The Pennsylvania State University The Graduate School ELEPHANTS WITHOUT BORDERS: HISTORICAL AND CONTEMPORARY GENETIC CONNECTIVITY IN TANZANIA A Dissertation in Biology by George Martin Gwaltu Lohay © 2019 George Martin Gwaltu Lohay Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2019 The dissertation of George Martin Gwaltu Lohay was reviewed and approved* by the following: Douglas R. Cavener Professor and Verne M. Willaman Dean, Eberly College of Science Dissertation Advisor Stephen W. Schaeffer Professor of Biology, Associate Department Head of Graduate Education Biology Ombudsperson Chair of Committee Katriona Shea Professor of Biology Alumni Professor in the Biological Sciences John E. Carlson Professor of Molecular Genetics Director of The Schatz Center for Tree Molecular Genetics George H. Perry Associate Professor of Anthropology and Biology Anna B. Estes Assistant Research Professor, The Huck Institutes of the Life Sciences Adjunct Professor, The Nelson Mandela African Institution of Science and Technology Special member *Signatures are on file in the Graduate School iii ABSTRACT African savanna elephants (Loxodonta africana) are ecologically important as ecosystem engineers and socio-politically as revenue earners for national economies and local communities. However, their population has declined due to poaching and loss of habitat as a result of an increase in the human population. Habitat loss and fragmentation makes most protected areas isolated because of blocking of wildlife corridors. This study covered four ecosystems (Serengeti, Tarangire-Manyara, Selous, and Ruaha) in Tanzania which have the largest elephant populations in the country to determine the extent of genetic diversity and population structure nuclear and mitochondrial DNA markers. We wanted to establish historical genetic connectivity using mitochondrial DNA and contemporary gene flow using microsatellite markers from DNA obtained non-invasively from fecal samples. We specifically wanted to determine if there is gene flow between the Serengeti and Tarangire-Manyara ecosystems and whether the genetic structure has substantially changed over the past 50 years. We assumed that the Greater Rift Valley between two ecosystems would also act a barrier to the gene flow. We collected 800 elephant fecal samples from the four ecosystems and performed genetic analyses at the Pennsylvania State University. Our results showed that the Serengeti elephants are genetically distinct from the Tarangire-Manyara. Elephants from Ngorongoro showed an admixture between the two ecosystems. We also identified that there was a higher genetic similarity of elephants between Ngorongoro and Lake Manyara compared to Lake Manyara and Tarangire. Also, Tarangire and Ruaha elephants shared the same population structure although they are more than 400 km apart. Within the Serengeti ecosystem, we identified two population clusters from south and north of the Serengeti. Our results suggest that even without any physical barriers, there is genetic differentiation. The analysis of nuclear and mitochondrial DNA showed significant population differentiation between the Ruaha and Selous ecosystems. We further found no evidence for female-mediated gene flow between Ruaha and Selous. Only 4% of elephants sampled in Ruaha shared a haplotype with the Selous Game Reserve. iv We also developed a novel fecal-centric approach to assess the age and sex structure of elephants and validated it with a rapid demographic assessment. We compared the sex ratio of elephants between Serengeti National Park, Ngorongoro Conservation Area and Maswa Game Reserve which have different protection status. In Serengeti, the sex ratio for adult age classes was skewed in favor of females whereas, in Ngorongoro, the sex ratio was skewed in favor of males for elephants older than 25 years. Although poaching is the main explanation for the observed sex ratio in Serengeti, we speculate that differential survival rates between males and female could explain the differences in sex ratio, particularly for young elephants. Our findings provide baseline information about historical connectivity using the mitochondrial DNA and recent gene flow (using nuclear markers) between protected areas in Tanzania. This information may be used to inform laws to protect the existing wildlife corridors or to restore the blocked corridors. We have highlighted some wildlife corridors that may have been or are still very important for the elephants based on our data; these would be suitable targets for conservation and restorations Keywords: African Savanna elephants, Population structure, genetic connectivity, corridors, fecal-centric, microsatellite markers, simple sequence repeats (SSRs), mitochondrial DNA (mtDNA), habitat loss, habitat fragmentation, wildlife corridors, age and sex structure, , Amelogenin gene (AMELX/Y) v TABLE OF CONTENTS LIST OF FIGURES ................................................................................................................. viii LIST OF TABLES ................................................................................................................... xi ACKNOWLEDGEMENTS ..................................................................................................... xii : Introduction ........................................................................................................... 1 Background ...................................................................................................................... 1 Elephant social structure .................................................................................................. 1 History of the elephant population in the Serengeti ecosystem ....................................... 3 Threats facing elephant conservation ............................................................................... 4 Poaching ........................................................................................................................... 4 Habitat loss and habitat fragmentation ............................................................................. 5 The role of a metapopulations in conservation ................................................................ 6 Genetic impacts of small population size and fragmentation .......................................... 7 Microsatellite or Simple Sequence Repeats (SSR) .......................................................... 7 Mitochondrial DNA ......................................................................................................... 8 Objectives......................................................................................................................... 9 References ........................................................................................................................ 12 : Genetic connectivity and population structure of African Savanna elephants in Tanzania ....................................................................................................................... 21 Abstract ............................................................................................................................ 21 Introduction ...................................................................................................................... 22 Materials and methods ..................................................................................................... 24 Description of study areas ................................................................................................ 24 The Serengeti ecosystem .................................................................................................. 25 Tarangire-Manyara ecosystem ......................................................................................... 26 Ruaha ecosystem .............................................................................................................. 28 Selous Ecosystem ............................................................................................................. 29 Field data sample collection ............................................................................................. 29 DNA isolation .................................................................................................................. 30 Microsatellite Analysis .................................................................................................... 31 PCR amplification and genotyping .................................................................................. 31 Genetic diversity and differentiation ................................................................................ 32 Population Structure ......................................................................................................... 33 Mitochondrial Sequencing and Analysis ......................................................................... 33 Results .............................................................................................................................. 34 Microsatellite analyses ..................................................................................................... 34 Mitochondrial DNA analysis ........................................................................................... 41 Discussion ........................................................................................................................ 50 Connectivity between