The American University in Cairo School of Sciences and Engineering Genetic Diversity Comparison among Invasive Fish Populations (Nemipterus randalli and Serranus cabrilla) from Mediterranean and Red Sea Coastal Wa- ters using Cytochrome c Oxidase Subunit I (COI) A Thesis Submitted to The Department of Biology in partial fulfilment of the requirements for the degree of a Master of Science in Biotechnology by Joel Ogwang under the supervision of Dr. Arthur Bos Dec. /2018 DEDICATION This thesis is dedicated to my parents, my mother Lily Awor and my late father Vitorino Ocen, and my siblings. I also dedicate this work to Mr. Alfred A. Olwit and his family. I con- sider Alfred more of a father than guardian because of his unwavering effort to support my academic journey. ii ACKNOWLEDGMENTS My supervisor, Dr. Arthur Bos was immensely supportive from the inception of this project and his effort never wavered at any point. Dr. M. Bariche (AUB) contributed samples from Lebanon. Dr. Ahmed Moustafa (AUC) provided invaluable support in the initial molecular analyses of this work. Mr. Amged Aouf (AUC) provided initial technical input to kick-start this project and handled most procurement related to this work. The laboratory attendants, Zain and Mohamed were immensely helpful. My friends offered a helping hand in the lab: Youssef, Mariam, Muziri, Eric Zadok, Yomna Moqidem, to mention a few. I am also grateful to the African Graduate Fellowship for the opportunity to conduct my graduate study in AUC. Finally, this work would have been impossible without the AUC Graduate Research Grant. iii Genetic Diversity Comparison among Invasive Fish Populations (Nemipterus randalli and Serranus cabrilla) from Mediterranean and Red Sea Coastal Wa- ters using Cytochrome c Oxidase Subunit I (COI) Joel Ogwang1 | AR Bos2 ABSTRACT Since the Suez Canal connected the Red Sea with the Mediterranean, several fish species have migrated between the two seas. Nemipterus randalli has crossed from the Red Sea to the Mediterranean (Lessepsian migration) whereas Serranus cabrilla is considered to have crossed in the reverse direction (anti-Lessepsian migration). Genetic variation between popu- lations of these fish species on either side of the Suez Canal might provide valuable infor- mation on their patterns of migration. In this study, 600 bp of cytochrome c oxidase subunit I (COI) sequences were used to compare genetic diversity of populations of N. randalli from the eastern Mediterranean with a population off the Red Sea coast near Hurghada, Egypt. For comparison, three other Nemipterus species were included. Similarly, genetic diversity of Serranus cabrilla from the Gulf of Suez was compared with populations in the eastern Medi- terranean Sea. A Maximum Likelihood (ML) tree was constructed using Molecular Evolu- tionary Genetics Analysis version 7 (MEGA7) software to visualize the evolutionary rela- tionships of S. cabrilla and Nemipterus species of the two seas. Population structures of N. randalli and S. cabrilla were assessed by constructing haplotype networks using PopART. Results from COI sequence divergence analysis revealed possible existence of cryptic species of N. bipunctatus in the Red Sea. Although the ML tree resolved Nemipterus species into four clades representing the four species analyzed, all N. randalli sequences from both seas formed a single clade. Genetic diversity analysis revealed that Mediterranean populations of N. randalli share one haplotype from the Red Sea and supported unidirectional multiple inva- sion events from the Red Sea to the Mediterranean Sea. Meanwhile, S. cabrilla sequences formed two phylogenetic clades representing the Gulf of Suez and eastern Mediterranean Sea populations. S. cabrilla from the Gulf of Suez also had a significantly reduced sequence di- vergence compared to Mediterranean Sea populations. In addition, none of the 17 haplotypes in the Mediterranean Sea was found among the 12 haplotypes in the Gulf of Suez. Together, these results provided evidence that the S. cabrilla population in the Gulf of Suez did not come from the Mediterranean Sea through the Suez Canal as was previously thought. Ac- cording to these results, reported cases of invasion on either side of the Suez Canal should be followed by genetic investigations on a species-by-species basis. Keywords – anti-Lessepsian migration, COI, haplotype diversity, invasive species, Les- sepsian migration, Nemipterus randalli, nucleotide diversity, Serranus cabrilla. 1Graduate student, Department of Biology, the American University in Cairo 2Supervisor and Chair, Department of Biology, the American University in Cairo iv TABLE OF CONTENTS List of Figures………………………....………………....………………...…….…………...vi List of Tables...…………………………………..……………………………….………….vii Introduction……………………..……………………………….…………………………….1 1.0 Literature Review………..……………………..………….…………..……………….….3 1.1 Lessepsian versus anti-Lessepsian Migration ……………………….……..…….……….3 1.2 Nemipterus randalli as a Lessepsian Migrant...…………………..….…………..………..4 1.3 Serranus cabrilla as an anti-Lessepsian migrant……...…………………...…..........….... 5 1.4 Hypotheses……………………………………...…..…………………..……………...….6 1.5 Study Aims and Objectives…………………………..…………...…………....…….……7 2.0 Experimental Design and Methods……………..…………………..……..………………8 2.1 Sample Collection and Handling………..………...…………...….……...…..…..……..…9 2.2 DNA extraction…..……………...…………......………..…………..……………..…...…9 2.3 Polymerase Chain Reaction (PCR) and Cycle Sequencing….…….….…...……....……..10 3.0 Data Analysis…………..………………………….……...…………………….………..11 3.1 Species Identification………………………………………………………...…......……11 3.2 DNA Barcoding…………………………………………………………………………..11 3.3 Metric Analysis……………………………………………………………….………….14 3.4 Molecular Evolutionary Analysis…………………….…...…………………………..….16 4.0 Results……..………….………………………...……………………………………..…19 4.1 Length-weight relationships……………………………………………….……………..19 4.2 Molecular Evolutionary Relationships………………………………………….………..22 5.0 Discussion …………………..………………………...…..……………….…………… 31 5.1 Evolutionary Relationships………………………………………………………………31 5.1.1 Phylogenetic Relationships: DNA Barcoding Perspective……………….……………31 5.1.2 Genetic Diversity………………………………………………………………………32 5.2 Length-weight relationships (LWRs)………………………………………...…………..35 6.0 Conclusion………………………... ………………..……………….………………….. 37 Declaration of Interest…………………………………………...…...………………………39 I References……………………………………………...………………..................................I II Appendix…………………………………...…………………....…………….....……….XV v LIST OF FIGURES Figure 1.0: Relationship between different players in the invasion process…………………..3 Figure 1.3: Geographical distribution of Serranus cabrilla……………………………….…..6 Figure 2.1: Map of the eastern Mediterranean and northern Red Sea………………………....9 Figure 3.1: Morphological Identities of Nemipterus spp. and Serranus cabrilla……….....…11 Figure 3.2: Schematic DNA Barcoding Pipeline…………………………………….………13 Figure 3.3.1: Metric Measurements of Fish………………………………………………….15 Figure 3.3.2: Length frequency distribution plots……………………………………………15 Figure 4.1.1: LWRs of four Nemipterus species……………………………………………..21 Figure 4.1.2: LWRs of Serranus cabrilla…………………………………...……………….22 Figure 4.2.1: Intraspecific COI sequence divergence within groups of Nemipterus spp. ...…23 Figure 4.2.2: Intraspecific COI sequence divergence within groups of Serranus cabrilla…..26 Figure 4.2.3: Maximum Likelihood tree for Nemipterus species…………………………….24 Figure 4.2.4: Maximum Likelihood tree for Serranus cabrilla…………………………...…27 Figure 4.2.5: Haplotype Network of Nemipterus species………………………………..…..29 Figure 4.2.6: Haplotype Network of Serranus cabrilla…………………………….………..30 vi LIST OF TABLES Table I: LWR parameters of Nemipterus species and Serranus cabrilla………………….…19 Table II: Empirical LWR lengths of Nemipterus spp. and Serranus cabrilla………….….....20 Table III: Percentage COI sequence divergence between groups of Nemipterus species…....23 Table IV: Percentage COI sequence divergence between groups of Serranus cabrilla…..…26 Table V: Genetic Diversity of Nemipterus randalli…………………………………….……28 Table VI: Genetic Diversity of Serranus cabrilla…………………………………….……...30 vii Introduction Invasive species are nonnative species that are introduced from other geographical locations mainly through travel and trade. Their widespread occurrences demand international inter- vention (Funk, 2015). Economically, they have been reported to cause damages worth bil- lions of dollars (Pimentel et al., 2000). For instance, they are responsible for significant eco- logical shifts in the Mediterranean (Belmaker et al., 2013) including a decline in native spe- cies; for example, the lionfish Pterois volitans feeds on and thus reduces the density of her- bivorous fish resulting in algal bloom that consequently affects the functioning of coral reefs (Hall-Spencer & Allen, 2015). Additionally, a recent analysis of freshwater fish catch data for the past three decades indicates that native fish species are on the decline whereas popula- tions of some invasive species are expanding (Mueller, & Geist, 2018). Worse still, the harm- ful effects of invasive species are expected to dramatically increase in the wake of increasing oceanic carbon dioxide levels due to climate change, with more adapted yet harmful species likely to phase out the less adapted ones (Hall-Spencer & Allen, 2015). Moreover, these harmful effects could worsen given the recent finding of a likely positive feedback mecha- nism where by one invasion reinforces another like a ripple effect (DiGiacopo