Phylogenetic Diversity of Cephalopoda (Animalia:Mollusca) Along the Saudi Arabian Red Sea Coastline Thesis by Gordon Byron In Partial Fulfillment of the Requirements For the Degree of Master of Science King Abdullah University of Science and Technology Thuwal, Kingdom of Saudi Arabia © December, 2016 Gordon Byron All rights reserved 2 EXAMINATION COMMITTEE PAGE The thesis of Gordon Byron is approved by the examination committee. Committee Chairperson: Michael Berumen Committee Co-Chair: Christian Voolstra Committee Member: Timothy Ravasi 3 ABSTRACT Phylogenetic Diversity of Cephalopoda (Animalia:Mollusca) Along the Saudi Red Sea Coastline Gordon Byron Although the Red Sea presents a unique environment with high temperature and salinity, it remains an area that is understudied. This lack of information is reflected in many areas, one which is biodiversity. Despite increasing work on biodiversity throughout the Red Sea and an increase in Cephalopoda studies, Cephalopoda in the Red Sea remain underrepresented, which is especially pronounced in molecular analyses. Members of the class Cephalopoda are considered to be major contributors to coral reef ecosystems, serving as part of the food chain and exhibiting population increases due to targeted teleost fisheries and global climate change. In order to assess the biodiversity of Cephalopoda in the Saudi Arabian Red Sea, 87 specimens were collected from 25 reef locations between 17°N and 28°N latitude, as well as from the largest fish market in the Kingdom of Saudi Arabia. Taxonomic identification of specimens was determined using morphological comparisons with previously reported species in the Red Sea and the molecular barcoding region Cytochrome Oxidase I. 84 Red Sea sequences were compared with sequences from GenBank and analyzed using a complement of Neighbor- Joining, Maximum-Likelihood, and Bayesian inference trees. Species complexes were also investigated for Sepia pharaonis and Sepioteuthis lessoniana, which had been previously reported. From 17 cuttlefish, our study yielded three species, two of which matched previously reported species in GenBank. In addition, two distinct clades of Sepia pharaonis were identified. Of 35 squid collected, four species were identified, one of which did not match any other accepted species in literature, while Sepioteuthis 4 lessoniana in the Red Sea formed a distinct clade. From 30 different specimens a total of five genera of Octopoda were present, forming six distinct species. Five Octopoda species collected did not match previously reported species, although many specimens were paralarvae or juveniles, so morphologically we could not compare to previously described species in the Red Sea. Cephalopoda fisheries in the Red Sea is low, and as their populations increase worldwide, this could be a viable fishery for Saudi Arabia. As such, further investigation into the role which cephalopods play in supporting biodiversity in the Red Sea is essential. 5 TABLE OF CONTENTS Examination Committee Page…..…………………………………………..……………..2 Abstract……………………………………………………………………………………3 Table of Contents………………………………………………………………………….5 List of Abbreviations……………………………………………………………………...6 List of Definitions….……………………………………………………………………...7 List of Illustrations………………………………………………………………………...8 List of Tables……………………………………………………………………………...9 Main Text 1. Introduction..…………………………………………………………………10 2. Materials and Methods……………………………………..………………...18 2.1 Collection and Locality…………………………………………………..18 2.2 Morphological Characteristics…………………………………………...19 2.3 Extraction/Sequencing/Barcoding……………………………………….22 2.4 DNA Analysis and DNA Taxonomy………………………………….....23 2.5 Clade Delineation………………………………………………………...27 3. Results………………………………………………………………………..28 3.1 Sepiidae…………………………………………………………………..28 3.1.1 Morphological Analysis………………………….………………28 3.1.1.1 Sepia pharaonis……………………………………………...29 3.1.1.2 Sepia gibba…………………………………………………...29 3.1.2 COI Barcoding.…………………………………………………..29 3.1.3 Clade Delineation…………………………………………….…..32 3.2 Myopsida and Oegopsida Squid…………………………………………35 3.2.1 Morphological Analysis……….…………………………………35 3.2.1.1 Sepioteuthis lessoniana…...………………………………….36 3.2.1.2 Uroteuthis singhalensis………………………………………37 3.2.1.3 Uroteuthis arabica…………………………………………...37 3.2.1.4 Abralia steindachneri………………………………………...38 3.2.2 COI Barcoding…………………………………………………...38 3.2.3 Clade Delineation………………………………………………...42 3.3 Octopus…………………………………………………………………..44 3.3.1 Morphological Analysis………………………………………….44 3.3.1.1 Abdopus….…………………………………………………...45 3.3.1.2 Callistoctopus………………………………………………..45 3.3.1.3 Cistopus……………………………………………………...46 3.3.1.4 Amphioctopus………………………………………………...46 3.3.1.5 Octopus cyanea………………………………………………48 3.3.2 COI Barcoding…………………………………………………...48 4. Discussion……………………………………………………………………53 4.1 Order Sepiidae…………………………………………………………...53 4.2 Suborder Myopsida and Oegopsida……………………………………...56 4.3 Order Octopoda…………………………………………………………..60 5. Conclusions…………………………………………………………………..66 6. References……………………………………………………………………67 6 LIST OF ABBREVIATIONS WoRMS – World Register of Marine Species SCUBA - Self-contained underwater breathing apparatus SST – Sea Surface Temperature ARMS – Autonomous Reef Monitoring Structures COI – Cytochrome Oxidase I PCR – Polymerase Chain Reaction KSA – Kingdom of Saudi Arabia ML – Mantle Length mm – millimeters A1 – Arm 1 A2 – Arm 2 A3 – Arm 3 A4 – Arm 4 CLI – Length of tenticular clubFLI – Length of greatest fin SS – Location of sucker series in manus FWI – Length of greatest total fin width GL – Length of shell GThI – Thickness of cuttlebone GWI – Width of shell HcLI – Length of hectocotylus HL – Length of head Htp – Number of pairs of hooks of tenticular club AA – Arm autotomy HWI – Greatest head width LLI – Length of ligula in Octopoda MWI – Greatest mantle width Nps – Number of pairs of suckers on hectocotylized arm NOP – Number of ocular photophores ZB – Zebra stripe banding on arm Ba-3 – Attachment of buccal connective to arm III WDI – Depth of deepest web sector DNA – Deoxyribonucleic Acid NCBI – National Center for Biological Information pmol – pico molar NJ – Neighbour Joining ML – Maximum Likelihood BI – Bayesian Inference 7 LIST OF DEFINITIONS Buccal connective - Muscular membrane that runs from the buccal support to the base of the adjacent arm Hectocotylus – The modified arm in males used for transferring spermatophores to the female Ligula – The portion of the sucker-free hectocotylus, which is generally elongate and longitudinally grooved Manus – The proximal, broad portion of the tenticular club Photophores – Organ that produces bioluminescent light. Photophores are often complex with various color filters, reflectors, light guides, lenses and chromatophores surrounding the site of light production, the photogenic region. The name of specific photophores often reflects their position on the animal. Tentacular club – The terminal, usually expanded part of the tentacle which bears suckers and/or hooks 8 LIST OF ILLUSTRATIONS Figure 2.1 (Sample location sites).....................................................................................19 Figure 3.1 (Sepiidae COI tree)…………………………………………………………...31 Figure 3.2 (Sepia pharaonis COI clade tree)……...……………………………………..34 Figure 3.3 (Myopsida and Oegopsida squid COI tree)……...…………………….……..40 Figure 3.4 (Sepioteuthis lessoniana COI clade tree)…….………...…………………….43 Figure 3.5 (Octopoda COI clade tree)…….…………….………………………………..50 9 LIST OF TABLES Table 1.1 (Red Sea Cephalopoda)………….....................................................................15 Table 2.1 (Abbreviations for morphological identification)……………………………..20 Table 2.2 (Sepiidae sequences)…….…………………………………..………………...24 Table 2.3 (Sepia pharaonis clade sequences)..……………………….………………….24 Table 2.4 (Myopsida and Oegopsida squid sequences)………………………………….25 Table 2.5 (Sepioteuthis lessoniana clade sequences)...……….………………..………..25 Table 2.6 (Octopoda sequences).….…...…...……………………………………………26 Table 3.1 (Morphometrics for Sepiidae)…………....……………………………………28 Table 3.2 (Genetic distances for Sepiidae)……...…………..…………………………...32 Table 3.3 (Genetic distances for Sepia pharaonis clades)……...………………..………35 Table 3.4 (Morphometrics for Sepioteuthis)………….....……………………………….36 Table 3.5 (Morphometrics for Uroteuthis)..………………..……………………………37 Table 3.6 (Morphometrics for Abralia)…….……………………………………………37 Table 3.7 (Genetic distances for Myopsida and Oegopsidasquid)………………………41 Table 3.8 (Genetic distances for Sepioteuthis lessoniana clades)...……………………..44 Table 3.9 (Morphometrics of Abdopus)…………………………………….….………...44 Table 3.10 (Morphometrics of Callistoctopus)………………………….……………….45 Table 3.11 (Morphometrics of Cistopus)……………………………………..………….46 Table 3.12 (Morphometrics of Amphioctopus)…………………………………………..46 Table 3.13 (Morphometrics of Octopus)………...………..……………………………..47 Table 3.14 (Genetic distances for Abdopus)………………………………..……………51 Table 3.15 (Genetic distances for Callistoctopus)..……………………………………...51 Table 3.16 (Genetic distances for Cistopus)…………………………………………..…51 Table 3.17 (Genetic distances for Amphioctopus)……………………………..………...52 10 1. Introduction Globally, coral reefs are considered hotspots for biodiversity, hosting an extensive variety of fish and invertebrate species (Roberts et al. 2002). Interest in the health of these fragile ecosystems created an interest in the study of biodiversity as it has been hypothesized as a factor related to ecosystem health (May 1986; Currie and Paquin 1987). Although there are many methods that can be used to assess the health of an ecosystem,