The Pennsylvania State University The Graduate School Department of Ecosystem Science and Management METHODOLOGIES IN ASSESSING BENTHIC MACROINVERTEBRATE COMMUNITIES A Thesis in Wildlife and Fisheries Science by Sara Jaye Mueller © 2016 Sara Jaye Mueller Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science December 2016 The thesis of Sara Mueller was reviewed and approved* by the following: Jay R. Stauffer Jr. Distinguished Professor of Ichthyology Thesis Advisor Elizabeth Boyer Associate Professor of Water Resources Gregory A. Hoover Senior Extension Associate, Department of Entomology Aaron Aunins Contractor, United States Geological Survey, Special Signatory Michael G. Messina Head of Department of Ecosystem Science and Management *Signatures are on file in the Graduate School iii ABSTRACT Benthic macroinvertebrates have been used for over a century in biomonitoring. Biomonitoring is the use of biological measures to determine the state of or to evaluate changes in the environment. This surveillance method is commonly used as part of water quality monitoring. Agencies vary in their approach in collecting, sorting, and identifying benthic macroinvertebrates. The objective of this study is to compare entire collections of benthic macroinvertebrates collected by researchers in 2015 to computer-simulated data of subsamples of 100, 200, and 300 organisms. The computer simulated data represent protocols used by the National Park Service and the Pennsylvania Department of Environmental Protection. The Brillouin Index was calculated for each site and subsample permutation. An ANOVA analysis of the Brillouin Index by site showed that there is a statistically significant difference (p<0.05) between the use of 100 and 200 organisms with the Brillouin Index for 200 being greater. Additionally, the ANOVA analysis showed that 45.5% (5/11) of the time, that there is a statistically significant difference (p<0.05) between the use of 200 and 300 organisms with the Brillouin Index for 300 being greater. Lastly, a one-sample t-test revealed that there is a statistically significant difference (p<0.05) between the use of 300 organisms (or 200 if 300 organisms were not obtained) and entire samples with the Brillouin Index for entire samples being greater at all but one site. These results indicate there could be valuable information missing from traditionally collected benthic macroinvertebrate samples for which DNA based technologies may improve macroinvertebrate bioassessments of aquatic ecosystems. Several recent studies have investigated the applicability of the mitochondrial genes cytochrome oxidase 1 (COI), 12S rDNA, and 16S rDNA for barcoding of various aquatic fauna including fish and iv aquatic invertebrates using artificial pools of individuals in the laboratory. Generally, molecular methods have shown an increase in taxonomic resolution compared to the morphological analyses. However, current limitations in barcoding primer design result in certain taxonomic groups being over or under- represented in the sequencing results. In addition, little work has been done to see how genetic based estimates of diversity based on sequencing of environmental DNA samples extracted from sediment or water samples compare to traditional morphological based analyses. This study tested 55 primer pairs targeting both mitochondrial 16S rDNA and COI genes. The COI primers, despite incorporation of numerous ambiguous nucleotides to facilitate application to wide taxonomic breadth, did not amplify several taxa tested. However testing of 16S primers amplified DNA missed by COI. The results of this study make a large contribution to the effort of building up a robust 16S rDNA barcode database for aquatic invertebrate metabarcoding, through the generation and curation of 74 unique sequences. v TABLE OF CONTENTS List of Figures .......................................................................................................................... v List of Tables ........................................................................................................................... vi Acknowledgements .................................................................................................................. vii Chapter 1 Introduction ............................................................................................................. 1 What is Biomonitoring? ................................................................................................... 1 Organisms in Biomonitoring ............................................................................................ 3 Fishes ....................................................................................................................... 3 Macrophytes ............................................................................................................. 4 Benthic Macroinvertebrates ..................................................................................... 4 Diatoms .................................................................................................................... 6 Other Organisms ...................................................................................................... 7 Multiple Organisms .................................................................................................. 7 New Trends in Biomonitoring ......................................................................................... 8 Literature Cited ................................................................................................................ 10 Chapter 2 Comparison of Historic Assemblages and Sampling Methods ............................... 14 Abstract ............................................................................................................................ 14 Introduction ...................................................................................................................... 15 Methods ............................................................................................................................ 16 Study Sites ................................................................................................................ 16 National Park Survey Methods ................................................................................ 19 Benthic Macroinvertebrate Sampling ...................................................................... 20 Analysis .................................................................................................................... 21 Results .............................................................................................................................. 24 Discussion ........................................................................................................................ 28 Literature Cited ................................................................................................................ 32 Chapter 3 Use of 16S mitochondrial rDNA for barcoding of Benthic Macroinvertebrates .... 36 Abstract ............................................................................................................................ 36 Introduction ...................................................................................................................... 37 Methods ............................................................................................................................ 40 COI Primer Design .................................................................................................. 40 Amplification and sequencing of invertebrate samples for reference library construction ................................................................................. 42 Results .............................................................................................................................. 44 Discussion ........................................................................................................................ 45 Literature Cited ................................................................................................................ 47 Appendix A. Comprehensive Benthic Macroinvertebrate List ................................................ 54 vi Appendix B. Comparison of Taxa Between Studies ................................................................ 63 Appendix C. Families used to create Primer Miner output ...................................................... 72 Appendix D. Primer Pairs Tested ............................................................................................ 77 Appendix E. DNA Library Sequences with BLAST results .................................................... 81 vii LIST OF FIGURES Figure 1. Overview of study sites throughout the National Capital Region Network National Parks. Park boundaries are indicated in green while survey sites are indicated by triangles. ................................................................... 18 Figure 2. Brillouin Index for each site across studies. Black bars represent the 2015 study, white bars represent National Park collections, light grey bars represent 100 simulated organisms, dark grey bars represent 200 simulated organisms, and medium grey bars represent 300 simulated organisms. ............................................. 22 Figure 3. Results of ANOVA test boxplots of Brillouin's Index for simulated data at each site. ................................ 23 Figure