USING MOLECULAR TECHNIQUES TO INVESTIGATE SOIL INVERTEBRATE COMMUNITIES IN TEMPERATE FORESTS A thesis submitted To Kent State University in partial Fulfillment of the requirements for the Degree of Master of Science By Dean James Horton December, 2015 © Copyright All rights reserved Thesis written by Dean James Horton B.S., Kent State University, 2012 M.S., Kent State University, 2015 Approved by Christopher B. Blackwood, Associate Professor, Ph.D., Department of Biological Sciences, Thesis Co-Advisor Mark W. Kershner, Associate Professor, Ph.D., Department of Biological Sciences, Thesis Co- Advisor Laura G. Leff, Chair Professor, Ph.D., Department of Biological Sciences James L. Blank, Dean, Ph.D., College of Arts and Sciences TABLE OF CONTENTS I. LIST OF FIGURES …………………………………….…………………… vi II. LIST OF TABLES ……………………………………..…………………….. x III. DEDICATION ………………………………………….………………….. xii IV. ACKNOWLEDGEMENTS ……………………………………………...... xiii V. CHAPTERS 1. GENERAL INTRODUCTION ………………………...………………... 1 REFERENCES …………………………………………..………………. 4 2. A PRIMER COMPARISON FOR MOLECULAR IDENTIFICATION OF INVERTEBRATE TAXA FROM SOIL AND LEAF LITTER ENVIRONMENTAL DNA ………………………………..…………… 11 1. ABSTRACT ………………………………………………………... 11 2. INTRODUCTION ………………………………………………….. 11 3. METHODS …………………………………………………………. 14 3.1. INDIVIDUAL VOUCHER SPECIMEN COLLECTION…….... 14 3.2. PRIMER TESTING ON INVERTEBRATE VOUCHER SPECIMENS………………………………………………...….. 14 3.3. eDNA IN ENVIRONMENTAL SAMPLES…………………… 16 4. RESULTS…………………………………………………………... 17 4.1. INVERTEBRATE VOUCHER SPECIMENS…………………. 17 4.2. ENVIRONMENTAL SOIL AND LEAF SAMPLES……….…. 18 5. DISCUSSION………………………………………………………. 19 iii 6. REFERENCES……………………………………………………… 21 3. HIGH-THROUGHPUT SEQUENCING REVEALS HIGH SOIL FAUNAL DIVERSITY AND SMALL-SCALE COMMUNITY TURNOVER IN TEMPERATE FORESTS…………………………….. 33 1. ABSTRACT………………………………………………………… 33 2. INTRODUCTION………………………………………………...… 34 3. METHODS…………………………………………………………. 38 3.1. STUDY AREA AND SAMPLING DESIGN………………..… 38 3.2. COMMUNITY CHARACTERIZATION……………..…….…. 39 3.3. DATA ANALYSIS…………………………………………..… 41 3.3.1. VARIABILITY AT THE LANDSCAPE SCALE AMONG SITES…………………………………………………………… 41 3.3.2. WITHIN-SITE SPATIAL ANALYSIS OF ANIMAL COMMUNITY TURNOVER…………………………………... 43 4. RESULTS…………………………………………………………... 44 4.1. HYPOTHESIS 1. ALPHA-DIVERSITY IN DIFFERENT HABITAT AND ECOSYSTEM TYPES…………………………… 44 4.2. HYPOTHESIS 2. EFFECTS OF ECOSYSTEM AND HABITAT TYPE ON COMMUNITY COMPOSITION AT THE FOREST STAND SCALE…………………………………………………….. 45 4.3. HYPOTHESIS 3. WITHIN-STAND VARIATION IN ANIMAL COMMUNITY COMPOSITION………………………………….... 46 5. DISCUSSION………………………………………………………. 47 iv 5.1. REGIONAL FAUNAL DIVERSITY PATTERNS…………….. 47 5.2. BETA DIVERSITY AT THE FOREST LANDSCAPE SCALE. 48 5.3. LOCAL (WITHIN-STAND) COMMUNITY TURNOVER…... 50 5.4. CONCLUSIONS……………………………………………….. 51 6. REFERENCES……………………………………………………… 52 v I. LIST OF FIGURES CHAPTER 2: Figure 1. Phylum-level community composition of taxa found within environmental samples when targeting 18S and COI genes. “Unclassified” sections of the histograms represent sequences that were not designated to at least phylum level, were assigned to taxonomic groups within alternative biological hierarchies (e.g. Bacteria, Fungi, Plantae), or were not classified by BLAST. Phylum is the lowest taxonomic level represented in this figure for clarity…………. 28 Figure 2. Animal community composition between leaf litter and soil environments analyzing the 18S dataset. Taxonomic groups shown are the lowest levels to which each 18S sequence was identified, ranging from phylum to genus. Leaf litter analysis represented 101 sequences, while soil analysis represented 10 sequences. These results were obtained through further analysis of the dataset displayed in Fig. 1………………………………………………………………….. 29 CHAPTER 3: Figure 1. α-diversity Hill metrics for each ecosystem. Histograms represent a. H0, b. H1, c. H2 and d. Hinf. Leaf habitats are represented by dark grey columns, and soil habitats are represented by light grey columns. Ecosystem types are represented by BOWO = Black Oak-White Oak, SMRO = Sugar Maple-Red Oak, and SMBW = Sugar Maple-Basswood. Error bars represent standard error……………………………………………………………………….…..……… 64 vi Figure 2. Redundancy analysis ordination of stand-level invertebrate community composition. Green coloration represents leaf communities, magenta coloration represents soil communities. Circles represent BOWO communities, squares represent SMRO communities, and triangles represent SMBW communities. Large symbols represent the centroids of each treatment…… 65 Figure 3. Community compositional network of taxonomic groups found in this study. Size of node represents relative abundance of OTUs assigned to that taxonomic group, including those identified to taxonomic groups at lower hierarchical levels. Green nodes represent taxonomic groups with significant correlations from indicator analyses with soil habitats, while purple nodes represent groups with significant correlations with leaf litter habitats. White nodes represent taxa that were not significantly associated with either soil or leaf litter habitats. Intensity of coloration represents the steepness of the rpb value………………………………... 66 Figure 4. Community compositional network of taxonomic groups found in the leaf litter habitat. Size of node represents relative abundance of OTUs assigned to that particular taxonomic group, including those which identified to taxonomic groups at lower hierarchical levels. Red nodes represent taxonomic groups with significant correlations from indicator analyses. Intensity of coloration represents the steepness of the rpb value. Diamond-shaped nodes represent indicator groups of the BOWO ecosystem, rectangle-shaped nodes represent indicator groups of the SMRO ecosystem, and triangle-shaped nodes represent indicator groups of the SMBW ecosystem. Circular nodes lack significant correlation to a particular ecosystem type……………………………………………………………………………………………… 68 vii Figure 5. Community compositional network of taxonomic groups found in the soil habitat. Size of node represents relative abundance of OTUs assigned to that particular taxonomic group, including those which identified to taxonomic groups at lower hierarchical levels. Red nodes represent taxonomic groups with significant correlations from indicator analyses. Intensity of coloration represents the steepness of the rpb value. Diamond-shaped nodes represent indicator groups of the BOWO ecosystem, rectangle-shaped nodes represent indicator groups of the SMRO ecosystem, and triangle-shaped nodes represent indicator groups of the SMBW ecosystem. Circular nodes lack significant correlation to a particular ecosystem type….…….. 70 Figure 6. Distance-decay plots for soil and leaf litter faunal communities based on geographic distance classes and Hellinger distance. Circles represent soil community points, and triangles represent leaf community points. Red symbols represent Hellinger distance values significantly lower than expected by chance (P < 0.05). a) BOWO, b) SMRO, c) SMBW…………………. 72 Figure 7. Principle Coodinates Analysis ordination based on results from Anderson’s test of multivariate homogeneity of variances on local community dispersion. Green coloration represents leaf communities, magenta coloration represents soil communities. Circles represent BOWO communities, squares represent SMRO communities, and triangles represent SMBW communities. Large symbols represent the centroids of each treatment. Axis 1 explained 18% of the variance in community composition, while Axis 2 explained 9.4% and Axis 3 explained 6.7%. Ellipses represent 95% confidence intervals around the centroids of each habitat- ecosystem type………………………………………………………………………………..… 73 viii Supplementary Figure 1. Rarefaction curves illustrating sampling coverage for a. regional faunal community analyses and b. local faunal community analyses. Colored lines represent separate environmental samples. The black line represents the 1:1 slope line for OTU:sequence number…………………………………………………………………………………………...74 ix I. LIST OF TABLES CHAPTER 2: Table 1. Comparison of invertebrate specimen identifications determined using traditional (morphological) and molecular (sequencing of 18S rRNA and mitochondrial COI genes) approaches. Numbers adjacent to Collembola identifications correspond to the number of individuals identified to that taxonomic rank. For all other taxa, there was only a single specimen included in the morphological and molecular identifications. The taxonomic rank for each identification is listed in parentheses following the rank (Codes = P: Phylum; sC: Subclass; O: Order; sO: Suborder; iO: Infraorder; SF: Superfamily; F: Family; sF: Subfamily; G: Genus; S: Species). The symbol ‘◊’ indicates taxonomic mismatch between morphological and sequence identifications. “No Amplification” indicates lack of DNA sequencing for that taxon for that particular gene as a result of insufficient PCR amplification. “No Hits” indicates lack of sequence match to sequences within the NCBI database. Assigned taxonomic ranks for molecular identifications were acquired from the NCBI taxonomic database…………………. 30 CHAPTER 3: Table 1. Distance classes to be generated by transect sampling and used to examine spatial structure. Distances in meters……………………………………………………..……………. 75 x Table 2. Mixed-model ANOVAs testing significance of ecosystem, habitat type, and interaction effects on α-diversity of forest stand communities. Corresponding F-values are reported for each ANOVA. H0 = OTU
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