Bray-??Curtis	 ?similarity	 ? indices.	 ?OTUs	 ?were	 ?ungrouped	 ?(top)	 ?and	 ?grouped	 ?(bottom)	 ?at	 ?the	 ?family	 ?level.	 ? ! !"#$%& ' ()**)+$' ,+)-./0+1"2'!!"!3452 '!#$%%!!!#$&'!(! #$)&! #$)*!+! #$),! #$*'!-! #$&)! #$,&!.! #$,/! #$,0!!! 6)7"8"92'!!"! #$&,! #$''!(! #$0/! #$),!+! #$&/! #$&*!-! #$*&! #$*)!.! #$1&! #$,%! 52 Table	 ? 3.4:	 ?P-??values	 ? from	 ?ANOVAs	 ?using	 ?occurrence	 ?of	 ?OTUs	 ?and	 ?number	 ?of	 ?reads	 ?from	 ? ectomycorrhizal	 ? fungi	 ? (EM),	 ? saprotrophic	 ? fungi	 ? (SAP)	 ? and	 ? EM:SAP.	 ? One-??way	 ?ANOVAs	 ? were	 ? performed	 ? separately	 ? for	 ? each	 ? window/functional	 ? group	 ?combination.	 ?P	 ?values	 ?<	 ?0.1	 ?are	 ?highlighted	 ?in	 ?bold.	 ? !!"#$%&''()#*+"%#,-'./%)0'123'%**)//4#*4'5)674/'%8'/4,$9':' !"# $%&'# $%'(##)*+# $%''# $%',##!"-)*+# $%,.# ;<;='>' !"# $%/.# ;<;='#)*+# $%&.# $%/'##!"-)*+# $%.0# ;<;?'@' !"# $%(1# $%2##)*+# $%(2# $%'2##!"-)*+# $%&2# $%,2#A' !"# $%&(# $%.1##)*+# $%//# ;<;?'#!"-)*+# $%2(# $%0(#B' !"# $%/2# $%&1 ##)*+# $%12# $%2,##!"-)*+# $%20# $%/0# 53  Figure	 ? 3.7:	 ? Boxplot	 ? of	 ? ectomycorrhizal	 ? (EM)	 ? to	 ? saprotrophic	 ? (SAP)	 ? fungal	 ? ratios	 ?based	 ?on	 ?number	 ?of	 ?fungal	 ?reads	 ?from	 ?high	 ?(H)	 ?and	 ?low	 ?(L)	 ?phosphatase	 ?areas	 ?from	 ?five	 ?(A-??E)	 ?root	 ?windows.	 ?(*)	 ?indicates	 ?p	 ?=	 ?0.05,	 ?while	 ?(?)	 ?indicates	 ?p	 ?=	 ?0.07	 ?between	 ?high	 ?and	 ?low	 ?phosphatase	 ?levels	 ?according	 ?to	 ?one-??way	 ?ANOVAs	 ?within	 ?the	 ?windows	 ?indicated.	 ?  54 Figure	 ?3.8:	 ?Boxplot	 ?of	 ?number	 ?of	 ?ectomycorrhizal	 ?(EM)	 ?fungal	 ?reads	 ?from	 ?high	 ?(H)	 ?and	 ?low	 ?(L)	 ?phosphatase	 ?areas	 ?from	 ?five	 ?(A-??E)	 ?root	 ?windows.	 ?(*)	 ?indicates	 ?p	 ?=	 ?0.05,	 ?between	 ?high	 ?and	 ?low	 ?phosphatase	 ?levels	 ?according	 ?to	 ?a	 ?one-??way	 ?ANOVA	 ?within	 ?the	 ?window	 ?indicated. Figure	 ?3.9:	 ?Boxplot	 ?of	 ?number	 ?of	 ?saprotrophic	 ?(SAP)	 ?fungal	 ?reads	 ?from	 ?high	 ?(H)	 ?and	 ?low	 ? (L)	 ? phosphatase	 ? areas	 ? from	 ? five	 ? (A-??E)	 ? root	 ? windows.	 ? (?)	 ? indicates	 ? p	 ? =	 ? 0.07,	 ?between	 ?high	 ?and	 ?low	 ?phosphatase	 ?levels	 ?according	 ?to	 ?a	 ?one-??way	 ?ANOVA	 ?within	 ?the	 ?window	 ?indicated.   55 3.4. DISCUSSION	 ?One	 ? of	 ? the	 ? current	 ? challenges	 ? in	 ? soil	 ? ecology	 ? is	 ? relating	 ? microbial	 ? community	 ?composition	 ?to	 ?specific	 ?ecosystem	 ?functions.	 ?The	 ?secretion	 ?of	 ?extracellular	 ?enzymes	 ?by	 ? soil	 ?microbes	 ? is	 ? a	 ? crucial	 ? step	 ? in	 ? the	 ? release	 ? of	 ?many	 ? nutrients	 ? that	 ?would	 ? be	 ?otherwise	 ? unavailable.	 ? Using	 ? the	 ? root	 ? window	 ? and	 ? soil	 ? imprinting	 ? approaches	 ?allowed	 ?me	 ?to	 ?specifically	 ?target	 ?areas	 ?of	 ?high	 ?enzyme	 ?activity	 ?in	 ?the	 ?soil	 ?profile,	 ?in	 ?situ,	 ?and	 ?associate	 ?those	 ?areas	 ?with	 ?fungal	 ?communities.	 ?My	 ?work	 ?showed	 ?that	 ?fine,	 ?mm-??scale	 ?phosphatase	 ?activities	 ?in	 ?soil	 ?were	 ?not	 ?associated	 ?with	 ?distinct	 ?groups	 ?of	 ?fungal	 ? taxa,	 ? but	 ? that	 ? the	 ? abundance	 ? and	 ? ratios	 ? of	 ? fungal	 ? functional	 ? groups	 ? varied	 ?with	 ? phosphatase	 ? activity	 ? in	 ? some	 ? windows.	 ? Across	 ? the	 ? site,	 ? however,	 ? fungal	 ?communities	 ?differed	 ?to	 ?a	 ?much	 ?greater	 ?extent	 ?between	 ?than	 ?within	 ?root	 ?windows.	 ?To	 ? my	 ? knowledge,	 ? this	 ? is	 ? the	 ? first	 ? study	 ? to	 ? investigate	 ? fungal	 ? communities	 ?associated	 ? with	 ? fine-??scale	 ? enzyme	 ? hotspots	 ? in	 ? soil	 ? using	 ? high-??throughput	 ?sequencing.	 ?3.4.1. Fungal	 ?communities	 ?and	 ?fine-??scale	 ?phosphatase	 ?activities	 ?Many	 ? studies	 ? have	 ? attempted	 ? to	 ? link	 ? enzyme	 ? activities	 ? in	 ? soil	 ? with	 ? changes	 ? in	 ?microbial	 ? taxa	 ? and	 ? communities.	 ? Both	 ? enzyme	 ? activity	 ? and	 ? microbial	 ? community	 ?composition	 ?are	 ?often	 ?strongly	 ? linked	 ? to	 ?substrate	 ?availability.	 ?For	 ?example,	 ?Lucas	 ?and	 ?Casper	 ?(2008)	 ?observed	 ?significant	 ?changes	 ?in	 ?EM	 ?fungal	 ?communities	 ?resulting	 ?from	 ?increased	 ?nitrogen	 ?availability.	 ?These	 ?changes	 ?were	 ?associated	 ?with	 ?increases	 ?in	 ? lignolytic	 ? enzymes	 ? and	 ? a	 ? decrease	 ? in	 ? phenol	 ? oxidase	 ? activity.	 ? Increases	 ? in	 ?available	 ?nitrogen	 ?also	 ?lead	 ?to	 ?reduced	 ?or	 ?altered	 ?fungal	 ?populations	 ?(Lilleskov	 ?et	 ?al.,	 ?2002,	 ? Dighton	 ? et	 ? al.,	 ? 2004;	 ? Frey	 ? et	 ? al.,	 ? 2004),	 ? as	 ? well	 ? as	 ? altered	 ? populations	 ? of	 ? 56 bacteria	 ?(Frey	 ?et	 ?al.,	 ?2004;	 ?Waldrop	 ?et	 ?al.,	 ?2004;	 ?Weand	 ?et	 ?al.,	 ?2010).	 ?Furthermore,	 ?phosphatase	 ?activities	 ?can	 ?be	 ?related	 ?to	 ?changes	 ?in	 ?substrate	 ?(Allison	 ?and	 ?Vitousek,	 ?2005),	 ?microbial	 ?community	 ?structure	 ?(Brooks,	 ?2010),	 ?and	 ?specific	 ?taxa	 ?(Conn	 ?and	 ?Dighton,	 ?2000;	 ?Nygren	 ?and	 ?Rosling,	 ?2009).	 ?I	 ?was	 ?intrigued,	 ?therefore,	 ?to	 ?find	 ?that	 ?the	 ?taxonomic	 ? composition	 ? of	 ? fungal	 ? communities	 ? in	 ? high	 ? and	 ? low	 ? phosphatase	 ?microsites	 ?was	 ?not	 ?different.	 ?	 ?There	 ? are	 ? several	 ? possible	 ? explanations	 ? for	 ? the	 ? lack	 ? of	 ? correlation	 ? between	 ?phosphatase	 ?activity	 ?and	 ?taxonomic	 ?composition	 ?of	 ?the	 ?fungal	 ?community.	 ?Fungi	 ?are	 ?remarkable	 ?in	 ?their	 ?ability	 ?to	 ?exhibit	 ?phenotypic	 ?plasticity,	 ?which	 ?is	 ?the	 ?ability	 ?of	 ?an	 ?organism	 ?to	 ?respond	 ?to	 ?environmental	 ?signals	 ?by	 ?altering	 ?morphology,	 ?physiological	 ?state,	 ?or	 ?behavior	 ?(West-??Eberhard,	 ?1989).	 ?Fungi	 ?are	 ?capable	 ?of	 ?producing	 ?a	 ?variety	 ?of	 ? extracellular	 ? enzymes,	 ? but	 ? whether	 ? or	 ? not	 ? they	 ? release	 ? them	 ? depends	 ? on	 ? the	 ?chemical	 ? properties	 ? of	 ? the	 ? surrounding	 ? medium.	 ? For	 ? example,	 ? the	 ? presence	 ? of	 ?glucose	 ?will	 ? inhibit	 ? the	 ? transcription	 ? of	 ? cellulase	 ? enzymes	 ? in	 ?A.	 ?niger	 ? (Hanif	 ? et	 ? al.,	 ?2004),	 ? and	 ? inorganic	 ? P	 ? concentrations	 ? regulate	 ? the	 ? transcription	 ? of	 ? secreted	 ?phosphatase	 ? genes	 ? in	 ?S.	 ?cerevisiae	 ? (Lenburg	 ? and	 ?O?Shea	 ?1996).	 ? Furthermore,	 ? both	 ?lab	 ?(Antibus	 ?et	 ?al.,	 ?1986;	 ?Baghel	 ?et	 ?al.,	 ?2009)	 ?and	 ?field	 ?studies	 ?(Ali	 ?et	 ?al.,	 ?2009;	 ?van	 ?Aerle	 ? and	 ? Plassard,	 ? 2010)	 ? have	 ? shown	 ? that	 ? fungi	 ? are	 ? capable	 ? of	 ? suppressing	 ? or	 ?inducing	 ?phosphatase	 ?activity	 ?depending	 ?on	 ?a	 ?variety	 ?of	 ?local	 ?nutrient,	 ?temperature,	 ?and	 ?pH	 ?conditions.	 ?The	 ?ability	 ?to	 ?produce	 ?extracellular	 ?phosphatase	 ?enzymes	 ?is	 ?not	 ?a	 ? rare	 ? trait	 ? among	 ? fungi,	 ? and	 ? has	 ? been	 ? observed	 ? among	 ? many	 ? different	 ? species	 ?(Alexander	 ?and	 ?Hardy,	 ?1981;	 ?Dighton,	 ?1983;	 ?Colpaert	 ?and	 ?van	 ?Laere,	 ?1996;	 ?Diedhiou	 ?et	 ?al.,	 ?2010).	 ?In	 ?Chapter	 ?2	 ?of	 ?this	 ?thesis,	 ?I	 ?presented	 ?evidence	 ?that	 ?high	 ?phosphatase	 ? 57 activities	 ? in	 ?mineral	 ?soils	 ?at	 ? this	 ?site	 ?occur	 ? in	 ?microsites	 ?with	 ?elevated	 ?carbon	 ?and	 ?nitrogen.	 ?These	 ?aspects	 ?of	 ?soil	 ?chemistry	 ?in	 ?high	 ?phosphatase	 ?microsites	 ?may	 ?have	 ?favoured	 ?the	 ?release	 ?of	 ?phosphatase	 ?enzymes	 ?by	 ?many	 ?fungal	 ?species.	 ?	 ?	 ?There	 ?may	 ?also	 ?have	 ?been	 ?some	 ?homogenizing	 ?of	 ?fungal	 ?taxa	 ?across	 ?soil	 ?microsites	 ?if	 ?DNA	 ?was	 ?extracted	 ?from	 ?fungal	 ?tissues	 ?not	 ?involved	 ?in	 ?nutrient	 ?release	 ?and	 ?uptake.	 ?Examples	 ? include	 ? exploratory	 ? hyphal	 ? strands	 ? that	 ? were	 ? passing	 ? through	 ? the	 ? soil,	 ?inactive	 ?hyphae,	 ?or	 ? fungal	 ?spores.	 ?For	 ?example,	 ?Amend	 ?et	 ?al.	 ? (2010)	 ?amplified	 ?and	 ?sequenced	 ? fungal	 ? spore	 ?DNA	 ? from	 ? air	 ? samples,	 ? and	 ?Hynes	 ? et	 ? al.	 ? (2010)	 ? amplified	 ?DNA	 ?from	 ?rhizomorphs	 ?colonizing	 ?mesh	 ?bags.	 ?	 ?Alternatively,	 ? high	 ? phosphatase	 ? areas	 ? could	 ? have	 ? also	 ? been	 ? areas	 ? of	 ? high	 ? fungal	 ?biomass	 ?relative	 ?to	 ?low	 ?phosphatase	 ?areas,	 ?without	 ?a	 ?change	 ?in	 ?species	 ?composition.	 ?Past	 ?studies	 ?have	 ?shown	 ?that	 ?phosphatase	 ?activity	 ?can	 ?be	 ?positively	 ?correlated	 ?with	 ?overall	 ?microbial	 ?biomass	 ?(Baldrian	 ?et	 ?al.,	 ?2010;	 ?Brocket	 ?et	 ?al.,	 ?2012).	 ?Because	 ?the	 ?samples	 ? were	 ? rarefied,	 ? and	 ? because	 ? read	 ? abundance	 ? is	 ? not	 ? a	 ? direct	 ? indicator	 ? of	 ?taxon	 ? abundance	 ? (Amend	 ? et	 ? al.	 ? 2010),	 ?we	 ? could	 ? not	 ? test	 ? for	 ? differences	 ? in	 ? fungal	 ?biomass	 ?with	 ?pyrosequencing.	 ?Because	 ?the	 ?soil	 ?samples	 ?were	 ?so	 ?small,	 ?I	 ?also	 ?could	 ?not	 ?subsample	 ?them	 ?to	 ?quantify	 ?fungal	 ?biomass	 ?on	 ?sub-??samples.	 ?Bacteria	 ? and	 ? archaea	 ? produce	 ? extracellular	 ? enzymes,	 ? including	 ? phosphatases,	 ?(Zimmerman	 ?et	 ?al.,	 ?2013)	 ?and,	 ?thus,	 ?high	 ?activities	 ?in	 ?soil	 ?may	 ?have	 ?been	 ?associated	 ?with	 ?these	 ?communities.	 ?Phosphatase-??producing	 ?bacteria	 ?have	 ?been	 ?well	 ?studied	 ?in	 ?agricultural	 ? soils	 ? in	 ? their	 ? role	 ? as	 ? plant	 ? growth	 ? promoting	 ? bacteria	 ? (Hayat	 ? et	 ? al.,	 ?2010),	 ?though	 ?their	 ?activity	 ?in	 ?forest	 ?soils	 ?is	 ?less	 ?clear.	 ?Although	 ?coniferous	 ?soils	 ?are	 ? 58 typically	 ? thought	 ? to	 ? be	 ? fungus-??dominated	 ? (Persson	 ? et	 ? al.,	 ? 1980),	 ? phosphatase	 ?activities	 ?in	 ?these	 ?soils	 ?can	 ?be	 ?correlated	 ?with	 ?bacterial	 ?numbers	 ?(Uroz	 ?et	 ?al.	 ?2013)	 ?or	 ? biomass	 ? (Baldrian	 ? et	 ? al.,	 ? 2010).	 ? Uroz	 ? et	 ? al.	 ? (2013)	 ? found	 ? that	 ? bacterial	 ?contribution	 ? to	 ? phosphatase	 ? activity	 ?was	 ?most	 ? apparent	 ? in	 ? conifer	 ? soils	 ?with	 ? low	 ?fungal	 ? to	 ? bacterial	 ? ratios,	 ? possibly	 ? because	 ? fungal	 ? hyphae	 ? can	 ? select	 ? against	 ?phosphatase	 ? releasing-??bacteria	 ? (Brooks	 ? et	 ? al.,	 ? 2011).	 ? I	 ? did	 ? not	 ? evaluate	 ? the	 ?contribution	 ?of	 ? bacteria	 ? and	 ?archaea	 ? to	 ?phosphatase	 ? activity	 ? in	 ?my	 ? study,	 ? but	 ? if	 ? it	 ?was	 ?substantial,	 ?then	 ?this	 ?could	 ?explain	 ?a	 ?variation	 ?in	 ?imprintable	 ?activity	 ?that	 ?was	 ?not	 ?related	 ?to	 ?a	 ?change	 ?in	 ?the	 ?taxonomic	 ?structure	 ?of	 ?the	 ?fungal	 ?community.	 ?Because	 ?my	 ?root	 ?windows	 ?were	 ?small,	 ?and	 ?because	 ?of	 ?the	 ?inherent	 ?heterogeneity	 ?of	 ?soil	 ? profiles,	 ? I	 ? was	 ? limited	 ? to	 ? sampling	 ? only	 ? three	 ? to	 ? five	 ? replicate	 ? samples	 ? per	 ?phosphatase	 ? treatment	 ? per	 ?window.	 ? Low	 ? sample	 ? size	 ? can	 ? result	 ? in	 ? higher	 ? sample	 ?variance,	 ?lower	 ?statistical	 ?power	 ?and	 ?thus,	 ?an	 ?increased	 ?type	 ?II	 ?error	 ?rate	 ?(failure	 ?to	 ?reject	 ?a	 ?false	 ?null	 ?hypothesis)(Peterman,	 ?1990).	 ?It	 ? is	 ?also	 ?possible	 ?that	 ?my	 ?samples	 ?taken	 ? from	 ? high	 ? and	 ? low	 ? phosphatase	 ? microsites	 ? were	 ? physically	 ? too	 ? large	 ? to	 ?capture	 ?differences	 ?in	 ?fungal	 ?communities.	 ?	 ?This	 ?seems	 ?unlikely,	 ?however,	 ?given	 ?that	 ?Brooks	 ? (2010)	 ? detected	 ? differences	 ? in	 ? terminal	 ? restriction	 ? fragment	 ? length	 ?polymorphism	 ?(T-??RFLP)	 ?fingerprints	 ?of	 ?the	 ?general	 ?fungal	 ?community	 ?between	 ?high	 ?and	 ?low	 ?phosphatase	 ?microsites	 ?sampled	 ?in	 ?the	 ?same	 ?way	 ?at	 ?similar	 ?forest	 ?sites.	 ?3.4.2. Ectomycorrhizal	 ?and	 ?saprotrophic	 ?fungi	 ?Several	 ?studies	 ?have	 ?shown	 ?that	 ?EM	 ?fungi	 ?tend	 ?to	 ?dominate	 ?deeper	 ?layers	 ?of	 ?the	 ?soil	 ?profile,	 ? while	 ? SAP	 ? fungi	 ? are	 ? confined	 ? mostly	 ? to	 ? upper	 ? litter	 ? layers,	 ? though	 ? their	 ? 59 ranges	 ? do	 ? overlap	 ? (Lindahl	 ? et	 ? al.,	 ? 2007;	 ? Clemmensen	 ? et	 ? al.,	 ? 2013).	 ? Saprotrophic	 ?fungi	 ?are	 ?generally	 ?better	 ?able	 ?than	 ?EM	 ?fungi	 ?to	 ?utilize	 ?complex	 ?carbon	 ?sources,	 ?such	 ?as	 ? lignin,	 ? found	 ? in	 ?surface	 ? litter	 ? (Rayner	 ?and	 ?Boddy,	 ?1998)	 ?because	 ? their	 ?genomes	 ?code	 ?for	 ?a	 ?wider	 ?range	 ?of	 ?oxidative	 ?enzymes	 ?(Floudas	 ?et	 ?al.	 ?2012).	 ?Mycorrhizal	 ?fungi	 ?receive	 ? their	 ? carbon	 ? from	 ? their	 ? host	 ? plants	 ? and	 ? do	 ? not	 ? depend	 ? on	 ? litter-??derived	 ?energy.	 ?Thus	 ? it	 ? is	 ?not	 ?surprising	 ?that	 ?my	 ?samples	 ?were	 ?dominated	 ?by	 ?EM	 ?fungi,	 ?as	 ?sampling	 ?occurred	 ?in	 ?the	 ?mineral	 ?soil.	 ?Nevertheless,	 ? EM	 ? and	 ? SAP	 ? fungi	 ? can	 ? co-??occur	 ? in	 ? the	 ? same	 ? soil	 ? layers	 ? and	 ? utilize	 ?many	 ?of	 ?the	 ?same	 ?substrates,	 ?so	 ?one	 ?would	 ?expect	 ?competitive	 ?interactions	 ?between	 ?these	 ? functional	 ? groups	 ? (Shaw	 ? et	 ? al.,	 ? 1995;	 ? Lindahl	 ? et	 ? al.,	 ? 2007;	 ? Zadworny	 ? et	 ? al.,	 ?2007).	 ? Based	 ? on	 ? the	 ? results	 ? of	 ? Brooks	 ? (2010),	 ? who	 ? found	 ? that	 ? high	 ? phosphatase	 ?microsites	 ?were	 ?associated	 ?with	 ?a	 ?lower	 ?EM	 ?fungal	 ?richness,	 ?as	 ?well	 ?as	 ?my	 ?findings	 ?(Chapter	 ?2)	 ? that	 ?high	 ?phosphatase	 ?sites	 ?are	 ?higher	 ? in	 ?C	 ?and	 ?N,	 ? I	 ?hypothesized	 ?that	 ?high	 ? phosphatase	 ? microsites	 ? would	 ? be	 ? areas	 ? of	 ? high	 ? SAP	 ? richness	 ? and	 ? relative	 ?abundance,	 ?relative	 ?to	 ? low	 ?phosphatase	 ?microsites.	 ?Although	 ?I	 ? found	 ?no	 ? indication	 ?of	 ?greater	 ?overall	 ?abundance	 ?of	 ?SAP	 ?fungi	 ?in	 ?high	 ?phosphatase	 ?sites,	 ?and	 ?indeed	 ?SAP	 ?read	 ? abundance	 ? was	 ? lower	 ? in	 ? high	 ? phosphatase	 ? sites	 ? at	 ? one	 ? window,	 ? I	 ? detected	 ?higher	 ? ratios	 ? of	 ? SAP:EM	 ? sequences	 ? at	 ? high-??phosphatase	 ? sites	 ? in	 ? two	 ? of	 ? the	 ? five	 ?windows.	 ? Saprotrophic	 ? fungi	 ? have	 ? a	 ? larger	 ? set	 ? of	 ? cell-??wall	 ? degrading	 ? enzymes	 ?(Martin	 ?et	 ?al.,	 ?2008;	 ?Nagendran	 ?et	 ?al.,	 ?2009;	 ?Floudas	 ?et	 ?al.	 ?2012),	 ?a	 ?greater	 ?capacity	 ?to	 ? degrade	 ? C-??rich	 ? litter	 ? (Colpaert	 ? and	 ? van	 ? Tichelen,	 ? 1996),	 ? and	 ? are	 ? the	 ? principal	 ?decomposers	 ?of	 ? lignin	 ?and	 ?cellulose	 ? in	 ? forests	 ?(Eriksson	 ?et	 ?al.,	 ?1990).	 ?Thus,	 ?higher	 ?SAP:EM	 ?may	 ?be	 ?expected	 ?in	 ?nutrient-??rich	 ?patches	 ?high	 ?in	 ?soil	 ?organic	 ?matter	 ?or	 ?high	 ? 60 C:N.	 ?In	 ? three	 ? of	 ? the	 ? five	 ? windows,	 ? there	 ? was	 ? no	 ? difference	 ? in	 ? SAP:EM	 ? fungal	 ? ratio	 ?between	 ?microsites	 ? differing	 ? in	 ? imprintable	 ? phosphatase	 ? activity.	 ? 	 ? Like	 ?my	 ? study,	 ?Brooks	 ?(2010)	 ?found	 ?differences	 ?in	 ?communities	 ?between	 ?microsites	 ?in	 ?about	 ?half	 ?of	 ?sampled	 ? windows	 ? (12	 ? windows).	 ? 	 ? In	 ? that	 ? case,	 ? a	 ? lower	 ? number	 ? of	 ? EM	 ? fungal	 ?signatures	 ? (equivalent	 ? to	 ? species	 ? richness)	 ? was	 ? detected	 ? in	 ? high	 ? than	 ? low	 ?phosphatase	 ?microsites	 ? in	 ? the	 ?mineral	 ? soil	 ? layers	 ? of	 ?windows	 ? from	 ? the	 ? two	 ? older	 ?stand-??age	 ? classes	 ? (>	 ? 65	 ? years	 ? old),	 ? but	 ? there	 ? was	 ? considerable	 ? variation	 ? from	 ?window	 ?to	 ?window.	 ?Such	 ?variation	 ? from	 ?window	 ?to	 ?window	 ? in	 ? the	 ?relationship	 ?of	 ?functional	 ? groups	 ? with	 ? phosphatase	 ? activity	 ? could	 ? be	 ? a	 ? result	 ? of	 ? priority	 ? effects,	 ?where	 ? the	 ? first	 ? fungus	 ? to	 ? colonize	 ?a	 ?nutrient-??rich	 ?patch	 ?will	 ? competitively	 ?exclude	 ?other	 ?fungi	 ?from	 ?colonizing	 ?the	 ?same	 ?patch.	 ?Furthermore,	 ?nutrient	 ?patches	 ?between	 ?windows	 ?may	 ? have	 ? slightly	 ? different	 ? soil	 ? chemistries,	 ? which	 ? could	 ? favor	 ? different	 ?fungal	 ? communities.	 ? Interestingly	 ? though,	 ? a	 ? pattern	 ?was	 ? observed	 ?with	 ? respect	 ? to	 ?which	 ?windows	 ?exhibited	 ?higher	 ?SAP:EM	 ?ratios	 ? in	 ?high	 ?phosphatase	 ? spots.	 ? 	 ?Those	 ?two	 ? windows,	 ? Windows	 ? A	 ? and	 ? B,	 ? also	 ? had	 ? significantly	 ? lower	 ? overall	 ? EM	 ? fungal	 ?richness	 ? than	 ? the	 ? other	 ? windows.	 ? The	 ? higher	 ? diversity	 ? of	 ? EM	 ? fungi	 ? in	 ? the	 ? other	 ?windows	 ?may	 ?have	 ?given	 ?a	 ?greater	 ?opportunity	 ?for	 ?EM	 ?fungi	 ?to	 ?successfully	 ?compete	 ?with	 ?SAP	 ?fungi	 ?in	 ?nutrient-??rich	 ?patches.	 ?	 ?The	 ? window	 ? that	 ? was	 ? most	 ? obviously	 ? different	 ? from	 ? the	 ? others	 ? in	 ? terms	 ? of	 ?functional	 ?groups	 ?was	 ?Window	 ?C.	 ?This	 ?window	 ?had	 ?the	 ?highest	 ?number	 ?of	 ?EM	 ?fungal	 ?reads,	 ?lowest	 ?SAP	 ?OTU	 ?richness	 ?and	 ?highest	 ?ratio	 ?of	 ?EM:SAP,	 ?both	 ?in	 ?terms	 ?of	 ?read	 ? 61 abundance	 ?and	 ?number	 ?of	 ?OTUs.	 ?	 ?This	 ?was	 ?the	 ?only	 ?window	 ?that	 ?had	 ?EM	 ?roots	 ?and	 ?associated	 ?hyphae	 ?profusely	 ?visible	 ?across	 ?the	 ?window	 ?(Figure	 ?A1).	 ?These	 ?clusters	 ?of	 ?hyphae	 ?were	 ?associated	 ?with	 ?many	 ?of	 ?the	 ?phosphatase	 ?hot	 ?spots	 ?on	 ?the	 ?imprint,	 ?so	 ? even	 ? though	 ? sampling	 ? was	 ? random	 ? among	 ? high	 ? phosphatase	 ? spots,	 ? it	 ? was	 ?inevitable	 ? that	 ? a	 ? high	 ? number	 ? of	 ? EM	 ? fungal	 ? sequences	 ? were	 ? present	 ? in	 ? these	 ?samples.	 ?	 ?This	 ?might	 ?explain	 ?the	 ?tendency	 ?for	 ?the	 ?EM:SAP	 ?ratio	 ?to	 ?show	 ?the	 ?opposite	 ?pattern	 ? with	 ? respect	 ? to	 ? phosphatase	 ? microsite	 ? compared	 ? to	 ? other	 ? windows.	 ?Regardless,	 ? the	 ?EM:SAP	 ? ratio	 ?was	 ?not	 ? significantly	 ? different	 ? between	 ?phosphatase	 ?microsites	 ?from	 ?this	 ?window	 ?(p=0.19).	 ?	 ?3.4.3. Differences	 ?in	 ?fungal	 ?communities	 ?between	 ?windows	 ?	 ?The	 ?factor	 ?that	 ?appeared	 ?to	 ?be	 ?most	 ?important	 ?in	 ?separating	 ?fungal	 ?communities	 ?in	 ?this	 ?study	 ?was	 ?sample	 ?window.	 ?The	 ?windows	 ?were	 ?located	 ?5-??15	 ?m	 ?apart	 ?and,	 ?hence,	 ?my	 ? results	 ? are	 ? consistent	 ?with	 ? studies	 ? showing	 ? that	 ? taxa	 ? and	 ? communities	 ? of	 ? EM	 ?fungi	 ?tend	 ?to	 ?be	 ?autocorrelated	 ?when	 ?sampled	 ?under	 ?distances	 ?of	 ?3-??4	 ?m	 ?(Lilleskov	 ?et	 ?al.	 ?2004;	 ?Pickles	 ?et	 ?al.,	 ?2010;	 ?Pickles	 ?et	 ?al.	 ?2012).	 ?This	 ?could	 ?be	 ?related	 ?to	 ?the	 ?size	 ?of	 ?an	 ? individual	 ? species	 ? or	 ? genet,	 ? or	 ? the	 ? distribution	 ? of	 ? fine	 ? roots	 ? of	 ? their	 ? hosts.	 ? For	 ?example,	 ?genets	 ?of	 ?Laccaria	 ?amethystina	 ?were	 ?found	 ?to	 ?be	 ?no	 ?larger	 ?than	 ?1.3	 ?m	 ?in	 ?a	 ?northern	 ?temperate	 ?forest	 ?(Hortal	 ?et	 ?al.,	 ?2012),	 ?and	 ?genets	 ?of	 ?Russula	 ?brevipes	 ?were	 ?less	 ?than	 ?3	 ?m	 ?on	 ?average	 ?in	 ?a	 ?coniferous	 ?forest	 ?(Bergemann	 ?and	 ?Miller	 ?2002).	 ?Genets	 ?of	 ?other	 ?species,	 ?such	 ?as	 ?Rhizopogon	 ?vinicolar	 ?and	 ?R.	 ?vesiculosus,	 ?which	 ?are	 ?common	 ?EM	 ?symbionts	 ?in	 ?these	 ?stands	 ?(Twieg	 ?et	 ?al.	 ?2007),	 ?can	 ?have	 ?genets	 ?exceeding	 ?10	 ?m	 ?in	 ?diameter	 ?(Beiler	 ?et	 ?al.	 ?2012).	 ?The	 ?windows	 ?were	 ?arranged,	 ?in	 ?order	 ?from	 ?Window	 ?A	 ?to	 ?Window	 ?E,	 ?down	 ?a	 ?slope,	 ?and	 ?there	 ?did	 ?appear	 ?to	 ?be	 ?more	 ?similarity	 ?in	 ?fungal	 ? 62 communities	 ? between	 ? Windows	 ? A	 ? and	 ? B	 ? and	 ? among	 ? Windows	 ? C	 ? through	 ? E.	 ?Differences	 ? in	 ? fungal	 ? communities	 ? between	 ? windows	 ? could,	 ? therefore,	 ? reflect	 ?differences	 ?in	 ?root	 ?abundance	 ?and	 ?identity,	 ?or	 ?edaphic	 ?conditions	 ?(Sato	 ?et	 ?al.,	 ?2012;	 ?Jarvis	 ? et	 ? al.	 ? 2013).	 ? Priority	 ? effects	 ? could	 ? have	 ? also	 ? played	 ? a	 ? role	 ? in	 ? shaping	 ?community	 ?structure	 ?between	 ?windows.	 ?For	 ?example,	 ?Fukami	 ?et	 ?al.	 ?(2010)	 ?showed	 ?that	 ? immigration	 ? history,	 ? regardless	 ? of	 ? nitrogen	 ? availability,	 ? strongly	 ? affected	 ?species	 ?richness	 ?and	 ?community	 ?structure	 ?of	 ?wood	 ?decaying	 ?fungi.	 ?Furthermore,	 ?the	 ?ability	 ?of	 ?an	 ?EM	 ?fungus	 ?to	 ?first	 ?colonize	 ?a	 ?root	 ?tip	 ?can	 ?lead	 ?to	 ?competitive	 ?exclusion	 ?of	 ?other	 ?EM	 ?fungi	 ?colonizing	 ?the	 ?same	 ?root	 ?tip	 ?(Kennedy	 ?et	 ?al.,	 ?2009).	 ?3.4.4. The	 ?fungal	 ?community	 ?detected	 ?by	 ?454	 ?pyrosequencing	 ?	 ?By	 ? using	 ? pyrosequencing,	 ? I	 ? was	 ? able	 ? to	 ? rapidly	 ? assess	 ? fungal	 ? community	 ?composition	 ? among	 ? high	 ? and	 ? low	 ? phosphatase	 ? microsites.	 ? After	 ? sequence	 ?processing,	 ?I	 ?was	 ?able	 ?to	 ?detect	 ?90-??155	 ?OTUs	 ?per	 ?sample.	 ?This	 ?number	 ?is	 ?relatively	 ?low	 ? compared	 ? to	 ? other	 ? studies	 ? examining	 ? fungal	 ? communities	 ? in	 ? soil	 ? with	 ?pyrosequencing	 ? (Bu?e	 ?et	 ? al.,	 ? 2009;	 ?Hartmann	 ?et	 ? al.,	 ? 2012),	 ? and	 ?may	 ?be	 ?due	 ? to	 ?my	 ?very	 ? small	 ? sample	 ? size.	 ? Furthermore,	 ? the	 ? average	 ? length	 ? of	 ? my	 ? ITS1	 ? sequences,	 ?316.6	 ?bp,	 ?was	 ?sufficient	 ?for	 ?identification	 ?of	 ?fungi	 ?at	 ?the	 ?genus	 ?level	 ?(Jumpponen	 ?et	 ?al.,	 ? 2010;	 ? Arfi	 ? et	 ? al.,	 ? 2012).	 ? Using	 ? T-??RFLP	 ? to	 ? characterize	 ? EM	 ? fungal	 ? hyphae	 ? from	 ?high	 ?and	 ?low	 ?phosphatase	 ?microsites	 ?from	 ?root	 ?windows,	 ?Brooks	 ?(2010)	 ?identified	 ?a	 ?maximum	 ? of	 ? 37	 ? EM	 ? fungal	 ? OTUs	 ? per	 ?microsite,	 ? with	 ? an	 ? average	 ? of	 ? 11	 ? EM	 ? fungal	 ?OTUs	 ?per	 ?microsite.	 ?Also,	 ?previous	 ?sequencing	 ?of	 ?EM	 ?fungal	 ?root	 ?tips	 ? from	 ?my	 ?site	 ?yielded	 ?66	 ?unique	 ?EM	 ?fungal	 ?OTUs	 ?(Twieg,	 ?2006).	 ?Using	 ?pyrosequencing,	 ?I	 ?found	 ?a	 ?maximum	 ?of	 ?71	 ?EM	 ?fungal	 ?OTUs	 ?per	 ?microsite,	 ?an	 ?average	 ?of	 ?43	 ?EM	 ?fungal	 ?OTUs	 ?per	 ? 63 microsite,	 ? and	 ? a	 ? total	 ? of	 ? 290	 ? unique	 ?EM	 ? fungal	 ?OTUs	 ? in	 ? total.	 ? Thus	 ? I	 ? uncovered	 ? a	 ?much	 ? larger	 ?EM	 ? fungal	 ? community	 ? than	 ? those	 ? found	 ?by	 ?Brooks	 ? (2010)	 ?and	 ?Twieg	 ?(2006).	 ?	 ? 	 ?	 ?Although	 ?pyrosequencing	 ? is	 ?a	 ?powerful	 ? tool	 ? in	 ?assessing	 ?microbial	 ?diversity,	 ? there	 ?are	 ?several	 ?methodological	 ?issues.	 ?Ten	 ?percent	 ?of	 ?my	 ?sequences	 ?were	 ?of	 ?low	 ?quality,	 ?including	 ? homopolymer	 ? runs	 ? exceeding	 ? 8	 ? and	 ? sequences	 ? under	 ? 100	 ? bp.	 ? These	 ?sequences	 ? were	 ? subsequently	 ? removed	 ? from	 ? the	 ? dataset	 ? during	 ? quality	 ? filtering.	 ?Furthermore,	 ? pyrosequencing	 ? relies	 ? on	 ? a	 ? critical	 ? clustering	 ? step,	 ? which	 ? clusters	 ?sequences	 ? into	 ? OTUs.	 ? There	 ? are	 ? a	 ? large	 ? number	 ? of	 ? clustering	 ? algorithms,	 ? yet	 ? no	 ?consensus	 ?on	 ?which	 ?algorithm	 ?is	 ?the	 ?most	 ?suitable.	 ?I	 ?compared	 ?four	 ?algorithms	 ?with	 ?my	 ? dataset:	 ? CD-??HIT	 ? (Li	 ? and	 ? Godzik,	 ? 2006),	 ? BLAST	 ? (Schloss	 ? et	 ? al.,	 ? 2009),	 ? uclust	 ?(Edgar,	 ? 2010),	 ? and	 ? usearch	 ? (Edgar,	 ? 2010).	 ? I	 ? chose	 ? CD-??HIT,	 ? because	 ? it	 ? resulted	 ? in	 ?fewer	 ?OTUs	 ?with	 ?unknown	 ?identification	 ?or	 ?no	 ?BLAST	 ?hits.	 ?Finally,	 ?read	 ?abundance	 ?can	 ? be	 ? a	 ? poor	 ? indicator	 ? of	 ? overall	 ? taxa	 ? abundance,	 ? particularly	 ? when	 ? comparing	 ?between	 ? species	 ? (Amend	 ? et	 ? al.,	 ? 2010,	 ? Engelbrekston	 ? et	 ? al.,	 ? 2010).	 ? This	 ? is	 ? because	 ?PCR	 ? bias	 ?may	 ? prefer	 ? the	 ? amplification	 ? of	 ? some	 ? taxa,	 ? and	 ? discriminate	 ? against	 ? the	 ?amplification	 ?of	 ?others.	 ?(Kanagawa,	 ?2003).	 ?Furthermore,	 ?different	 ?species	 ?can	 ?have	 ?different	 ? copy	 ?numbers	 ? of	 ? the	 ? target	 ? gene,	 ? leading	 ? to	 ?preferential	 ? amplification	 ?of	 ?certain	 ? species	 ? (van	 ? Wintzingerode	 ? et	 ? al.,	 ? 1997;	 ? Amend	 ? et	 ? al.,	 ? 2010).	 ? Relative	 ?abundance	 ? is	 ? still	 ?being	 ?used	 ?as	 ?a	 ?proxy	 ? for	 ?microbial	 ?abundance	 ? (Clemmensen	 ?et	 ?al.,	 ?2013;	 ?Coince	 ?et	 ?al.,	 ?2013;	 ?Uroz	 ?et	 ?al.,	 ?2013),	 ?although	 ?occurrence-??based	 ?estimates	 ?of	 ? community	 ? diversity	 ? may	 ? be	 ? more	 ? powerful	 ? than	 ? abundance	 ? when	 ? examining	 ?pyrosequencing	 ?data.  64 4. CONCLUSION	 ?4.1. GENERAL	 ?DISCUSSION	 ? 	 ?Phosphatase	 ? plays	 ? an	 ? important	 ? role	 ? in	 ? the	 ? breakdown	 ? of	 ? organic	 ? phosphorus	 ? in	 ?forest	 ?soils.	 ?Past	 ?studies	 ?have	 ?examined	 ?how	 ?nutrient	 ?conditions	 ?affect	 ?phosphatase	 ?activities	 ? in	 ? bulk	 ? soil	 ? (Turner	 ? and	 ? Haygarth,	 ? 2005;	 ? Allison	 ? and	 ? Vistousek,	 ? 2005;	 ?Hernandez	 ?and	 ?Hobbie,	 ?2010;	 ?Burke	 ?et	 ?al	 ?2012),	 ?but	 ?almost	 ?no	 ?work	 ?has	 ?been	 ?done	 ?at	 ?fine	 ?scales.	 ?In	 ?addition	 ?to	 ?the	 ?previous	 ?work	 ?of	 ?Brooks	 ?(2010),	 ?the	 ?examination	 ?of	 ?phosphatase	 ? activitiy	 ? in	 ? situ,	 ? at	 ? fine	 ? mm-??scales	 ? is	 ? a	 ? major	 ? contribution	 ? of	 ? the	 ?research	 ? presented	 ? in	 ? this	 ? thesis.	 ? Furthermore,	 ? although	 ? Brooks	 ? (2010)	 ? found	 ? a	 ?lower	 ? richness	 ? of	 ? EM	 ? fungal	 ? taxa	 ? in	 ? high	 ? rather	 ? than	 ? low	 ?phosphatase	 ?microsites,	 ?overall	 ? fungal	 ? communities	 ? associated	 ? with	 ? these	 ? areas	 ? still	 ? remain	 ? largely	 ?uncharacterized.	 ?Using	 ?the	 ?root	 ?window	 ?approach	 ?developed	 ?by	 ?Dong	 ?et	 ?al.	 ?(2007),	 ?I	 ?was	 ?able	 ?to	 ?investigate	 ?both	 ?the	 ?nutrient	 ?status	 ?and	 ?fungal	 ?communities	 ?associated	 ?with	 ? fine-??scale	 ? phosphatase	 ? activities	 ? in	 ? soil	 ? microsites	 ? from	 ? a	 ?mixed	 ? Douglas-??fir	 ?(Pseudotsuga	 ?menziesii	 ?(Mirb.)	 ? Franco)	 ? and	 ? paper	 ? birch	 ? (Betula	 ?papyrifera	 ?Marsh)	 ?stand	 ?in	 ?the	 ?southern	 ?interior	 ?of	 ?British	 ?Columbia.	 ?Two	 ?objectives	 ?were	 ?addressed	 ?in	 ?Chapter	 ?2.	 ?The	 ?first	 ?was	 ?to	 ?compare	 ?soil	 ?nutrient	 ?status	 ? in	 ? high	 ? and	 ? low	 ? phosphatase	 ?microsites.	 ? I	 ? predicted	 ? that	 ? high	 ? phosphatase	 ?activities	 ?would	 ?be	 ?associated	 ?with	 ?microsites	 ?high	 ?in	 ?C	 ?and	 ?N,	 ?and	 ?low	 ?in	 ?inorganic	 ?P.	 ?My	 ?results	 ?indicate	 ?that	 ?high	 ?phosphatase	 ?microsites	 ?were	 ?associated	 ?with	 ?higher	 ?percent	 ?total	 ?carbon	 ?and	 ?nitrogen,	 ?and	 ?marginally	 ?higher	 ?C:N	 ?than	 ?low	 ?phosphatase	 ?microsites.	 ?However,	 ?levels	 ?of	 ?organic,	 ?inorganic,	 ?and	 ?total	 ?phosphorus	 ?were	 ?similar.	 ? 65 The	 ?second	 ?objective	 ?was	 ?to	 ?test	 ?whether	 ?the	 ?addition	 ?of	 ?nutrients	 ?to	 ?soil	 ?profiles	 ?in	 ?situ	 ? would	 ? influence	 ? phosphatase	 ? activity.	 ? I	 ? predicted	 ? that	 ? additions	 ? of	 ? C	 ? and	 ? N	 ?would	 ?stimulate	 ?microbes	 ?to	 ?produce	 ?phosphatase.	 ?I	 ?found	 ?that	 ?additions	 ?of	 ?labile	 ?C	 ?and	 ?N,	 ? alone	 ? and	 ? in	 ? combination,	 ? did	 ?not	 ? stimulate	 ? activity.	 ?However,	 ? a	 ? follow-??up	 ?study	 ?in	 ?our	 ?lab	 ?found	 ?that	 ?additions	 ?of	 ?simple	 ?and	 ?complex	 ?C	 ?and	 ?N	 ?did	 ?stimulate	 ?activity.	 ?	 ?I	 ?concluded	 ?that	 ?phosphatase	 ?activity	 ?is	 ?affected	 ?by	 ?the	 ?availability	 ?of	 ?C	 ?and	 ?N	 ?in	 ?my	 ?sites.	 ?	 ?Chapter	 ?3	 ? addressed	 ?a	 ? third	 ?objective:	 ? to	 ? characterize	 ? fungal	 ? communities	 ? in	 ?high	 ?and	 ? low	 ?phosphatase	 ?microsites	 ?using	 ?pyrosequencing.	 ?First,	 ? I	 ?predicted	 ? that	 ?high	 ?phosphatase	 ?microsites	 ?would	 ?be	 ?associated	 ?with	 ?different	 ?fungal	 ?communities	 ?than	 ?low	 ? phosphatase	 ? microsites.	 ? My	 ? results	 ? indicate	 ? that,	 ? when	 ? examined	 ? as	 ?assemblages	 ?of	 ?OTUs,	 ?fungal	 ?communities	 ?were	 ?not	 ?different	 ?between	 ?high	 ?and	 ?low	 ?phosphatase	 ? microsites,	 ? though	 ? communities	 ? did	 ? differ	 ? between	 ? windows.	 ?However,	 ? the	 ?number	 ?of	 ? sequences	 ?as	 ?OTUs	 ?grouped	 ?by	 ? functional	 ? group	 ?differed	 ?between	 ? microsites	 ? in	 ? some	 ? windows.	 ? I	 ? had	 ? predicted	 ? that	 ? high	 ? phosphatase	 ?microsites	 ?would	 ?have	 ?lower	 ?richness	 ?of	 ?EM	 ?fungi	 ?than	 ?low	 ?phosphatase	 ?microsites.	 ?I	 ? found	 ? that,	 ? across	 ? all	 ?windows,	 ? the	 ? richness	 ?of	 ?EM	 ? fungal	 ? taxa	 ?was	 ?not	 ?different	 ?between	 ?high	 ?and	 ?low	 ?phosphatase	 ?microsites.	 ?Third,	 ? I	 ?predicted	 ?that	 ?the	 ?richness	 ?and	 ?abundance	 ?of	 ?SAP	 ? fungal	 ? taxa	 ?would	 ?be	 ?greater	 ? in	 ?high	 ? than	 ? low	 ?phosphatase	 ?microsites.	 ? I	 ? did	 ?not	 ? find	 ?differences	 ? in	 ? the	 ? richness	 ? and	 ?abundance	 ?of	 ? SAP	 ? fungal	 ?taxa	 ? between	 ? high	 ? and	 ? low	 ? phosphatase	 ?microsites	 ? across	 ? all	 ?windows;	 ? however,	 ?the	 ? abundance	 ? of	 ? SAP	 ? fungal	 ? taxa	 ? was	 ? higher	 ? in	 ? low	 ? than	 ? high	 ? phosphatase	 ?microsites	 ? in	 ? one	 ? window.	 ? Lastly,	 ? I	 ? predicted	 ? that	 ? the	 ? ratio	 ? of	 ? SAP	 ? to	 ? EM	 ? fungal	 ? 66 sequences	 ? would	 ? be	 ? higher	 ? in	 ? high	 ? than	 ? low	 ? phosphatase	 ? microsites.	 ? My	 ? results	 ?indicate	 ? that	 ? the	 ? SAP:EM	 ? was	 ? different	 ? between	 ? high	 ? and	 ? low	 ? phosphatase	 ?microsites	 ?across	 ?all	 ?windows,	 ?but	 ?varied	 ?from	 ?window	 ?to	 ?window.	 ?Specifically,	 ?the	 ?SAP:EM	 ?based	 ?on	 ?number	 ?of	 ? sequences	 ?was	 ?higher	 ? in	 ?high	 ? than	 ? low	 ?microsites	 ? in	 ?windows	 ? A	 ? and	 ? B.	 ? These	 ? two	 ? windows	 ? had	 ? significantly	 ? low	 ? EM	 ? OTU	 ? richness.	 ?	 ?Therefore	 ? I	 ? concluded	 ? that	 ? the	 ? relative	 ? ability	 ? of	 ? EM	 ? and	 ? SAP	 ? fungi	 ? to	 ? colonize	 ?organic	 ?microsites	 ? in	 ? soil	 ?depends	 ?on	 ? the	 ?number	 ?of	 ?EM	 ? fungal	 ? species	 ?present.	 ? 	 ? I	 ?hypothesize	 ? that	 ?some	 ?EM	 ?species	 ?are	 ?better	 ?able	 ? to	 ?compete	 ?with	 ?SAP	 ? fungi	 ? than	 ?others.	 ?4.2. STRENGTHS	 ?AND	 ?WEAKNESSES	 ? 	 ?Soil	 ? is	 ? a	 ? highly	 ? heterogeneous	 ? medium,	 ? where	 ? dynamic	 ? fine-??scale	 ? microsite	 ?processes	 ? are	 ? important	 ? for	 ? regulating	 ?whole-??soil	 ? processes	 ? (Schimel	 ? and	 ?Bennet,	 ?2004).	 ? The	 ? mineralization	 ? of	 ? organic	 ? nutrients	 ? depends	 ? on	 ? individual	 ? microsite	 ?characteristics,	 ? such	 ? as	 ? nutrient	 ? availability	 ? or	 ?microbial	 ? community	 ? composition,	 ?which	 ?can	 ?vary	 ?throughout	 ?the	 ?soil	 ?from	 ?microsite	 ?to	 ?microsite.	 ?A	 ?major	 ?strength	 ?of	 ?this	 ? investigation	 ? was	 ? using	 ? the	 ? enzyme	 ? imprinting	 ? method	 ? (Dong	 ? et	 ? al.	 ? 2007)	 ?combined	 ? with	 ? targeted	 ? sampling	 ? (Brooks,	 ? 2010),	 ? which	 ? allowed	 ? me	 ? to	 ? detect	 ?significant	 ? fine	 ?mm-??scale	 ? differences	 ? in	 ? soil	 ? nutrient	 ? status	 ? between	 ?high	 ? and	 ? low	 ?phosphatase	 ?microsites.	 ?	 ?Another	 ?strength	 ?of	 ?this	 ?investigation	 ?was	 ?the	 ?use	 ?of	 ?high	 ?throughput	 ?sequencing.	 ?I	 ?was	 ?able	 ?to	 ?uncover	 ?a	 ?much	 ?larger	 ?diversity	 ?of	 ? fungi	 ? in	 ?these	 ?sites	 ?than	 ?previously	 ?described	 ?by	 ?Brooks	 ?(2010)	 ?using	 ?TRFLP.	 ?However,	 ?due	 ?to	 ?large	 ?gaps	 ?in	 ?fungal	 ?ITS	 ? 67 databases,	 ?many	 ? sequences	 ? could	 ? not	 ? be	 ? described.	 ? Furthermore,	 ? because	 ? of	 ? time	 ?and	 ?financial	 ?constraints,	 ?I	 ?was	 ?only	 ?able	 ?to	 ?sequence	 ?DNA	 ?from	 ?fungal	 ?sources,	 ?and	 ?not	 ?other	 ?soil	 ?microbes.	 ?Bacteria	 ? likely	 ?play	 ?a	 ? large	 ?role	 ? in	 ?phosphatase	 ?activity	 ? in	 ?soil,	 ? as	 ? many	 ? soil	 ? bacteria	 ? have	 ? phosphatase-??encoding	 ? genes	 ? (Zimmerman	 ? et	 ? al.,	 ?2013),	 ? and	 ? phosphatase	 ? activity	 ? in	 ? soil	 ? has	 ? been	 ? highly	 ? correlated	 ? with	 ? bacterial	 ?biomass	 ?(Brocket	 ?et	 ?al.,	 ?2012).	 ?While	 ?I	 ?did	 ?measure	 ?abundance	 ?of	 ?fungal	 ?sequences	 ?in	 ? high	 ? and	 ? low	 ? phosphatase	 ? areas,	 ? I	 ?was	 ? unable	 ? to	 ? quantify	 ? fungal	 ? and	 ? bacterial	 ?biomass.	 ?It	 ?was	 ?surprising	 ?to	 ?find	 ?that,	 ?even	 ?though	 ?high	 ?phosphatase	 ?microsites	 ?had	 ?higher	 ?C	 ?and	 ?N,	 ? I	 ? could	 ?not	 ?stimulate	 ?phosphatase	 ?activity	 ?with	 ?additions	 ?of	 ? labile	 ?C	 ?and	 ?N.	 ?One	 ?possible	 ?reason	 ?why	 ?I	 ?did	 ?not	 ?see	 ?changes	 ?in	 ?activity	 ?after	 ?injections	 ?was	 ?that	 ?it	 ?rained	 ?on	 ?the	 ?day	 ?of	 ?injections	 ?as	 ?well	 ?as	 ?the	 ?first	 ?sampling	 ?day	 ?(24hrs).	 ?Although	 ?we	 ?did	 ?our	 ?best	 ?to	 ?cover	 ?up	 ?the	 ?root	 ?windows	 ?with	 ?tarps,	 ?some	 ?water	 ?may	 ?have	 ?made	 ?its	 ?way	 ?into	 ?the	 ?root	 ?windows	 ?causing	 ?the	 ?nutrient	 ?injections	 ?to	 ?run	 ?down	 ?into	 ?the	 ?soil	 ? profiles.	 ? This	 ?may	 ? have	 ? caused	 ? changes	 ? in	 ? detectable	 ? phosphatase	 ? activity	 ? on	 ?the	 ?imprints,	 ?thereby	 ?leading	 ?to	 ?erroneous	 ?intensity	 ?readings.	 ?This	 ?idea	 ?is	 ?supported	 ?by	 ?the	 ?blurry	 ?images	 ?that	 ?I	 ?observed	 ?from	 ?this	 ?series	 ?of	 ?imprints.	 ?Although	 ? the	 ?small	 ? sample	 ?size	 ?of	 ?soils	 ? from	 ?high	 ?and	 ? low	 ?phosphatase	 ?microsites	 ?allowed	 ?me	 ? to	 ? test	 ? important	 ? aspects	 ? of	 ? soil	 ? micro-??scale	 ? processes,	 ? it	 ? also	 ? led	 ? to	 ?several	 ?complications.	 ?For	 ?example,	 ?I	 ?was	 ?not	 ?able	 ?to	 ?run	 ?separate	 ?analyses	 ?on	 ?the	 ?same	 ? soil	 ? sample	 ? because	 ? each	 ? sample	 ? was	 ? so	 ? small.	 ? Therefore,	 ? samples	 ? for	 ?different	 ? analyses	 ? (DNA,	 ?P,	 ? C&N)	 ?had	 ? to	 ?be	 ? collected	 ? separately.	 ? Furthermore,	 ? the	 ? 68 small	 ?sample	 ?size	 ?reduced	 ?the	 ?statistical	 ?power	 ?for	 ?some	 ?analyses.	 ?For	 ?soil	 ?nutrient	 ?status,	 ?pH,	 ?and	 ?soil	 ?moisture,	 ?I	 ?had	 ?to	 ?combine	 ?samples	 ?from	 ?each	 ?window	 ?together	 ?to	 ?have	 ?appropriate	 ?sample	 ?sizes.	 ?Thus	 ?I	 ?did	 ?not	 ?have	 ?the	 ?ability	 ?to	 ?look	 ?for	 ?variation	 ?between	 ?microsites	 ?within	 ?windows,	 ?as	 ?with	 ?the	 ?DNA	 ?analysis	 ?where	 ?samples	 ?were	 ?not	 ? combined.	 ? Lastly,	 ? small	 ? sample	 ? size	 ? may	 ? have	 ? caused	 ? issues	 ? with	 ? the	 ? pH	 ?analysis.	 ?Although	 ?we	 ?measured	 ?pH	 ?of	 ? soil	 ? from	 ?microsites	 ? in	 ? solution	 ?using	 ?a	 ?pH	 ?electrode,	 ?it	 ?was	 ?difficult	 ?to	 ?measure	 ?because	 ?soil	 ?samples	 ?were	 ?so	 ?small.	 ?4.3. FUTURE	 ?DIRECTIONS	 ?AND	 ?SIGNIFICANCE	 ?Our	 ? lab	 ? performed	 ? a	 ? follow-??up	 ? study	 ? (Lidher,	 ? unpublished)	 ? to	 ? determine	 ? if	 ? other	 ?forms	 ?of	 ?carbon	 ?and	 ?nitrogen	 ?could	 ?stimulate	 ?phosphatase	 ?activity	 ?in	 ?soil	 ?using	 ?the	 ?root	 ? window	 ? approach.	 ? Cellulose,	 ? chitin,	 ? and	 ? sodium	 ? acetate	 ? and	 ? ammonium	 ?chloride	 ? all	 ? stimulated	 ? activity.	 ? Furthermore,	 ? there	 ? was	 ? no	 ? correlation	 ? between	 ?microbial	 ?biomass	 ?and	 ?phosphatase	 ?activity.	 ?These	 ?results,	 ?along	 ?with	 ?the	 ?results	 ?of	 ?my	 ? study	 ? showing	 ? that	 ? high	 ? phosphatase	 ? microsites	 ? are	 ? sites	 ? of	 ? high	 ? C	 ? and	 ? N,	 ?suggest	 ? that	 ? high	 ? phosphatase	 ? sites	 ? are	 ? not	 ? necessarily	 ? sites	 ? of	 ? high	 ? microbial	 ?biomass,	 ?but	 ?are	 ?instead	 ?sites	 ?driven	 ?by	 ?high	 ?C	 ?and	 ?N.	 ?	 ?I	 ? found	 ? that,	 ?when	 ?examined	 ?as	 ? assemblages	 ?of	 ?OTUs,	 ? fungal	 ? communities	 ? in	 ?high	 ?and	 ?low	 ?phosphatase	 ?were	 ?not	 ?different	 ?across	 ?all	 ?windows.	 ?I	 ?propose	 ?that	 ?bacteria	 ?could	 ? be	 ? contributing	 ? to	 ? phosphatase	 ? activity.	 ? High-??throughput	 ? sequencing	 ? of	 ?bacterial	 ?16s	 ?genes	 ?could	 ?be	 ?used	 ?to	 ?determine	 ?bacterial	 ?community	 ?composition,	 ?in	 ?high	 ? and	 ? low	 ? phosphatase	 ? areas.	 ? Furthermore,	 ? analyses	 ? of	 ? fungal	 ? and	 ? bacterial	 ?transcripts	 ?using	 ?qPCR	 ?could	 ? indicate	 ?which	 ?members	 ?of	 ?the	 ?microbial	 ?community	 ? 69 are	 ?actively	 ?producing	 ?phosphatase.	 ?Another	 ?interesting	 ?aspect	 ?of	 ?this	 ?investigation	 ?was	 ?that,	 ?when	 ?OTUs	 ?were	 ?designated	 ?into	 ?functional	 ?groups,	 ?I	 ?found	 ?lower	 ?EM:SAP	 ?based	 ? on	 ? abundance	 ? in	 ? high	 ? phosphatase	 ? areas	 ? in	 ? windows	 ? A	 ? and	 ? B,	 ? and	 ? lower	 ?overall	 ?EM	 ?OTU	 ?richness	 ?in	 ?these	 ?windows.	 ?The	 ?lower	 ?overall	 ?EM	 ?fungal	 ?occurrence	 ?may	 ? have	 ? led	 ? to	 ? SAP	 ? fungi	 ? being	 ?more	 ? competitive	 ? in	 ? high	 ? phosphatase	 ? areas	 ? in	 ?these	 ?windows.	 ?Although	 ? I	 ? found	 ? no	 ? differences	 ? in	 ? fungal	 ? communities	 ? between	 ? high	 ? and	 ? low	 ?phosphatase	 ? areas,	 ? I	 ? did	 ? find	 ? significant	 ? differences	 ? in	 ? OTU	 ? composition	 ? between	 ?windows.	 ? I	 ? proposed	 ? that	 ? distance	 ? appeared	 ? to	 ? be	 ? the	 ? most	 ? important	 ? factor	 ? in	 ?determining	 ? fungal	 ? community	 ? composition	 ? in	 ? these	 ? forests.	 ? By	 ? measuring	 ? the	 ?distances	 ?between	 ? samples	 ?within	 ?windows,	 ? and	 ? the	 ?distances	 ?between	 ?windows,	 ?one	 ? could	 ? determine	 ? at	 ? what	 ? distance	 ? fungal	 ? communities	 ? or	 ? individual	 ? taxa	 ? are	 ?autocorrelated.	 ? This	 ? information	 ? would	 ? be	 ? important	 ? to	 ? forest	 ? soil	 ? ecologists,	 ? to	 ?better	 ?understand	 ?drivers	 ?of	 ?fungal	 ?community	 ?composition.	 ?I	 ? attempted	 ? to	 ?address	 ? the	 ?hypothesis	 ? from	 ?Brooks	 ? (2010)	 ? that	 ?SAP	 ? fungi	 ?may	 ?be	 ?more	 ? highly	 ? associated	 ?with	 ? high	 ? phosphatase	 ? patches	 ? than	 ? EM	 ? fungi.	 ? Although	 ? I	 ?found	 ?the	 ?richness	 ?and	 ?abundance	 ?of	 ?EM	 ?and	 ?SAP	 ?fungal	 ?sequences	 ?to	 ?be	 ?similar	 ?in	 ?high	 ?and	 ? low	 ?phosphatase	 ?sites	 ?overall,	 ? this	 ?hypothesis	 ? could	 ?be	 ? further	 ? tested	 ?by	 ?measuring	 ? fungal	 ? biomass.	 ? If	 ? a	 ? small	 ? number	 ? of	 ? SAP	 ? individuals	 ? dominated	 ? high	 ?phosphatase	 ? sites,	 ? their	 ? biomass	 ?may	 ? be	 ? larger	 ? and	 ? not	 ? represented	 ? by	 ? sequence	 ?abundance.	 ?	 ? 70 Understanding	 ? fine-??scale	 ? soil	 ? processes	 ? is	 ? important	 ? for	 ? determining	 ? how	 ? these	 ?processes	 ? affect	 ? ecosystems	 ? at	 ? larger	 ? scales.	 ? In	 ? this	 ? study,	 ? 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   85 APPENDIX	 ?A:	 ?Soil	 ?profile	 ? Figure	 ?A.1:	 ?Soil	 ?profile	 ?from	 ?window	 ?C.	 ?

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