The Pennsylvania State University The Graduate School Eberly College of Science DNA PRESERVATION UNDER EXTREME CONDITIONS A Dissertation in Biology and Astrobiology by Kristine Korzow Richter © 2014 Kristine Korzow Richter Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2014 The dissertation of Kristine Korzow Richter was reviewed and approved* by the following: Beth Shapiro Associate Professor, Ecology and Evolutionary Biology, UC Santa Cruz Dissertation Co-Adviser Jennifer L. Macalady Associate Professor of Geosciences Dissertation Co-Adviser, Co-Chair of Committee James H. Marden Professor of Biology Co-Chair of Committee Stephen W. Schaeffer Professor of Biology Christopher House Professor of Geosciences, Director PSARC, Director PA Space Grant Consortium Douglas R. Cavener Professor of Biology Head of Department of Biology *Signatures are on file in the Graduate School ii Abstract Over the past thirty years the field of ancient DNA (aDNA) has expanded from looking at short DNA sequences from historic animals in museums to reconstructing entire genomes of extinct organisms. The preservation conditions of DNA in bone are well researched; however, ancient DNA is found in more places than in bone. The research herein discusses several of these other environments: microbial DNA from cells in the deep subsurface, microbial DNA trapped in salt crystals, and DNA transferred to objects during use. In addition, extreme conditions of preservation in bones are briefly addressed. Possible recovery of DNA biomarkers of a 34.5 million year old hydrothermal system indicates that ancient environments may leave traces for millennia in microbial communities. Discovery of Native American DNA preserved in moccasins and cordage for 700 years leads to a potentially large source of information on the genetic composition of ancient populations. Development and analysis of the Depressed Rate Test (DRT) shows the potential for authenticating ancient claims using sequence data only for when isotopic dating is unavailable. Finally, unsuccessful recovery of DNA using tradition methods from bone samples from species living 100,000-200,000 years ago addresses the limitations of traditional methods. However, new methods may allow DNA recovery from these samples. Examining DNA preservation under different conditions helps to inform sampling strategies, analytical techniques, and authentication of DNA data from extreme environments here on Earth and on other planets or moons in the Solar System. iii Table of Contents List of Figures ............................................................................................................................................. vii List of Tables ................................................................................................................................................ x Acknowledgments ....................................................................................................................................... xii Chapter 1: Introduction ................................................................................................................................ 1 DNA as a window to the past .......................................................................................................... 2 Concerns with aDNA research ......................................................................................................... 4 DNA degradation through time .......................................................................................... 4 Co-extracted compounds inhibit reactions .......................................................................... 8 DNA contamination ............................................................................................................ 9 Characterizing DNA under extreme conditions ............................................................................. 13 Determining the authenticity of geologically ancient DNA ............................................. 14 Detecting biomarkers from an ancient hydrothermal system ........................................... 15 Recovering aDNA from touched objects .......................................................................... 18 The limit of recoverable DNA in bones ............................................................................ 19 Ancient DNA and astrobiology implications .................................................................... 20 References ...................................................................................................................................... 23 Chapter 2: A computational test of the authenticity of geologically ancient DNA .................................... 41 Introduction .................................................................................................................................... 42 Materials and Methods ................................................................................................................... 46 Data Sets ........................................................................................................................... 46 Analysis ............................................................................................................................ 50 Results ............................................................................................................................................ 52 Simulated Data .................................................................................................................. 52 Horse Data ........................................................................................................................ 55 Bacterial and Archaeal ‘Test’ Data .................................................................................. 55 Discussion ...................................................................................................................................... 56 References ...................................................................................................................................... 60 Chapter 3: Biosignatures of thermophilic microorganisms from a temporary hydrothermal system 34.5 million years ago in the Chesapeake Bay Impact Structure ................................................................ 65 Introduction .................................................................................................................................... 66 Impact craters and biomarkers .......................................................................................... 66 Chesapeake Bay impact structure ..................................................................................... 67 Materials and Methods ................................................................................................................... 70 Coring and contamination control .................................................................................... 70 DNA extraction ................................................................................................................. 70 PCR amplification, library preparation and sequencing ................................................... 72 Data processing and analysis ............................................................................................ 74 Results ............................................................................................................................................ 76 DNA extraction, amplification and library preparation .................................................... 76 Sequencing results and metagenomic composition .......................................................... 77 Taxonomy analysis ........................................................................................................... 78 Genes Indicative of Metabolic Potential ........................................................................... 85 Discussion ...................................................................................................................................... 87 Low biomass samples – difficulties resulting in high fragmentation ............................... 87 Community composition and biomarker identification .................................................... 88 Biomarkers of an ancient hydrothermal system ................................................................ 90 iv Astrobiology and DNA biomarkers .................................................................................. 92 References ...................................................................................................................................... 93 Chapter 4: Identifying human haplotypes from DNA transferred from objects during use ..................... 102 Introduction .................................................................................................................................. 103 DNA transferred to objects ............................................................................................. 103 Political and cultural challenges of working with human remains ................................. 103 Challenges of DNA recovery from transferred objects .................................................. 104 Cave Site ......................................................................................................................... 105 Materials and Methods ................................................................................................................