頭足綱動物控制不斷變化的體色與動作來做偽裝與溝通，而視覺溝通對於他們在生殖行為上扮演著非常重要的腳色。在軟絲 (萊氏擬烏賊) 中，不同體型雄軟絲會利用不同的體色與動作來吸引雌性的注意，而不同的交配方式會造成精莢放置於雌性身體的不同位置，進而導致授精成功率上的差異。但是，目前的研究在交配時的體色與動作，且生殖行為上為何會演化出如此多樣的交配方式，仍是所知甚少。因此，為了探究軟絲的視覺溝通與其生殖策略，我們首先記錄了所有從野外與實驗室內產卵場雌雄軟絲交配時的體色變化。生殖行為過程可分為常見的五大類：平行游動、雄性護雌、雄性間競爭、雄性下位交配與雄性上位交配，並總共區分出27種體色單元。進一步分析每個行為下的體色單元時序變化結果顯示，不僅每個行為皆是由數個體色單元所組成，且每種體色單元也會在不同行為下展現。其生殖行為可由特定數個體色單元的組成即可表示，顯示軟絲此體色變化模式為非常有效率的行為溝通模式。另外，我們借由調整配對雄性的體型來控制其交配策略，用以探討雄性不同交配姿勢與雌性選擇對於交配成功率的影響。結果顯示，雄性會快速地改變自身的交配策略以應對雌性的交配選擇。雌性會利用體色變化的視覺訊息以傳達拒絕雄性某特定的交配姿勢，迫使雄性改採另一種策略來成功交配。此外，借由分析不同體型雄性生殖構造來探討在精子競爭層面是否有型值上與雄性交配策略上的關係。目前的研究結果，雄性軟絲無論是在精莢的型值或是精子的大小上，皆無呈現二型性。綜合本結果與前結果雄性可依雌性選擇快速改變交配姿勢，顯示雄性萊氏擬烏賊在生殖策略上具有可塑性。軟絲雄性對於不同交配策略的高可塑性，將使本交配型為一雌多雄的軟絲雄性能不斷的在兩種交配策略中切換，以維持生殖成功率。本研究借由研究軟絲的生殖時期的行為體色、雌性選擇、雄性生殖策略選擇與觀察生殖構造形質，希望能對於頭足綱生物的視覺溝通與生殖策略上有更進一步的了解。

Cephalopods use dynamic body patterns for camouflage and communication. This complex body patterning also plays an important role in their reproductive behavior. In the oval squid Sepioteuthis lessoniana, males of different body sizes use distinct mating body patterns and postures to interact with females, this results in different spermatophore planting sites and fertilization success. However, little is known about the significance of these body patterns in courtship behavior, and the evolution of these mating postures in reproductive strategy. To study visual communication and reproductive strategy in S. lessoniana, we first characterized body patterns of males and females during courtship (i.e., ethogram of distinct chromatic component expression) at spawning grounds in wild and in captivity. Five behaviors were commonly seen among these reproductively active squids, namely parallel swimming, male guarding, male-male fighting, male-parallel mating, and male-upturn mating. Total of 27 chromatic components were identified in these five reproductive behaviors. By analyzing the dynamic body patterning time series associated with each behavior, the results not only revealed that each behavior is composed of multiple chromatic components, but the findings also showed that each component is often associated with multiple behaviors. The present study thus demonstrates that dynamic body pattering, by expressing unique sets of key components, is an efficient behavioral information in communication among individuals in the oval squid. To assess mating success of different male copulation postures and female choices in the mating behavior, we conducted a captivity experiment in which males displayed alternative mating types and the correlation between male mating success and female choice was examined. We found that male squid changed their adopting mating tactics according to female mate choice. This finding suggests that females use body patterns to show their mating preference visually and that males alter their mating tactics accordingly. In addition, we examined the sperm morphology from different sizes of males to investigate if there is any morphological difference in sperm competition. We found that there was no distinct size dimorphism in relation to either spermatophore size or sperm morphology in oval squids. This suggests that males change mating tactic flexibly according to male-male competition and female choice outcomes, thus morphological specialization is not evolved in this species. This behavioral plasticity may benefit the reproductive success of these males and maintain the dynamic stability of two alternative mating types in this polyandrous species. By studying the body patterning, female choice, male alternative mating types, and diverse reproductive structure in oval squids, this study sheds light on visual communication and reproductive strategy of cephalopods.

Abstract ………………..…………………………………...……………………….I
摘要 ……….....………………………..…..………………………….……………III
謝詞 ……….....……………………………………………………………………..V
Contents …………………………………………………………………………VII
Chapter 1: Introduction 1
1.1 Visual communication in cephalopods 2
1.1.1 Visual communication during reproductive behaviors 2
1.2 Reproductive behaviors in cephalopods 3
1.2.1 Diverse mating behaviors in Loliginidae 3
1.2.2 Female choice in Loliginidae 4
1.3 Sperm competition during reproductive behaviors 5
1.4 Sepioteuthis lessoniana 6
1.5 Specific aims 6
Chapter 2: Quantitative analysis of dynamic body patterning reveals the grammar of visual signals during the reproductive behavior of the oval squid Sepioteuthis lessoniana 9
2.1 Materials and methods 10
2.1.1 Animals 10
2.1.2 Analyzing the dynamic body patterns 11
2.2 Results 14
2.3 Discussion 21
Chapter 3: Female choice leads to a switch in oval squid male mating tactics 27
3.1 Materials and methods 28
3.1.1 Animals and captive rearing condition 28
3.1.2 Video recording and image analysis 28
3.2 Results 30
3.3 Discussion 34
Chapter 4: The overlapping reproductive traits of the two male mating types of the oval squid Sepioteuthis lessoniana 38
4.1 Materials and methods 39
4.1.1 Sample collection 39
4.1.2 Morphological analysis of the spermatophore and sperm 40
4.2 Results 42
4.3 Discussion 45
Chapter 5: Overall conclusion and general discussion 51
References 54
Tables 60
Figures 62
Supplementary figures 88
Movies 98
Appendix

Akmajian, A., Demers, R. A., Farmer, A. K. and Harnish, R. M. (2010). Linguistics: An Introduction to Language and Communication. Cambridge, Mass.: MIT Press.
Andersson, M. B. (1994). Sexual Selection. Princeton, NJ: Princeton University Press.
Birkhead, T. R., Hosken, D. J. and Pitnick, S. S. (2009). Sperm Biology: An Evolutionary Perspective. London: Academic press.
Birkhead, T. R., Martinez, J. G., Burke, T. and Froman, D. P. (1999). Sperm mobility determines the outcome of sperm competition in the domestic fowl. Proceedings of the Royal Society of London B: Biological Sciences 266, 1759-1764.
Boal, J. G. (1996). Absence of social recognition in laboratory-reared cuttlefish, Sepia officinalis L (Mollusca: Cephalopoda). Animal Behaviour 52, 529-537.
Boal, J. G. (1997). Female choice of males in cuttlefish (Mollusca : Cephalopoda). Behaviour 134, 975-988.
Bolhuis, J. J. and Everaert, M. (2013). Birdsong, Speech, and Language: Exploring The Evolution of Mind and Brain. Cambridge, MA: MIT press.
Boyle, P. R. and Ngoile, M. A. K. (1993). Assessment of maturity state and seasonality of reproduction in Loligo forbesi (Cephalopoda: Loliginidae) from Scottish waters. Recent advances in cephalopod fisheries biology. Tokai University Press, Tokyo, 37-48.
Buresch, K. C., Maxwell, M. R., Cox, M. R. and Hanlon, R. T. (2009). Temporal dynamics of mating and paternity in the squid Loligo pealeii. Marine Ecology Progress Series 387, 197-203.
Byrne, R. A., Griebel, U., Wood, J. B. and Mather, J. A. (2003). Squid say it with skin: a graphic model for skin displays in Caribbean reef squid (Sepioteuthis sepioidae). Free University of Berlin, Germany,.
Caldwell, R. L., Ross, R., Rodaniche, A. and Huffard, C. L. (2015). Behavior and body patterns of the larger pacific striped octopus. PLoS ONE 10, e0134152.
Chen, C. S., Chen, J. Y. and Lin, C. W. (2015). Variation in life-history traits for micro-cohorts of Sepioteuthis lessoniana in the waters off northern Taiwan. Fisheries Science 81, 53-64.
Cheng, S. H., Anderson, F. E., Bergman, A., Mahardika, G. N., Muchlisin, Z. A., Dang, B. T., Calumpong, H. P., Mohamed, K. S., Sasikumar, G. and Venkatesan, V. (2014). Molecular evidence for co-occurring cryptic lineages within the Sepioteuthis cf. lessoniana species complex in the Indian and Indo-West Pacific Oceans. Hydrobiologia 725, 165-188.
Chiao, C. C., Chubb, C. and Hanlon, R. T. (2015). A review of visual perception mechanisms that regulate rapid adaptive camouflage in cuttlefish. Journal of Comparative Physiology a-Neuroethology Sensory Neural and Behavioral Physiology 201, 933-945.
Cummins, S. F., Boal, J. G., Buresch, K. C., Kuanpradit, C., Sobhon, P., Holm, J. B., Degnan, B. M., Nagle, G. T. and Hanlon, R. T. (2011). Extreme Aggression in Male Squid Induced by a beta-MSP-like Pheromone. Current Biology 21, 322-327.
DeMartini, D. G., Ghoshal, A., Pandolfi, E., Weaver, A. T., Baum, M. and Morse, D. E. (2013). Dynamic biophotonics: female squid exhibit sexually dimorphic tunable leucophores and iridocytes. Journal of Experimental Biology 216, 3733-3741.
DiMarco, F. P. and Hanlon, R. T. (1997). Agonistic behavior in the squid Loligo pealei (Loliginidae, Teuthoidea): fighting tactics and the effects of size and resource value. Ethology 103, 89-108.
Eberhard, W. G. (1982). Beetle horn dimorphism: making the best of a bad lot. The American Naturalist 119, 420-426.
Fitzpatrick, J. L., Montgomerie, R., Desjardins, J. K., Stiver, K. A., Kolm, N. and Balshine, S. (2009). Female promiscuity promotes the evolution of faster sperm in cichlid fishes. Proceedings of the National Academy of Sciences 106, 1128-1132.
Gage, M. J. G., Macfarlane, C. P., Yeates, S., Ward, R. G., Searle, J. B. and Parker, G. A. (2004). Spermatozoal traits and sperm competition in Atlantic salmon: Relative sperm velocity is the primary determinant of fertilization success. Current Biology 14, 44-47.
Gomendio, M. and Roldan, E. R. S. (2008). Implications of diversity in sperm size and function for sperm competition and fertility. International Journal of Developmental Biology 52, 439-447.
Gross, M. R. (1996). Alternative reproductive strategies and tactics: diversity within sexes. Trends in Ecology & Evolution 11, 92-98.
Hall, K. C. and Hanlon, R. T. (2002). Principal features of the mating system of a large spawning aggregation of the giant Australian cuttlefish Sepia apama (Mollusca : Cephalopoda). Marine Biology 140, 533-545.
Hanlon, R. T. (1982). The functional organization of chromatophores and iridescent cells in the body patterning of Loligo plei (Cephalopoda, Myopsida). Malacologia 23, 89-119.
Hanlon, R. T. (1988). Behavioral and body patterning characters useful in taxonomy and field identification of cephalopods. Malacologia 29, 247-265.
Hanlon, R. T. (1996). Evolutionary games that squids play: fighting, courting, sneaking, and mating behaviors used for sexual selection in Loligo pealei. Biological Bulletin 191, 309-310.
Hanlon, R. T., Maxwell, M. R. and Shashar, N. (1997). Behavioral dynamics that would lead to multiple paternity within egg capsules of the squid Loligo pealei. Biological Bulletin 193, 212-214.
Hanlon, R. T., Maxwell, M. R., Shashar, N., Loew, E. R. and Boyle, K. L. (1999). An ethogram of body patterning behavior in the biomedically and commercially valuable squid Loligo pealei off Cape Cod, Massachusetts. Biological Bulletin 197, 49-62.
Hanlon, R. T. and Messenger, J. B. (1996). Cephalopod Behaviour. Cambridge: Cambridge University Press.
Hanlon, R. T., Naud, M. J., Shaw, P. W. and Havenhand, J. N. (2005). Behavioural ecology: transient sexual mimicry leads to fertilization. Nature 433, 212.
Hanlon, R. T., Smale, M. J. and Sauer, W. H. H. (1994). An ethogram of body patterning behavior in the squid Loligo vulgaris reynaudii on spawning grounds in South Africa. Biological Bulletin 187, 363-372.
Hanlon, R. T., Smale, M. J. and Sauer, W. H. H. (2002). The mating system of the squid Loligo vulgaris reynaudii (Cephalopoda, Mollusca) off South Africa: fighting, guarding, sneaking, mating and egg laying behavior. Bulletin of Marine Science 71, 331-345.
Hirohashi, N., Alvarez, L., Shiba, K., Fujiwara, E., Iwata, Y., Mohri, T., Inaba, K., Chiba, K., Ochi, H., Supuran, C. T. et al. (2013). Sperm from sneaker male squids exhibit chemotactic swarming to CO2. Current Biology 23, 775-781.
Hirohashi, N. and Iwata, Y. (2013). The different types of sperm morphology and behavior within a single species: why do sperm of squid sneaker males form a cluster? Communicative & Integrative Biology 6, e26729.
Hirohashi, N., Iwata, Y., Sauer, W. H. and Kakiuchi, Y. (2014). Respiratory CO2 mediates sperm chemotaxis in squids. In Sexual Reproduction in Animals and Plants, pp. 13-21: Springer.
Hirohashi, N., Tamura-Nakano, M., Nakaya, F., Iida, T. and Iwata, Y. (2016). Sneaker male squid produce long-lived spermatozoa by modulating their energy metabolism. Journal of Biological Chemistry 291, 19324-19334.
Huffard, C. L., Caldwell, R. L. and Boneka, F. (2008). Mating behavior of Abdopus aculeatus (d'Orbigny 1834) (Cephalopoda : Octopodidae) in the wild. Marine Biology 154, 353-362.
Huffard, C. L., Caldwell, R. L. and Boneka, F. (2010). Male-male and male-female aggression may influence mating associations in wild octopuses (Abdopus aculeatus). Journal of Comparative Psychology 124, 38.
Immler, S., Pryke, S. R., Birkhead, T. R. and Griffith, S. C. (2010). Pronounced within-individual plasticity in sperm morphometry across social environments. Evolution 64, 1634-43.
Iwata, Y., Ito, K. and Sakurai, Y. (2008). Effect of low temperature on mating behavior of squid Loligo bleekeri. Fisheries Science 74, 1345-1347.
Iwata, Y., Munehara, H. and Sakurai, Y. (2005). Dependence of paternity rates on alternative reproductive behaviors in the squid Loligo bleekeri. Marine Ecology Progress Series 298, 219-228.
Iwata, Y. and Sakurai, Y. (2007). Threshold dimorphism in ejaculate characteristics in the squid Loligo bleekeri. Marine Ecology Progress Series 345, 141-146.
Iwata, Y., Sakurai, Y. and Shaw, P. (2015). Dimorphic sperm-transfer strategies and alternative mating tactics in loliginid squid. Journal of Molluscan Studies 81, 147-151.
Iwata, Y., Shaw, P., Fujiwara, E., Shiba, K., Kakiuchi, Y. and Hirohashi, N. (2011). Why small males have big sperm: dimorphic squid sperm linked to alternative mating behaviours. BMC Evolutionary Biology 11, 236.
Izuka, T., Segawa, S., Okutani, T. and Numachi, K. (1994). Evidence on the existence of three species in the oval squid Sepioteuthis lessoniana complex in Ishigaki Island, Okinawa, southwestern Japan, by isozyme analyses. Venus (Japanese Journal of Malacology) 53, 217-228.
Jantzen, T. M. and Havenhand, J. N. (2003a). Reproductive behavior in the squid Sepioteuthis australis from South Australia: ethogram of reproductive body patterns. Biological Bulletin 204, 290-304.
Jantzen, T. M. and Havenhand, J. N. (2003b). Reproductive behavior in the squid Sepioteuthis australis from South Australia: interactions on the spawning grounds. Biological Bulletin 204, 305-317.
Jereb, P. and Roper, C. F. E. (2005). Cephalopods of the World : an Annotated and Illustrated Catalogue of Cephalopod Species Known to Date. Rome: Food and Agriculture Organization of the United Nations.
Langridge, K. V., Broom, M. and Osorio, D. (2007). Selective signalling by cuttlefish to predators. Current Biology 17, R1044-R1045.
Lin, C. Y. and Chiao, C. C. (2017). Female choice leads to a switch in oval squid male mating tactics. Biological Bulletin 233, 219-226.
Lin, C. Y., Tsai, Y. C. and Chiao, C. C. (2017). Quantitative analysis of dynamic body patterning reveals the grammar of visual signals during the reproductive behavior of the oval squid Sepioteuthis lessoniana. Frontiers in Ecology and Evolution, section Behavioral and Evolutionary Ecology 5.
Manning, A. and Dawkins, M. S. (2012). An Introduction to Animal Behaviour. Cambridge: Cambridge University Press.
Marcus, G. F. (2006). Language: startling starlings. Nature 440, 1117-1118.
Mather, J. (2016). Mating games squid play: reproductive behaviour and sexual skin displays in Caribbean reef squid Sepioteuthis sepioidea. Marine and Freshwater Behaviour and Physiology 49, 359-373.
Messenger, J. B. (1991). Photoreception and vision in molluscs. In Vision and Visual Dysfunction. Vol. 2: Evolution of the Eye and Visual System, eds. J. R. Cronly-Dillon and R. L. Gregory), pp. 364-397. London: Macmillan Press.
Moynihan, M. (1985). Communication and Noncommunication by Cephalopods. Bloomington: Indiana University Press.
Moynihan, M. and Rodaniche, A. F. (1982). The behavior and natural history of the caribbean reef squid Sepioteuthis sepioidea with a consideration of social, signal, and defensive patterns for difficult and dangerous environments. Fortschritte der Verhaltensforschung 25, 9-150.
Naud, M. J., Hanlon, R. T., Hall, K. C., Shaw, P. W. and Havenhand, J. N. (2004). Behavioural and genetic assessment of reproductive success in a spawning aggregation of the Australian giant cuttlefish, Sepia apama. Animal Behaviour 67, 1043-1050.
Naud, M. J., Sauer, W. H., McKeown, N. J. and Shaw, P. W. (2016). Multiple mating, paternity and complex fertilisation patterns in the chokka squid Loligo reynaudi. PLoS ONE 11, e0146995.
Packard, A. (1982). Morphogenesis of chromatophore patterns in cephalopods: Are morphological and physiological ‘units’ the same? Malacologia 23, 193-201.
Packard, A. and Hochberg, F. G. (1977). Skin patterning in octopus and other genera. In Symposia of the Zoological Society of London, vol. 38, pp. 191-231. London.
Packard, A. and Sanders, G. (1969). What the octopus shows to the world. Endeavour 28, 92-99.
Packard, A. and Sanders, G. D. (1971). Body patterns of Octopus vulgaris and maturation of the response to disturbance. Animal Behaviour 19, 780-790.
Parker, G. A. (1970). Sperm competition and its evolutionary consequences in the insects. Biological Reviews 45, 525-567.
Pepperberg, I. M. (2009). The Alex Studies: Cognitive and Communicative Abilities of Grey Parrots. Cambridge: Harvard University Press.
Roper, C. F. E. and Hochberg, F. G. (1988). Behavior and systematics of cephalopods from Lizard Island, Australia, based on color and body patterns. Malacologia 29, 153-194.
Sato, N., Kasugai, T. and Munehara, H. (2013). Sperm transfer or spermatangia removal: Postcopulatory behaviour of picking up spermatangium by female Japanese pygmy squid. Marine Biology 160, 553-561.
Sato, N., Kasugai, T. and Munehara, H. (2014). Female pygmy squid cryptically favour small males and fast copulation as observed by removal of spermatangia. Evolutionary Biology 41, 221-228.
Scheel, D., Godfrey-Smith, P. and Lawrence, M. (2016). Signal use by octopuses in agonistic interactions. Current Biology 26, 377-382.
Shashar, N. and Hanlon, R. T. (2013). Spawning behavior dynamics at communal egg beds in the squid Doryteuthis (Loligo) pealeii. Journal of Experimental Marine Biology and Ecology 447, 65-74.
Simmons, L. W., Tomkins, J. L. and Hunt, J. (1999). Sperm competition games played by dimorphic male beetles. Proceedings of the Royal Society B-Biological Sciences 266, 145-150.
Staudinger, M. D., Hanlon, R. T. and Juanes, F. (2011). Primary and secondary defences of squid to cruising and ambush fish predators: variable tactics and their survival value. Animal Behaviour 81, 585-594.
Taborsky, M. (1998). Sperm competition in fish: 'bourgeois' males and parasitic spawning. Trends in Ecology & Evolution 13, 222-227.
Wada, T., Takegaki, T., Mori, T. and Natsukari, Y. (2005). Alternative male mating behaviors dependent on relative body size in captive oval squid Sepioteuthis lessoniana (Cephalopoda, Loliginidae). Zoological Science 22, 645-651.
Wada, Y. and Kobayashi, T. (1995). On an iteroparity of the oval squid Sepioteuthis lessoniana. Nippon Suisan Gakkaishi 61, 151-158.
Wood, J. B., Pennoyer, K. E. and Derby, C. D. (2008). Ink is a conspecific alarm cue in the Caribbean reef squid, Sepioteuthis sepioidea. Journal of Experimental Marine Biology and Ecology 367, 11-16.