BARCODING, METABARCODING, AND EXPERIMENTAL ANALYSES OF COMMUNITY DYNAMICS AND ENVIRONMENTAL CONDITIONS AFFECTING PREDATION OF LARVAL LAKE STURGEON IN THE BLACK RIVER, MICHIGAN By Justin Waraniak A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Fisheries and Wildlife–Master of Science 2017 ABSTRACT BARCODING, METABARCODING, AND EXPERIMENTAL ANALYSES OF COMMUNITY DYNAMICSAND ENVIRONMENTAL CONDITIONS AFFECTING PREDATION OF LARVAL LAKE STURGEON IN THE BLACK RIVER, MICHIGAN By Justin Waraniak The larval stage of most fishes is characterized by high levels of mortality and is likely a bottleneck to recruitment for many populations. Predation is an important source of mortality for the larval stage of many fish species, and is a possible factor driving high mortality in some populations. Lake sturgeon are a species of conservation concern in the Great Lakes region, with many populations experiencing little to no natural recruitment and high mortality rates during the vulnerable egg and larval early life stages. Predation of larval lake sturgeon, and larval fishes generally, has been difficult to quantify with morphological diet analyses due to rapid digestion times in the gastrointestinal (GI) tracts of predators. This study developed and utilized alternative molecular genetic methods to detect larval lake sturgeon in the diets of predator fishes, as well as conducting an experiment to further examine findings of the molecular diet analysis. Sturgeon-specific barcoding analysis of the COI mtDNA region quantified the predation frequency predation of larval lake sturgeon and revealed increased abundance of alternative prey and abiotic factors that lowered visibility could reduce predation of larval lake sturgeon. Metabarcoding analysis of predator diets using universal 18S rRNA primers revealed seasonal dietary shifts of predators when larval lake sturgeon were present in the prey community compared to after lake sturgeon larvae were no longer available, but that lake sturgeon larvae made up a small portion of the overall diets of predator fishes. Experimental manipulations of relative prey abundance quantified how the prey community could affect predator preferences. This study exemplifies the utility and improved accuracy of molecular tools in detecting predation of larval fish and other soft-bodied prey compared to morphological analyses, as well as the importance of the biotic community and environmental factors influencing predation mortality in larval fishes. Copyright by JUSTIN WARANIAK 2017 ACKNOWLEDGEMENTS This project was funded by the Michigan Department of Natural Resources, the Great Lakes Fisheries Trust, and the US Fish and Wildlife Service Coastal Program. Special thanks to the 2015 and 2016 field crews at the Black River Sturgeon Rearing Facility, Dr. Kim Scribner, Dr. Edward Baker, Dr. Gary Mittelbach, Dr. James Bence, Dr. Brian Maurer, Dr. Terrence Marsh, Danielle Blumstein, Shaley Valentine, Jeanette Kanefsky, and Sturgeon for Tomorrow – Black Lake Chapter. iv TABLE OF CONTENTS LIST OF TABLES ...................................................................................................................................... vii LIST OF FIGURES ..................................................................................................................................... ix THESIS INTRODUCTION...........................................................................................................................1 LITERATURE CITED .....................................................................................................................6 CHAPTER 1 ................................................................................................................................................12 BARCODING PCR PRIMERS DETECT LARVAL LAKE STURGEON (ACIPENSER FULVESCENS) IN DIETS OF PISCINE PREDATORS.......................................................................................................12 ABSTRACT....................................................................................................................................12 INTRODUCTION ..........................................................................................................................13 MATERIALS AND METHODS....................................................................................................16 Study area and sample collection ...................................................................................16 DNA extraction.................................................................................................................19 Primer design ...................................................................................................................19 PCR optimization, primer specificity, and sensitivity ..................................................20 Statistical analysis ............................................................................................................22 RESULTS .......................................................................................................................................24 Primer performance ........................................................................................................24 Larval lake sturgeon predation by species ....................................................................24 Larval lake sturgeon predation rates in different habitats defined by substrate ......26 DISCUSSION .................................................................................................................................28 LITERATURE CITED ...................................................................................................................33 CHAPTER 2 ................................................................................................................................................37 MOLECULAR DIET ANALYSIS REVEALS PREDATOR-PREY COMMUNITY DYNAMICS AND ENVIRONMENTAL FACTORS AFFECT PREDATION OF LARVAL LAKE STURGEON (ACIPENSER FULVESCENS) IN A NATURAL SYSTEM.......................................................................37 ABSTRACT....................................................................................................................................37 INTRODUCTION ..........................................................................................................................38 MATERIALS AND METHODS....................................................................................................41 Study area and sample collection ...................................................................................41 DNA extraction and amplification .................................................................................44 Statistical analysis ............................................................................................................46 Model description .................................................................................................46 Model selection.....................................................................................................47 Analysis of variable effects ...................................................................................48 RESULTS .......................................................................................................................................49 Field and molecular data analysis ..................................................................................49 Model selection and importance of variables ................................................................50 Analysis of variable effects..............................................................................................52 DISCUSSION .................................................................................................................................57 LITERATURE CITED ...................................................................................................................63 v CHAPTER 3 ................................................................................................................................................70 18S METABARCODING DIET ANALYSIS OF THE PREDATORY FISH COMMUNITY IN THE BLACK RIVER, CHEBOYGAN COUNTY, MICHIGAN, ACROSS SEASONAL CHANGES IN PREY AVAILABILITY .........................................................................................................................................70 ABSTRACT....................................................................................................................................70 INTRODUCTION ..........................................................................................................................71 MATERIALS AND METHODS....................................................................................................74 Study area and sample collection ...................................................................................74 DNA extraction and sequencing .....................................................................................79 DNA sequence processing................................................................................................80 Statistical analysis ............................................................................................................81 RESULTS .......................................................................................................................................84