對於廣泛分佈的物種，它們的族群間基因交流容易因為海洋中各種因素
而中斷，導致族群分化。過去研究認為複雜的沿岸流系統，長江沖淡水以及更
新世冰期週期海平面變化是造成中國沿岸海洋生物分化的可能因素。髭縞虾虎
(Tridentiger barbatus)是一種廣泛分佈于中國，韓國和日本沿岸的底棲性虾虎，
其族群遺傳結構亦可能受到上述因素的影響。為研究其族群遺傳結構，本研究
從中國沿岸 14 個樣點採集共 258 尾髭縞虾虎個體，并擴增其細胞色素 b，視紫
紅質 (Rhodopsin)和 RAG1 基因序列片段進行分析。以貝葉斯分析法(Bayesian
analysis) 重建的親緣關係樹和 TCS 法重構的單倍型網絡圖均显示髭縞虾虎內部
存在兩個分化較深的譜系(southern lineage 和 northern lineage)，這兩個系譜的分
佈模式與中國沿岸夏季洋流模式類似，意味著洋流對髭縞蝦虎魚親緣地理模式
可能有重要影響。細胞色素 b 基因單倍型分佈，pairwise Fst 和 AMOVA 表明
southern lineage 內亦存在分化，可能與近岸洋流和潛在的地理屏障有關。兩系
譜間分化時間約為一百六十五萬年前, 可追溯到更新世時期。兩系譜的 Tajima’
D 和 Fu’s Fs 值均為顯著的負值，表明兩系譜在過去存在突然擴張。貝葉斯天際
線(Bayesian skyline plot)分析結果顯示 southern lineage 和 northern lineage 的突然
擴張時間分別為一萬年前和一萬五千年前，均發生于末次冰期(The last glacial
period)結束后(約 2 萬年前)。這意味著更新世冰期可能對髭縞虾虎的分化和族群
規模有重要影響。

Gene exchange of widespread marine species populations may be easily interrupted
by various conditions in the ocean, which may result in divergences. Previous studies
suggested that complex coastal current system, Changjiang Diluted Water and
Pleistocene Glacial Cycle are possible factors causing divergence of marine
organisms in Coastal China. Tridentiger barbatus is a benthic goby widely spread in
coastal China, Korea and Japan. In this study, we collected 258 individuals of T.
barbatus from 14 sites along the coast of China to explore its genetic structure.
Fragments of cyt b, Rh and RAG1 from all individuals were obtained and analyzed.
Bayesian trees and TCS network revealed two deeply divided lineages (southern
lineage and northern lineage) in T. barbatus, distributions of the two lineages
appeared to match summer coastal current pattern in China. It implied that currents
may have important effect on the phylogeographic pattern of T. barbatus. Cyt b
haplotype distributions and AMOVA showed further divisions within southern
lineage, which may be related to currents and barriers in coastal China. Divergence
time of the two lineages was 1.65 Mya, which may be dated back to Pleistocene
glacial cycles. Tajima’ D and Fu’ Fs statistics were significantly negative in both
southern and northern lineage, suggesting that the two lineages might have undergone
sudden expansions in past. Bayesian skyline plot showed that sudden expansion times
of southern and northern lineage were 0.010 Mya and 0.015 Mya, respectively, which
happened at the end of the last glacial period (about 0.02 Mya). It implied that
Pleistocene glaciation may be important to divergence and population size of T.
barbatus.

Content
論文審定書..................................................................................................................... i
Acknowledgement ......................................................................................................... ii
摘要.............................................................................................................................. iii
Abstract ......................................................................................................................... iv
Contents ......................................................................................................................... v
List of Figures ............................................................................................................... vi
List of Tables ............................................................................................................... vii
1. Introduction ................................................................................................................ 1
2. Material and methods ................................................................................................. 4
2.1. Sampling and DNA extraction............................................................................. 4
2.2 PCR and sequencing ............................................................................................ 4
2.3 Genetic diversities and genealogy........................................................................ 5
2.4 Molecular dating and historical demography ...................................................... 6
3. Results ........................................................................................................................ 8
3.1 Genetic diversity and genealogy .......................................................................... 8
3.2 Population differentiations within lineages ......................................................... 9
3.3 Molecular dating and historical demography .................................................... 10
4. Discussions .............................................................................................................. 11
4.1 Phylogeographic pattern within T. barbatus ..................................................... 11
4.2 Population differentiation within southern lineage ........................................... 12
4.3 Divergence and historical demography ............................................................. 13
Reference ..................................................................................................................... 16

List of Figures
Fig 1. Sampling sites and summer hydrographical conditions. . ................................. 27
Fig 2. TCS haplotype networks. . ................................................................................ 28
Fig 3. Distributions of southern lineage and northern lineage. ................................... 29
Fig 4. Bayesian trees of concatenated haplotypes. ...................................................... 30
Fig 5. Bayesian trees of cyt b haplotypes. . ................................................................. 31
Fig 6. Bayesian trees of RAG1 and Rh haplotypes. . .................................................. 32
Fig 7. Pairwise Fst matrices of southern lineage and northern lineage. . .................... 33
Fig 8. Isolation by distance. ........................................................................................ 34
Fig 9. Bayesian skyline plot. ....................................................................................... 35

List of Tables
Table 1. Primers. ......................................................................................................... 36
Table 2. Diveristy indexes of southern lineage and northern lineage. ......................... 37
Table 3. Pairwise Fst matrices of southern lineage and northern lineage. .................. 38
Table 4. AMOVA. ....................................................................................................... 39

Reference
Akihito, A., Iwata., Kobayashi, T., Ikeo, K., Imanishi, T., Ono, H., Umehara, Y., Hamamatsu, C., Sugiyama, K., Ikeda, Y., Sakamoto, K., Fumihito, A., Ohno, S., Gojobori, T., 2000. Evolutionary aspects of gobioid fishes based upon a phylogenetic analysis of mitochondrial cytochrome b genes. Gene 259, 5–15.
Bakun A. 2006. Fronts and eddies as key structures in the habitat of marine fish larvae: opportunity, adaptive response and competitive advantage. Scientia Marina, 70(S2): 105-122.
Bay, L. K., Crozier, R. H. & Caley, M. J. 2006. The relationship between population genetic structure and pelagic larval duration in coral reef fishes on the Great Barrier Reef. Marine Biology, 149(5), 1247-1256.
Beldade, R., Heiser, J. B., Robertson, D. R., Gasparini, J. L., Floeter, S. R. & Bernardi, G. 2009. Historical biogeography and speciation in the Creole wrasses (Labridae, Clepticus). Marine biology, 156(4), 679-687.
Bohonak, A. J. 2002. IBD (Isolation By Distance): a program for analyses of isolation by distance. Journal of Heredity 93: 153-154.
Bouckaert, R., Heled, J., Kühnert, D., Vaughan, T., Wu, C-H., Xie, D., Suchard, M. A., Rambaut, A. & Drummond, A. J. 2014. BEAST 2: A Software Platform for Bayesian Evolutionary Analysis. PLoS Computational Biology, 10(4), e1003537.
Bradbury, I. R., Laurel, B., Snelgrove, P. V., Bentzen, P., & Campana, S. E. 2008. Global patterns in marine dispersal estimates: the influence of geography, taxonomic category and life history. Proceedings of the Royal Society of London B: Biological Sciences, 275(1644), 1803-1809.
Chen, W.J., Bonillo, C. & Lecointre, G. 2003. Repeatability of clades as a criterion of reliability: a case study for molecular phylogeny of Acanthomorpha (Teleostei) with larger number of taxa. Molecular Phylogenetics and Evolution 26, 262–288.
Clark, P. U., Dyke, A. S., Shakun, J. D., Carlson, A. E., Clark, J., Wohlfarth, B., Mitrovica, X.J., Hostetler, S. W. & McCabe, A. M. 2009. The last glacial maximum. science, 325(5941), 710-714.
Clement, M., Snell, Q., Walker, P., Posada, D. & Crandall, K. 2002. TCS: Estimating gene genealogies. Parallel and Distributed Processing Symposium, International Proceedings, 2, 184.
Cowen, R. K. & Sponaugle, S. 2009. Larval dispersal and marine population connectivity. Annual Review of Marine Science 2009;1:443–66.
Cui, R., Pan, Y., Yang, X. & Wang, Y. 2013. A new barbeled goby from south China (Teleostei: Gobiidae). Zootaxa, 3670(2), 177-192.
Darriba, D., Taboada, G. L., Doallo, R., & Posada, D. 2012. JModelTest 2: more models, new heuristics and parallel computing. Nature methods, 9(8): 772.
Díaz-Jaimes, P., Uribe-Alcocer, M., Rocha-Olivares, A., García-de-León, F. J., Nortmoon, P., & Durand, J. D. 2010. Global phylogeography of the dolphinfish (Coryphaena hippurus): the influence of large effective population size and recent dispersal on the divergence of a marine pelagic cosmopolitan species. Molecular Phylogenetics and Evolution, 57(3), 1209-1218.
Dong, Y. W., Wang, H. S., Han, G. D., Ke, C. H., Zhan, X., Nakano, T. & Williams, G. A. 2012. The impact of Yangtze River discharge, ocean currents and historical events on the biogeographic pattern of Cellana toreuma along the China coast. PLoS One, 7(4), e36178.
Dôtu, Y. 1957. The bionomics and life history of the goby, Triaenopogon barbatus (Günther) in the innermost part of Ariake Sound. Science Bulletin of the Faculty of Agriculture, Kyushu University 16:261-274. (In Japanese).
Drummond, A. J., Suchard, M. A., Xie, D. & Rambaut, A. 2012. Bayesian phylogenetics with BEAUti and the BEAST 1.7.Molecular biology and evolution, 29(8), 1969-1973.
Ely, B., Viñas, J., Bremer, J. R. A., Black, D., Lucas, L., Covello, K., Labrie, A. V. & Thelen, E. 2005. Consequences of the historical demography on the global population structure of two highly migratory cosmopolitan marine fishes: the yellowfin tuna (Thunnus albacares) and the skipjack tuna (Katsuwonus pelamis). BMC Evolutionary Biology, 5(1), 19.
Excoffier L. & Lischer H. E. L. 2010. Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Molecular ecology resources, 10(3): 564-567.
Floeter, S. R., Rocha, L. A., Robertson, D. R., Joyeux, J. C., Smith‐Vaniz, W. F., Wirtz, P., Edwards A. J., Barreiros J. P., Ferreira C. E. L., Gasparini J. L., Brito A., Falcón J. M., Bowen B. W., & Brito, A. 2008. Atlantic reef fish biogeography and evolution. Journal of Biogeography, 35(1), 22-47.
Fu, Y. X. & Li, W. H. 1993. Statistical tests of neutrality of mutations. Genetics, 133(3), 693-709.
García, G. 2012. Phylogeography from South-Western Atlantic Ocean: Challenges for the Southern Hemisphere. Current Topics in Phylogenetics and Phylogeography of Terrestrial and Aquatic Systems. (Anamthawat-Jónsson, K., ed), pp. 13–32. Rijeka: InTech.
Han, Z. Q., Gao, T. X., Yanagimoto, T. & Sakurai, Y. 2008. Deep phylogeographic break among white croaker Pennahia argentata (Sciaenidae, Perciformes) populations in North-western Pacific. Fisheries Science, 74(4), 770-780.
Han, Z., Yanagimoto, T., Zhang, Y. & Gao, T. 2012. Phylogeography study of Ammodytes personatus in Northwestern Pacific: Pleistocene isolation, temperature and current conducted secondary contact. PLoS One, 7(5), e37425.
Han, Z., Zheng, W., Zhu, W., Yu, C., Shui, B. & Gao, T. 2015. A barrier to gene flow in the Asian paddle crab, Charybdis japonica, in the Yellow Sea. ICES Journal of Marine Science, 72(5), 1440-1448.
Hasegawa, M., Kishino, H. & Yano, T. A. 1985. Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. Journal of molecular evolution, 22(2), 160-174.
Heled, J. & Drummond, A. J. 2008. Bayesian inference of population size history from multiple loci. BMC Evolutionary Biology, 8(1), 289.
Hohenlohe, P. A. 2004. Limits to gene flow in marine animals with planktonic larvae: models of Littorina species around Point Conception, California. Biological Journal of the linnean Society, 82(2), 169-187.
Huang, W. C., Chang, J. T., Liao, C., Tawa, A., Iizuka, Y., Liao, T. Y. & Shiao, J. C. 2018. Pelagic larval duration, growth rate, and population genetic structure of the tidepool snake moray Uropterygius micropterus around the southern Ryukyu Islands, Taiwan, and the central Philippines. PeerJ, 6, e4741.
Hwang, J. H., Van, S. P., Choi, B. J., Chang, Y. S., & Kim, Y. H. 2014. The physical processes in the Yellow Sea. Ocean & coastal management, 102, 449-457.
Iglésias, S. P., Frotté, L., & Sellos, D. Y. 2016. Gobius salamansa, a new species of goby (Gobiidae) from the Cape Verde Islands supported by a unique cephalic lateral line system and DNA barcoding. Ichthyological research, 63(3), 356-369.
Isobe, A. 2008. Recent advances in ocean-circulation research on the Yellow Sea and East China Sea shelves. Journal of oceanography, 64(4), 569-584.
Johns, G. C. & Avise, J. C. 1998. A comparative summary of genetic distances in the vertebrates from the mitochondrial cytochrome b gene. Molecular Biology and Evolution, 15(11), 1481-1490.
Kelly, R. P. & Palumbi, S. R. 2010. Genetic structure among 50 species of the northeastern Pacific rocky intertidal community. PLoS One, 5(1), e8594.
Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of molecular evolution, 16(2), 111-120.
Kumar, S., Stecher, G. & Tamura, K. 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular biology and evolution, 33(7), 1870-1874.
Larmuseau, M. H., Van Houdt, J. K., Guelinckx, J., Hellemans, B. & Volckaert, F. A. 2009. Distributional and demographic consequences of Pleistocene climate fluctuations for a marine demersal fish in the north‐eastern Atlantic. Journal of Biogeography, 36(6), 1138-1151.
Levin, L. A. 2006. Recent progress in understanding larval dispersal: new directions and digressions. Integrative and comparative biology, 46(3), 282-297.
Li, G., Li, P., Liu, Y., Qiao, L., Ma, Y., Xu, J., & Yang, Z. 2014. Sedimentary system response to the global sea level change in the East China Seas since the last glacial maximum. Earth-Science Reviews, 139, 390-405.
Liu J. X., Gao T. X., Wu S. F. & Zhang Y. P. 2007. Pleistocene isolation in the Northwestern Pacific marginal seas and limited dispersal in a marine fish, Chelon haematocheilus (Temminck & Schlegel, 1845). Molecular Ecology, 16(2): 275-288.
Lobel P. S. & Robinson A. R. 1986.Transport and entrapment of fish larvae by ocean mesoscale eddies and currents in Hawaiian waters. Deep Sea Research Part A. Oceanographic Research Papers, 33(4): 483-500.
López, J. A., Chen, W. J. & Ortí, G. 2004. Esociform phylogeny. Copeia, 2004(3), 449-464.
Luiz, O. J., Madin, J. S., Robertson, D. R., Rocha, L. A., Wirtz, P. & Floeter, S. R. 2012. Ecological traits influencing range expansion across large oceanic dispersal barriers: insights from tropical Atlantic reef fishes. Proc. R. Soc. B, 279(1730), 1033-1040.
Magsino, R. M. & Juinio-Meñez, M. A. 2008. The influence of contrasting life history traits and oceanic processes on genetic structuring of rabbitfish populations Siganus argenteus and Siganus fuscescens along the eastern Philippine coasts. Marine Biology, 154(3), 519-532.
Miller, M. A., Pfeiffer, W. & Schwartz, T. 2010. "Creating the CIPRES Science Gateway for inference of large phylogenetic trees" in Proceedings of the Gateway Computing Environments Workshop (GCE), 14 Nov. 2010, New Orleans, LA pp 1 - 8.
Ni, G., Li, Q., Kong, L. & Yu, H. 2014. Comparative phylogeography in marginal seas of the northwestern Pacific. Molecular Ecology, 23(3), 534-548.
Ni, G., Li, Q., Kong, L. & Zheng, X. 2012. Phylogeography of bivalve Cyclina sinensis: testing the historical glaciations and Changjiang River outflow hypotheses in northwestern Pacific. PLoS One, 7(11), e49487.
Ni, G., Li, Q., Ni, L., Kong, L. & Yu, H. 2015. Population subdivision of the surf clam Mactra chinensis in the East China Sea: Changjiang River outflow is not the sole driver. PeerJ, 3, e1240.
Palumbi, S. R. 1994. Genetic divergence, reproductive isolation, and marine speciation. Annual Review of Ecology and Systematics, 25, 547–572.
Qiu, F., Li, H., Lin, H., Ding, S. & Miyamoto, M. M. 2016. Phylogeography of the inshore fish, Bostrychus sinensis, along the Pacific coastline of China. Molecular phylogenetics and evolution, 96, 112-117.
Riginos, C. & Victor, B. C. 2001. Larval spatial distributions and other early life–history characteristics predict genetic differentiation in eastern Pacific blennioid fishes. Proceedings of the Royal Society of London B: Biological Sciences, 268(1479), 1931-1936.
Riginos, C., Douglas, K. E., Jin, Y., Shanahan, D. F. & Treml, E. A. 2011. Effects of geography and life history traits on genetic differentiation in benthic marine fishes. Ecography, 34(4), 566-575.
Rocha L. A., Bass A. L., Robertson D. R. & Bowen B.W. 2002. Adult habitat preferences, larval dispersal, and the comparative phylogeography of three Atlantic surgeon fishes (Teleostei: Acanthuridae). Molecular Ecology, 11, 243–252.
Rocha, L. A. 2003. Patterns of distribution and processes of speciation in Brazilian reef fishes. Journal of Biogeography, 30(8), 1161-1171.
Rocha-Olivares, A. & Vetter, R. D. 1999. Effects of oceanographic circulation on the gene flow, genetic structure, and phylogeography of the rosethorn rockfish (Sebastes helvomaculatus). Canadian Journal of Fisheries and Aquatic Sciences, 56(5), 803-813.
Ronquist, F., Teslenko, M., Van Der Mark, P., Ayres, D. L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard M. A. & Huelsenbeck, J. P. 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic biology, 61(3), 539-542.
Rozas, J., Ferrer-Mata, A., Sanchez-DelBarrio, J. C., Guirao-Librado P., Ramos-Onsins S. E., Sanchez-Garcia, A., 2017. Dna SP v6: DNA sequence polymorphism analysis of large data. Molecular Biology and Evolution. (in press)
Sale, P. F. 1980. The ecology of fishes on coral reefs. Oceanography and Marine Biology, 18, 367–421.
Santos, S., Hrbek, T., Farias, I. P., Schneider, H. & Sampaio, I. 2006. Population genetic structuring of the king weakfish, Macrodon ancylodon (Sciaenidae), in Atlantic coastal waters of South America: deep genetic divergence without morphological change. Molecular Ecology, 15(14), 4361-4373.
Schunter, C., Carreras-Carbonell, J., Planes, S., Sala, E., Ballesteros, E., Zabala, M., Harmelin, J. G., Harmelin-Vivien, M., Macpherson E. & Pascual, M. 2011. Genetic connectivity patterns in an endangered species: The dusky grouper (Epinephelus marginatus). Journal of Experimental Marine Biology and Ecology, 401(1-2), 126-133.
Schwarz, G. 1978. Estimating the dimension of a model. The annals of statistics, 6(2), 461-464.
Selkoe, K. A. & Toonen, R. J. 2011. Marine connectivity: a new look at pelagic larval duration and genetic metrics of dispersal. Marine Ecology Progress Series, 436, 291-305.
Sevilla, R. G., Diez, A., Norén, M., Mouchel, O., Jérôme, M., Verrez‐Bagnis, V., Pelt, H. V., Favre-Krey, L., Krey, G., The Fishtrace Consortium & Bautista, J. M. 2007. Primers and polymerase chain reaction conditions for DNA barcoding teleost fish based on the mitochondrial cytochrome b and nuclear rhodopsin genes. Molecular Ecology Resources, 7(5), 730-734.
Shanks, A. L., Grantham, B. A. & Carr, M. H. 2003. Propagule dispersal distance and the size and spacing of marine reserves. Ecological applications, S159-S169.
Shanks, A. L. 2009. Pelagic larval duration and dispersal distance revisited. The biological bulletin, 216(3), 373-385.
Shen, K. N., Jamandre, B. W., Hsu, C. C., Tzeng, W. N. & Durand, J. D. 2011. Plio-Pleistocene sea level and temperature fluctuations in the northwestern Pacific promoted speciation in the globally-distributed flathead mullet Mugil cephalus. BMC Evolutionary Biology, 11(1), 83.
Shu, Y., Xue, H., Wang, D., Xie, Q., Chen, J., Li, J., Chen R., He Y. K. & Li, D. 2016. Observed evidence of the anomalous South China Sea western boundary current during the summers of 2010 and 2011. Journal of Geophysical Research: Oceans, 121(2), 1145-1159.
Song, N., Zhang, X. M., Sun, X. F., Yanagimoto, T. & Gao, T. X. 2010. Population genetic structure and larval dispersal potential of spottedtail goby Synechogobius ommaturus in the north‐west Pacific. Journal of Fish Biology, 77(2), 388-402.
Tajima, F. 1989. The effect of change in population size on DNA polymorphism. Genetics, 123(3), 597-601.
Von der Heyden, S., Bowie, R. C. K., Prochazka, K., Bloomer, P., Crane, N. L. & Bernardi, G. 2011. Phylogeographic patterns and cryptic speciation across oceanographic barriers in South African intertidal fishes. Journal of evolutionary biology, 24(11), 2505-2519.
Wang, B., Li, Y., & Yuan, D. 2013. Effects of topography on the sub-tidal circulation in the southwestern Huanghai Sea (Yellow Sea) in summer. Acta Oceanologica Sinica, 32(3), 1-9.
Waples, R. S. 1987. A multispecies approach to the analysis of gene flow in marine shore fishes. Evolution, 41(2), 385-400.
Weersing, K. & Toonen, R. J. 2009. Population genetics, larval dispersal, and connectivity in marine systems. Marine Ecology Progress Series, 393, 1-12.
White, C., Selkoe, K. A., Watson, J., Siegel, D. A., Zacherl, D. C. & Toonen, R. J. 2010. Ocean currents help explain population genetic structure. Proceedings of the Royal Society of London B: Biological Sciences, rspb20092214.
Wolanski, E. 2017. Bounded and unbounded boundaries–Untangling mechanisms for estuarine-marine ecological connectivity: Scales of m to 10,000 km–A review. Estuarine, Coastal and Shelf Science, 198, 378-392.
Wonham, M. J., Carlton, J. T., Ruiz, G. M. & Smith, L. D. 2000. Fish and ships: relating dispersal frequency to success in biological invasions. Marine Biology, 136(6), 1111-1121.
Wu, H. L. & Zhong, J. S. 2008. Fauna sinica, ostichthyes, perciformes (V), gobioidei. Science, Beijing: 643-657 (in Chinese).
Xing, L., Zhao, M., Zhang, H., Zhao, X., Zhao, X., Yang, Z. & Liu, C. 2012. Biomarker evidence for paleoenvironmental changes in the southern Yellow Sea over the last 8200 years. Chinese Journal of Oceanology and Limnology, 30(1), 1-11.
Xuereb, A., Benestan, L., Normandeau, E., Daigle, R. M., Curtis, J. M., Bernatchez, L. & Fortin, M. J. 2018. Asymmetric oceanographic processes mediate connectivity and population genetic structure, as revealed by RAD seq, in a highly dispersive marine invertebrate (Parastichopus californicus). Molecular ecology, 27(10), 2347-2364.