Phylogenetics, Biology, and Ecology of Two Unclassified Species of Small Near-Shore Octopuses in Hawai'i
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Phylogenetics, biology, and ecology of two unclassified species of small near-shore octopuses in Hawai‘i Presented to the Faculty of the Tropical Conservation Biology and Environmental Science Graduate Program University of Hawai‘i at Hilo In partial fulfillment of the requirement for the degree of Master of Science In Tropical Conservation Biology and Environmental Science By Adam L. Daw Hilo, Hawai‘i May, 2016 Thesis Committee: Maria Haws, Chairperson Kevin Hopkins Christine Huffard Acknowledgements This project would not be possible without the support of my committee members, Maria Haws, Kevin Hopkins, and Christine Huffard who provided invaluable encouragement and critics through out the research and writing of this thesis. I would like to thank the other groups with the University of Hawaii at Hilo that assisted me in various forms including the Pacific Aquaculture and Coastal Resources Center for allowing me to use their facilities, Anne Veillet of the Genetics Core Facility and Tara Holitzki of the Analytical Laboratory for assisting me in processing samples, and the faculty of the Tropical Conservation Biology and Environmental Science Department. This would also not be possible without the help of many people that assisted in collecting octopus, in particular Senifa Annandale and Keile‘a Carlson. Finally, I would like to express my gratitude to family and friends who have supported along the way. ii © Adam L. Daw, 2016 iii Abstract The current understanding of the biology and ecology of octopuses has focused on a limited number of species compared to the high diversity of species currently described. Within the tropical – subtropical Pacific there have been multiple studies describing the phylogenetic relationships among the near-shore octopus species, in particular within the southern and eastern regions. As we start to better understand species diversity through phylogenetic analysis, there is still limited information describing the biology and ecology of these species. In this study the phylogenetic relationships of two unclassified species of octopuses (Abdopus sp. 1 “Crescent octopus” and Octopus cf. vitiensis) inhabiting the near-shore coral reef ecosystem of Hawai‘i were compared to the octopus diversity of the tropical – subtropical Pacific. In order to better understand the physiology and biology of these two species, the growth rates of wild caught specimens and the effect of temperature on the onset of hatching were observed. Finally δ15N and δ13C stable isotope analyses were used to compare the trophic niche of four octopus species (Octopus cyanea, Callistoctopus ornatus, Abdopus sp. 1, and Octopus cf. vitiensis) that utilize the near-shore coral reef habitat in order to determine if there is a difference between species utilizing the same habitat. The results of the phylogenetic analysis indicate that Abdopus sp. 1 is a distinct species and is closely related to Octopus laqueus. Octopus cf. vitiensis is closely related to but distinct from Octopus oliveri, there were no genetic sequences of the type specimen or other non-Hawaiian Octopus cf. vitiensis available to conduct comparisons to determine if the Hawaiian population is the same or a distinct species. Growth observations of the two species indicate that Octopus. cf. vitiensis is sexual dimorphic with males obtaining a smaller maximum mass and mantle length (observed max 74.2g, 47.9mm) compared to females (observed max 133.0g, 60.0mm). There was no observed difference in size and mass between males and females of Abdopus sp. 1 in this study, possibly due to small sample size. The timing of the onset of hatching of the two species was negatively correlated with temperature, ranging from 27 days at 28°C to 57 days at 20°C for Octopus cf. vitiensis. There was no difference in iv egg developmental time between species at corresponding temperatures. Results from stable isotope analysis indicate that habitat/location has a greater effect on trophic niche than octopus size for the species and sizes analyzed. Differences in δ15N between species were variable between habitats/locations. This indicates that these species are opportunistic feeders, with prey selection based on what prey are more prevalent or easy to capture in a specific location. v Table of Contents Acknowledgements………………………………………………… ii Abstract………………………………………………………....….. iv List of Tables………………………………………………………..vii List of Figures……………………………………………………… viii List of Appendices…………………………………………………..xi Introduction………………………………………………………… 1 Methods……………………………………………………………..7 Results………………………………………………………............ 14 Discussion………………………………………………………….. 19 Conclusion…………………………………………………………. 28 Notes……………………………………………………………….. 31 References…………………………………………………………. 32 Tables………………………………………………………............ 38 Figures……………………………………………………………... 47 Appendix A………………………………………………………… 75 vi List of Tables Table 1. Primer sequences used for analysis ..........................................38 Table 2. PCR cycling regime .................................................................38 Table 3. Large dataset octopus species ..................................................39 Table 4. Recovered DNA sequence lengths ...........................................42 Table 5. List of Genbank accession numbers for DNA sequences obtained in this study ...............................................................42 Table 6. Loci and sequence lengths used in phylogenetic analysis .......43 Table 7. Pairwise estimate of cox1 evolutionary divergence .................43 Table 8. Division of eggs into temperature trials ...................................44 Table 9. Number of individuals of each species collected from each site ....................................................................................44 Table 10. Mean δ15N and δ13C values for octopus species found in literature and from this study ...................................................45 vii List of Figures Figure 1. Map of Hawai‘i Island, Hawai‘i with collection sites .............47 Figure 2. Phylogram of large cox1 data set utilizing Maximum Likelihood analysis ..................................................................48 Figure 3. Phylogram of large cox1 data set utilizing Bayesian analysis ..... ..................................................................................................49 Figure 4. Phylogram of small cox1 data set utilizing Maximum Likelihood analysis ..................................................................50 Figure 5. Phylogram of small cox1 data set utilizing Bayesian analysis .... ..................................................................................................51 Figure 6. Phylogram of cox1+16s concatenated data set utilizing Bayesian analysis .....................................................................52 Figure 7. Phylogram of cox1+16s concatenated data set utilizing Maximum Likelihood analysis ................................................53 Figure 8. Phylogram of cox1+cox3 concatenated data set utilizing Bayesian analysis .....................................................................54 Figure 9. Phylogram of cox1+cox3 concatenated data set utilizing Maximum Likelihood analysis ................................................55 viii Figure 10. Plot of mantle length versus mass for Octopus cf. vitiensis ....56 Figure 11. Plot of mantle length versus mass for Abdopus sp. 1 ..............57 Figure 12. Plot of mantle length to mass ratio by sex for Abdopus sp. 1 and Octopus cf. vitiensis ..........................................................58 Figure 13. Plot of recorded masses of Octopus cf. vitiensis by sex ..........59 Figure 14. Change in mass of individual Octopus cf. vitiensis in captivity by sex .......................................................................................60 Figure 15. Plot of recorded masses of Abdopus sp. 1 by sex ....................61 Figure 16. Change in mass of individual Abdopus sp. 1 in captivity by sex ............................................................................................62 Figure 17. Comparison of the onset of hatching between Octopus cf. vitiensis and Abdopus sp. 1 at multiple temperatures ..............63 Figure 18. Plot of the onset of hatching versus temperature for Octopus cf. vietiensis ...................................................................................64 Figure 19. Comparison of δ15N values of three octopus species at site 1 ..... ..................................................................................................65 Figure 20. Comparison of δ15N values for Octopus cf. vitiensis between sites 1 and 2 ..............................................................................66 ix Figure 21. Comparison of δ15N values for Octopus cyanea between sites 2 and 3 .........................................................................................67 Figure 22. Comparison of δ15N values for Octopus cf. vitiensis and Octopus cyanea at site 2 ..........................................................68 Figure 23. Comparison of δ13C values for octopuses between sites 1, 2, and 3 .........................................................................................69 Figure 24. Comparison of δ15N values by species ....................................70 Figure 25. Comparison of the effect of mass on δ15N values of octopus species ......................................................................................71 Figure 26. Comparison of δ15N versus δ13C