MICROBIAL FUNCTIONAL ACTIVITY AND DIVERSITY PATTERNS AT MULTIPLE SPATIAL SCALES A Dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Larry M. Feinstein August, 2012 Dissertation written by Larry M. Feinstein B.S., Wright State University, 1999 Ph.D., Kent State University, 2012 Approved by , Chair, Doctoral Dissertation Committee Christopher B. Blackwood , Members, Doctoral Dissertation Committee Laura G. Leff Mark W. Kershner Gwenn L. Volkert Accepted by , Chair, Department of Biological Sciences James L. Blank , Dean, College of Arts and Science John R. D. Stalvey ii TABLE OF CONTENTS LIST OF FIGURES……………………………………………...............……………….iv LIST OF TABLES...............................................................................................................x ACKNOWLEDGMENTS................................................................................................xiii 5 CHAPTER 1. Introduction……………….........……………………………………...……..…....1 References..................................................................................................11 2. Assessment of Bias Associated with Incomplete Extraction of Microbial DNA from Soil.........................................................................………………..……….18 10 Abstract.......…….………………………………………………..….…...18 Introduction............…………………………………..……………..……19 Methods......……………………………………………………….…...…21 Results...............................................................................................….....27 Discussion.........………………………………………………………….43 15 Referneces.........……………………………………………………….....47 3. Taxa-area Relationship and Neutral Dynamics Influence the Diversity of Fungal Communities on Senesced Tree Leaves................................................................55 Abstract...........………………………………................…………...…...55 Introduction...............……………………………………....................…54 iii 20 Methods...........………………………………………...…………………59 Results...........………………………………………….......……………..63 Discussion...........…………………………………………...........………85 References.............………………………………...............……………..90 4. The Spatial Scaling of Fungal Diversity..............................................................101 25 Abstract............……………………………............................................101 Introduction................………….........................................…………….102 Methods..........…………..........................................…………………....105 Results..........……………………….................................................…...113 Discussion...........………….................................................……………128 30 References............………………………….................................……...134 5. The Impact of Litter Diversity on Microbial Enzyme Activity, Biomass, and Leaf Litter Decomposition...........................................................................................146 Abstract...........……………….............………………............................146 Introduction...............………………..................................................….147 35 Methods...........……………...........……………………………….…….152 Results............…………...........................……………………………...159 Discussion............………................……………………………………175 References..............…………..........……………………………………183 6. Synthesis..............................................................................................................199 40 References................................................................................................206 iv LIST OF FIGURES CHAPTER 2. Assessment of Bias Associated with Incomplete Extraction of Microbial DNA from Soil Figure 1: Cumulative DNA yield in successive extractions conducted using four 45 cell lysis treatments. A) organic soil; B) clay soil; C) sand soil............................29 Figure 2. PFGE gel of 1st and 6th extractions for clay and sand soils. The top row of numbers refers to extraction treatment, the bottom row refers to extraction step. Lane 9 contains lambda ladder PFG marker (visible bands are 48.5 kbp and 98 kb). Lane 10 contains lambda PstI marker (visible bands are 11.5, 5.1, 4.6, 2.8, 50 and 2.6 kb).............................................................................................................32 Figure 3. Cumulative microbial gene copies/g soil in successive extractions. A) bacterial 16S gene; B) fungal 18S gene.................................................................34 Figure 4. Microbial gene copies/ng DNA for each extraction (not cumulative or weighted average). Significant differences are described in the text. A) bacterial 55 16S gene; B) fungal 18S gene................................................................................36 Figure 5. Weighted average ratio of fungal to bacterial ribosomal gene copies. Significant differences are described in the text....................................................38 Figure 6. Canonical principal components plot of T-RFLP profiles derived from redundancy analysis. Soil type and extraction are significant as described in text. 60 A) bacterial profiles; B) fungal profiles. “Wted ave-3” is the weighted average of v the first three T-RFLP profiles; “wted ave-6” is the weighted average of all six T- RFLP profiles.........................................................................................................40 CHAPTER 3. Taxa-area Relationship and Neutral Dynamics Influence the Diversity of Fungal Communities on Senesced Tree Leaves 65 Supplementary Figure S1(a). Relative abundance charts show proportion of community occupied by dominant (multi-colored) and non-dominant (black) OTUs on vernal pool leaves...................................................................................65 Supplementary Figure S1(b). Relative abundance charts show proportion of community occupied by dominant (multi-colored) and non-dominant (black) 70 OTUs on upland leaves..........................................................................................66 Supplementary Figure S1(c). Relative abundance charts show proportion of community occupied by dominant (multi-colored) and non-dominant (black) OTUs on riparian leaves........................................................................................67 Supplementary Figure S2(a). Rarefaction curves for diversity indices for 75 individual leaves located at Upland Sites L6 and 14.............................................69 Supplementary Figure S2(b). Rarefaction curves for diversity indices for individual leaves located at Riparian Sites J1 and Q1...........................................70 Supplementary Figure S2(c). Rarefaction curves for diversity indices for individual leaves located at Vernal Pool Sites J3 and H6......................................71 80 Figure 1. Mean OTU richness (S), Shannon diversity (H’), and Simpson diversity (1/D) values for large vs. small sugar maple and beech leaves gathered from 3 vi temperate forest habitats (n=2 leaves per bar). Analysis of sugar maple and beech leaves together resulted in a significant species X size interaction (see text). P values shown with each chart indicate significance of leaf size within either sugar 85 maple or beech (size X habitat interactions were never significant).....................73 Figure 2. Fungal taxa-area relationship detected on maple leaves (P < 0.05) but not beech. Regression lines are shown for maple upland, vernal pool, and riparian habitat TARs. For maple leaves, model selection using AICc resulted in a model where habitat TARs have identical z-values (slope; 0.22 ± 0.07) and different c- 90 values (Y-axis intercepts)......................................................................................76 Figure 3. Redundancy analysis ordination of fungal community composition at the six field sites. Habitat is indicated by symbol shape, with site names shown in legend. Because leaf type explained little variation in community composition (5%), each symbol represents the centroid of the four leaves sampled at each site. 95 ................................................................................................................................81 CHAPTER 4. The Spatial Scaling of Fungal Diversity Figure 1. Graphical representation of step distance matrix showing connections between leaves collected at each Jennings Woods site........................................108 Figure 2. Distance decay plots showing the average Hellinger distance between 100 communities at increasing step distances for each of the six sites in this study. Habitat abbreviations (VP=vernal pool, Rip=riparian, Up=upland), site ID, and the standardized Mantel statistic (rM) are shown for each plot............................114 vii Figure 3. Collectors curve analysis quantifying new TRFs with each randomly selected fungal community. Plots show the set of all randomly selected 105 communities at each site (white circles) and communities randomly selected at each depth within the forest floor leaf pack that the communities were located in (black symbols). Symbols for layers of the leaf pack that communities were sampled from: top layer: X; 2nd layer: square; 3rd layer: triangle; 4th layer: diamond; 5th layer: star; 6th layer circle...............................................................121 110 Figure 4. Collectors curve analysis quantifying the difference between new and previous fungal community composition. The community composition with