珊瑚礁生態系因為受到許多不可逆轉的天然災害、人為及全球暖化的因素而面臨嚴重的威脅。台灣的珊瑚群聚在熱帶及亞熱帶地區皆有分布，大約有317個物種, 也受到威脅。受到干擾的珊瑚群聚主要依靠成功的生殖來維持及回復。因此，為執行有效的策略來管理珊瑚礁生態系，珊瑚生殖的研究乃為重要。過去關於石珊瑚的有性生殖之研究主要集中在熱帶區產之物種。在台灣，目前有56種石珊瑚其包括一模式物種 - 腎型真葉珊瑚的有性生殖之特徵曾被描述過。 對其它珊瑚物種於此方面的認知還顯不足。因此本論文的研究目標為 1）以深入的野外潛水採樣 (2013年至2016年間進行)、觀察及組織切片的方式來紀錄在台灣北部地區的珊瑚群聚物種之有性生殖; 2）利用組織切片的方式和水族缸飼養方式來確認台灣北部地衣微孔珊瑚的有性生殖的特徵，包括性別，受精方式及生殖腺發育過程，3）利用腎型真葉珊瑚當模式物種來了解Dmrt基因家族的在珊瑚有性生殖過程中扮演的角色。
從第一個研究(2013年到2016收集的資料)的結果顯示， 排放精卵珊瑚的物種及數量在年間有顯著的差異。這四年內一共觀察到六科19屬43種珊瑚排放精卵。而排放精卵在七月及／或八月的晚上八點到十點之間 。而且同一株珊瑚不同步排放精卵的現象也很顯著。
從第二個研究結合組織切片和水族養殖觀察之結果，我確認了微孔地衣珊瑚是雌雄異體及孵育型。生殖腺發育及受精時間有季節性。在組織切片的觀察過程，發現兩個從未發現的生殖型態特微：在早期生殖腺發育時，雌雄同體的個體，其卵和精細胞在精巢內一起發育，以及在精巢內發現黑色素顆粒細胞。
從第三個研究中, 我成功在腎型針葉珊瑚找到並描述七個Dmrt基因(EaDmrtA, EaDmrtB, EaDmrtC, EaDmrtE, EaDmrtF, EaDmrtG, 和 EaDmrtH) ，基因表現分析結果只有EaDmrtE具在卵發育時具專一性的表現。根據演化分析結果，此在卵發育過程具專一性表現的EaDmrtE，和在多孔軸孔珊瑚的DM1 (目前鑑定為DmrtA; 可能也和有性生殖有關)，及脊椎動物性別決定基因Dmrt1的其親源關係並不相近。EaDmrtE的基因表現在未受精的卵及初期受精卵維持高度的表現，但是在受精的後期顯著下降，因此推測EaDmrtE為母性因子在卵細胞發育過程及初期受精卵發育時扮演重要的角色。而在早期動物演化的過程中， 就有和生殖有關的Dmrt基因的存在，且這樣的基因可能經過多次的演化。雌性專一性表現的EaDmrtE 除了可能應用在鑑定珊瑚的性別，且提供一個分子生物研究的基因材料，來研究並建立生殖基因調控的機制。

Coral reef ecosystem is now facing severe threats due to irreversible disturbances caused by natural disasters, anthropogenic effects and global warming. Coral communities in Taiwan are found in both subtropical and tropical regions and contain about 317 species that are also under threats. The persistence and recovery of coral communities depend partly on the success of reproduction after the disturbances. It is, therefore, important to study their reproductive biology in order to propose the effective strategy for the management of the coral reef ecosystem. Most of the previous studies documenting the sexual reproduction in scleractinian corals, however, concerned mainly the tropical-living species. To date, among the reported species in Taiwan, only 56 species including a model species, Euphyllia ancora, have been investigated. Little was known from other species. The objectives of this dissertation are 1) to study the coral spawning in the subtropical coral communities in northern Taiwan by comprehensive field samplings and observations conducted from 2013 and 2016, and by the assessments from histological analyses of the collected samples; 2) to characterize the sexual system, mode of reproduction as well as gametogenesis of Porites lichen living in Pitoujiiao, northern Taiwan by histological analyses and in situ aquarium observations; 3) to investigate the possible roles of the Dmrt gene family in coral reproduction by using Euphyllia ancora as a model.
First, field observations and histological examinations revealed a large annual variation of spawning pattern among species and individuals. Forty-three species classified into 19 genera and six families spawned asynchronously in the nights (between 8 pm to 10 pm) July and/or August from 2013 to 2014. In addition, corals spawned asynchronously within the same colony.
Second, the combined results from the histological examinations and others revealed that the gonochoric P. lichen is a polygamodioecious brooder that displays a seasonal pattern of sexual reproduction in gametogenesis and embryogenesis. In addition, I also reported two new morphological configurations during the early stage of gametogenesis with oocytes being developed within spermaries in bisexual polyps and melanin granular cells being clustered into spermaries.
Third, I successfully isolated and characterized seven paralogous Dmrt genes (EaDmrtA, EaDmrtB, EaDmrtC, EaDmrtE, EaDmrtF, EaDmrtG, and EaDmrtH) from the gonochoric E. ancora. I found that EaDmrtE is predominantly expressed in oocytes. This oocyte-specific DmrtE is not closely related to neither the DM1 gene (i.e. currently known as DmrtA; its function is possibly related to sexual reproduction) of Acropora millepora nor to Dmrt1 of vertebrates based on the result of phylogenetic analysis; these two genes are involved in sexual reproduction, especially in sex determination (e.g. vertebrate Dmrt1 gene). High levels of EaDmrtE transcripts were detected in unfertilized mature eggs, and that were retained in newly formed zygotes and, then, the levels of expression decreased during embryonic development. I suggest that this newly discovered gene possibly plays an important role during oogenesis and early embryogenesis in corals as a maternal factor. These results strongly support a complex evolutionary history for the sexual reproduction-associated Dmrt genes with the possibility that such kinds of the genes have originated multiple times early in the evolution of metazoans. The identification of female-specific genes (E. ancora DmrtE) not only provides useful biomarkers to determine the sex of gonochoric corals, but also provide materials for molecular studies on the characterization of the genetic pathways underlying the sexual reproduction in corals.

Acknowledgments

中文摘要……………………………………………………………………………...I
Abstract………………………………………………………………..…………….III
Chapter 1 General Introduction…………………………………………………….1
1.1 Sexual reproduction in scleractinian corals……………………………………..1
1.2 Gametogenesis and spawning in corals…………………………………………3
1.3 Coral communities in Taiwan…………………………………………………..4
1.4 Studies of sexual reproduction in corals in Taiwan…………………………….6
1.5 Molecular mechanisms of sexual reproduction in scleractinian corals………...7
1.6 Objectives of this dissertation…………….…………………………………….8
Chapter 2 Multi-species Spawning of Scleractinian Corals in Pitoujiiao and Longdong, Northern Taiwan……………………………………………………….13
2.1 Introduction…………….…………………..……………………………...…..13
2.2 Materials and methods……..……………..………………………………..….14
2.2.1 Study sites……………………………………………………………...14
2.2.2 Coral spawning by field survey………………….……………………..14
2.2.3 Multiple spawning among conspecific colonies……………………….15
2.2.4 Inference of spawning period by histological analysis………………...15
2.2.5 Seawater temperatures and typhoon occurrence……………………….16
2.2.6 Analytical methods……………………………………….…………….16
2.2.6.1 Species identification……………………………………………...16
2.2.6.2 Histological analysis………………………………………………16
2.2.6.3 Seawater temperature...……………………………………………17
2.3 Results and Discussion………………………….....………………………...17
2.3.1 Spawning time window and related to the full moon from 2013 to 2016…………………………………………………………………..………17
2.3.2 Annual variation in spawning pattern from 2014 to 2016…………….18
2.3.3 Multiple spawning in conspecific species and within colony……....…19
2.3.4 Spawning periods inferred by histological analyses…………...….…..19
2.3.5 Temperature…………………………………………………….……..20
2.3.6 Typhoon disturbance during spawning and post-spawning…….…..…21
Chapter 3 Characterization of sexual reproduction of a scleractinian, Porites lichen Pitoujiiao, northern Taiwan……………………….……………………...31
3.1 Introduction….……………………………………….……..………..……….31
3.2 Materials and methods….………………………………………..……….…..31
3.2.1 Sample collections……………………………………………….……31
3.2.2 Histological analysis…………………………………..………………32
3.2.3 In situ observation of coral reproduction in aquarium………….……..32
3.3 Analytic methods…………………………………………………...…………33
3.4 Results and Discussion………………………...…………………….…….….33
3.4.1 Sexual system …………………….…………..……………………….33
3.4.2 Gametogenesis of P. lichen……………………………………………34
3.4.3 Germ cell development in bisexual colonies………………….……….36
3.4.4 mode of reproduction and reproductive cycle in P. lichen………….....37
3.4.5 Melanin granular cells…………………………………………….……37
Chapter 4 A novel female-specific and sexual reproduction-associated Dmrt gene discovered in the stony coral, Euphyllia ancora (Cnidarian, Anthozoa)…..46
4.1 Introduction…………………………………………….……………………..46
4.2 Materials and Methods……………………………………….……………….48
4.2.1 Coral collection in the field and different time points from adult colony……………………………………………………………………..….48
4.2.2 Collection for specific tissues……………………………...…………..49
4.2.3 Analytical methods……………………………………….…………….49
4.2.3.1 cDNA synthesis, polymerase chain reaction amplification, cloning and sequencing …………………………..………………………..49
4.2.3.2 Quantitative RT-PCR analysis……………………………………51
4.2.3.3 Phylogenetic analyses and comparison of sequence similarity…...53
4.2.3.4 Northern Bloting and in situ hybridization……….…………...…..54
4.3 Results………………………………………………………………………...55
4.3.1 Isolation and characterization of EaDmrt paralogs……………...……..55
4.3.2 Phylogeny of metazoan Dmrt genes…………………………….……..56
4.3.3 Temporal gene expression of EaDmrts………………………………..56
4.3.4 Spatial gene expression for EaDmrts……………………….…………57
4.3.5 Northern blot analysis and in situ hybridization of EaDmrtE transcripts…………………………………………………………………….57
4.3.6 EaDmrtE as a maternal factor…………………………….……………57
4.4 Discussion………………………………...………………………………….58
4.4.1 Female-specific Dmrt genes in metazoans……………………………..58
4.4.2 Transcriptional regulations in oogenesis……………………………….59
4.4.3 Maternal inheritance of Dmrts…………………………………………60
4.4.4 Homology of cnidarian sexual reproduction-associated Dmrt genes..…61
Chapter 5 Conclusion and perspectives……………………………………..…..77
References……………………………………………………….…………………..80
Appendix I. Coral species studied in Taiwan in Chapter 2…………………………..98
Appendix II. Primers used for the classical PCR amplification, 5'' and 3'' RACE, and q-PCR in chapter 4……………………………………………………………………105
Appendix III. NCBI sequences and reference for chapter 4………………..………110
Appendix IV. References for Fig. 4.1 in Chapter 4…………………………..…….120 
Figure content
Figure 2.1 Study sites……………………………………………………………..….24
Figure 2.2.1 Abundance of spawning corals in Merulinidae in July and August from 2014 to 2016………………………………………………………………..….25
Figure 2.2.2 Abundance of spawning corals in Poritidae, Agaricidae, Fungiidae, Psammocoridae, and Lobophyllidae in July and August from 2014 to 2016……………………………………………………………………………26
Figure 2.3 Multiple spawning in Favites pentagona, Astrea curta, Favites stylifera and Favites halicora from 20 to 28 July 2016…………………………..…….27
Figure 2.4 Inference of the coral spawning by histological analyses in 2014…….…28
Figure 2.5 Sea water temperature from January 2014 to September 2016 in Pitoujiiao, northern Taiwan……………………………………………………………….29
Figure 2.6 Typhoon occurrences in July, August (during spawning) and September (after spawning) from 2014 to 2016…………………….…………….……….30
Figure 3.1 Study sites and a colony of Porites lichen………………………..………40
Figure 3.2 Histological examinations of the sexual system of P. lichen………….…41
Figure 3.3 Spermatogenesis in male colonies in 2015……………………………….42
Figure 3.4 Oogenesis in female colonies in 2015……………………………………43
Figure 3.5 Germ cell development in bisexual colonies…………………….……….44
Figure 3.6 Melanin granular cells in P. lichen………………………………...……..45
Figure 4.1 Summary of the available data on the diversity, tissue-specific expression patterns, and functions of the Dmrt members in metazoan adults and embryos………………………………………………………………………..64
Figure 4.2 The schematic diagrams of the Euphyllia ancora Dmrt transcripts…………………………………..……………………………….…66
Figure 4.3 The 50% majority-rule consensus tree is resulted from the Bayesian phylogenetic analysis with best-fit JTT+Gamma model based on the amino-acid sequences from the DM domains o the metazoan Dmrts……………..……….67
Figure 4.4 The 50% majority-rule consensus tree resulting from the Bayesian phylogenetic analysis with best-fit JTT+Gamma model based on the amino-acid sequences from the DM domains of the cnidarian Dmrts………………….….69
Figure 4.5 Quantification of the relative expression of EaDmrts in gonad tissues during spermatogenesis and oogenesis of gonad tissues by quantitative RT-PCR analysis………………………………………………………………….…….71
Figure 4.6 Quantification of the relative expression of EaDmrts in the male and female tissues that were collected in March…………………….…………….73
Figure 4.7 Expression of EaDmrtE in ovarian tissues……………………..………..75
Figure 4.8 Relative expression of EaDmrtE during E. ancora embryonic development …………………………………………………………………….…………...76
 
Table content
Table 1.1 Coral species studied in Taiwan……………………………………………9
Table 2.1 Numbers of spawning colony and species from 2014 to 2016………..…..23
Table 3.1 Criteria for oogenic and spermatogenic stages for P. lichen…………...…39
Table 4.1 Comparison of similarity of E. ancora Dmrt amino acid sequences (full sequence and DM/DMA domains)………………………………………………..….63

Abascal F, Zardoya R, Posada D (2005) ProtTest selection of best-fit models of protein evolution. Bioinformatics 21:2104-2105
Arrigoni R, Stefani F, Pichon M, Galli P, Benzoni F (2012) Molecular phylogeny of the robust clade (Faviidae, Mussidae, Merulinidae, and Pectiniidae): An Indian Ocean perspective. Mol Phylogenet Evol 65:183-193
Babcock RC, Bull GD, Harrison PL, Heyward AL, Oliver JK, Wallace CC, Willis BL (1986) Synchronous spawning of 105 scleractinian coral species on the Great Barrier Reef. Mar Biol 90:397-394
Baird AH, Guest JR, Willis BL (2009) Systematic and Biogeographical Patterns in the Reproductive Biology of Scleractinian Corals. Annu Rev Ecol Evol Syst 40:551-571
Bastidas C, Cróquer A, Zubillaga AL, Ramos R, Kortnik V, Weinberger C, Márquez LM (2005) Coral mass- and split-spawning at a coastal and an offshore Venezuelan reefs, southern Caribbean. Hydrobiologia 541:101-106
Bauman AG, Baird AH, Cavalcante GH (2011) Coral reproduction in the world’s warmest reefs: southern Persian Gulf (Dubai, United Arab Emirates). Coral Reefs 30:405-413
Bellefroid EJ, Leclère L, Saulnier A, Keruzore M, Sirakov M, Vervoort M, De Clercq S (2013) Expanding roles for the evolutionarily conserved Dmrt sex transcriptional regulators during embryogenesis. Cell Mol Life Sci 70:3829-3845
Billeter JC, Villella A, Allendorfer JB, Dornan AJ, Richardson M, Gailey DA, Goodwin SF (2006) Isoform-specific control of male neuronal differentiation and behavior in Drosophila by the fruitless gene. Curr Biol 16:1063-1076
Bouwmeester J, Berumen ML, Baird AH (2012) Daytime broadcast spawning of Pocillopora verrucosa on coral reefs of the central Red Sea. Galaxea 13:23-24
Bouwmeester J, Baird AH, Chen CJ, Guest JR, Vicentuan KC, Berumen ML (2014) Multi-species spawning synchrony within scleractinian coral assemblages in the Red Sea. Coral Reefs 34:65-77
Brennan MD, Weiner AJ, Goralski TJ, Mahowald AP (1982) The follicle cells are a major site of vitellogenin synthesis in Drosophila melanogaster synthesis. Dev Biol 236:225-236
Bronstein O, Loya Y (2011) Daytime spawning of Porites rus on the coral reefs of Chumbe Island in Zanzibar, Western Indian Ocean (WIO). Coral Reefs 30:441-441
Burtis KC, Coschigano KT, Baker BS, Wensink PC (1991) The doublesex proteins of Drosophila melanogaster bind directly to a sex-specific yolk protein gene enhancer. EMBO J 10:2577-2582
Burtis KC, Baker BS (1989) Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced mRNAs encoding related sex specific polypeptides. Cell 56:997-1010
Cairns SD (1999) Species Richness of recent scleractinia. Atoll Res Bull 459: 1-46
Charnov EL, Smith JM, Bull JJ (1976) Why be an hermaphrodite? Nature 263:125-126
Chen CA (1999) Analysis of scleractinian distribution in Taiwan indicating a pattern congruent with sea surface temperatures and currents: examples from Acropora and Faviidae Corals. Zool Stud38:119-129
Chen CA, Keshavmurthy S (2009) Taiwan as a connective stepping stone in the Kuroshio Traiangle and the conservation of coral ecosystems under the impacts of climate change. Kuroshio Science 3:15-22
Chornesky EA, Peters EC (1987) Sexual reproduction and colony growth in scleractinian coral Porites astreoides. Biol Bull 172:161-177
Chui APY, Wong MC, Liu SH, Lee GW, Chan SW, Lau PL, Leung SM, Jr. PA (2014) Gametogenesis, embryogenesis, and fertilization ecology of Platygyra acuta in marginal nonreefal coral communities in Hong Kong. J Mar Biol [doi: 10.1155/2014/953587]
Dai CF, Soong K, Fan TY (1993) Sexual reproduction of corals in Northern and Southern Taiwan. Proc 7th Int Coral Reef Symp 1:448-455
Dai CF (1997) Assessment of the present health of coral reefs in Taiwan. In: Grigg RW, Birkeland C (eds) Status of coral reefs in the Pacific. University of Hawaii, Hawaii, pp123-131
Dai CF, Fan TY, Yu JK (2000) Reproductive isolaton and genetic differentiation of a scleractinian coral Mycedium elephantotus. Mar Ecol Prog Ser 201:179-187
Dai CF, Soong K, Chen CA, Fan TY, Hsieh HJ, Jeng MS, Chen CH, Horng S (2004) Status of coral reefs of Taiwan. GCRM Report of East China Sea Region 3:153-163
Dai CF, Horng S (2009) Scleractinia fauna of Taiwan I. The complex group Taipei, Taiwan: National Taiwan University pp114
Denis V, Loubeyres M, Doo SS, de Palmas S, Keshavmurthy S, Hsieh HJ, Chen CA (2014) Can benthic algae mediate larval behavior and settlement of the coral Acropora muricata? Coral Reefs 33:431-440
De-Grandi A, Calvari V, Bertini V, Bulfone A, Peverali G, Camerino G, Borsani G, Guioli S (2000) The expression pattern of a mouse doublesex-related gene is consistent with a role in gonadal differentiation. Mech Dev 90:323-326

Dunn CW, Giribet G, Edgecombe GD, Hejnol A (2014) Animal phylogeny and its evolutionary implications. Annu Rev Ecol Evol Syst 45:371-395
Fan TY, Dai CF (1998) Sexual reproduction of the scleractinian coral Merulina ampliata in southern Taiwan. Bull Mar Sci 62:897-904
Fan TY, Dai CF (1999) Reproductive plasticity in the reef coral Echinopora lamellosa. Mar Ecol Prog Ser 190:297-301
Fan TY, Li JJ, Le SX, Fang LS (2002) Lunar periodicity of larval release by Pocilloporid corals in southern Taiwan. Zool Stud 41:288-294
Fukami H, Chen CA, Budd AF, Collins A, Wallace C, Chunag YY, Chen C, Dai CF, Iwao K, Shappard C, Knowlton N (2008) Mitochondrial and nuclear genes suggest that stony corals are monophyletic but most families of stony corals are not (Order Scleractinia, Class Anthozoa, Phylum Cnidaria) PLoS One 3:e3222
Ghiselin MT (1969) The evolution of hermaphroditism among animals. Q Rev Biol 44:189-208
Gittenberger A, Reijnen BT, Hoeksema BW (2011) A molecularly based phylogeny reconstruction of mushroom corals (Scleractinia: Fungiidae) with taxonomic consequences and evolutionary implication for life history traits. Contrib Zool 80:107-132
Glynn PW, Colley SB, Eakin CM, Smith DB, Cortés J, Gassman NJ, Guzmán HM, Rosario JBD, Feingold JS (1994) Reef coral reproduction in the eastern Pacific: Costa Rica, Panamá, and Galápagos Islands (Ecuador). II. Poritidae. Mar Biol 118:191-208
Graham NA, McClanahan TR, MacNeil MA, Wilson SK, Polunin NV, Jennings S, Chabanet P, Clark S, Spalding MD, Letourneur Y, Bigot L, Galzin R, Ohman MC, Garpe KC, Edwards AJ, Sheppard CR (2008) Climate warming, marine protected areas and the ocean-scale integrity of coral reef ecosystems. PLoS One 3:e3039
Guan G, Kobayashi T, Nagahama Y (2000) Sexually dimorphic expression of two types of DM (Doublesex/Mab-3)-domain genes in a teleost fish, the Tilapia (Oreochromis niloticus). Biochem Biophys Res Commun 272:662-666
Guest JR, Baird AH, Goh BPL, Chou LM (2005) Reproductive seasonality in an equatorial assemblage of scleractinian corals. Coral Reefs 24:112-116
Guest JR, Baird AH, Goh BPL, Chou LM (2012) Sexual systems in scleractinian corals: an unusual pattern in the reef-building species Diploastrea heliopora. Coral Reefs 31:705-713
Graham EM, Baird AH, Connolly SR (2008) Survival dynamics of scleractinian coral larvae and implications for dispersal. Coral reefs 27:529-539
Greenstein BJ, Pandolfi JM (2008) Escaping the heat: range shifts of reef coral taxa in coastal western Australia. Global Change Biol 14:513-528
Guindon S, Gascuel O (2003) A simple fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696-704
Hanafy MH, Aamer MA, Habib M, Rouphael AB, Baird AH (2010) Synchronous reproduction of corals in the Red Sea. Coral Reefs 29:119-124
Harrison PL (1988) Pseudo-gynodioecy: an unusual breeding system in the scleractinian coral Galaxea fascicularis. Proc 6th Int Coral Reef Symp 2:699-705
Harrison PL, Wallace CC (1990) Reproduction, dispersal and recruitment of Scleractinia corals. In: Dubinsky Z (ed), Ecosystems of the world, vol 25: Coral Reefs. Elsevier Science Publication Amsterdam, pp133-207
Harrison PL (2011) Sexual Reproduction of Scleractinian Corals. In: Dubinsky Z, Stambler N (eds) Coral reefs: An ecosystem in transition. Springer, London, New York, pp59-85
Hayashibara T, Shimoike K, Kimura T, Hosaka S, Heyward A, Harrison P, Kudo K, Omori M (1993) Patterns of coral spawning at Akajima Island, Okinawa, Japan. Mar Ecol Prog Ser 101:253-262
Ho MJ (2014) Studies on the coral communities in Yenliao Bay, northern Taiwan. Ph.D. thesis, National Taiwan University, pp14
Ho MJ, Dai CF (2014) Coral recruitment of a subtropical coral community at Yenliao Bay, northern Taiwan. Zool Stud 53:5
Hsieh HJ, Hsien YL, Jeng MS, Tsai WS, Su WC, Chen CA (2008) Tropical fishes killed by the cold. Coral Reefs 27:599-599
Huang D, Licuanan WY, Baird AH, Fukami H (2011) Cleaning up the ‘Bigmessidae’: molecular phylogeny of scleractinian corals from Faviidae, Merulinidae, Pectiniidae and Trachyphylliidae. BMC Evol Biol 11:37
Huang D, Licuanan WY, Hoeksema BW, Chen CA, Ang PO, Huang H, Lane DJW, Vo ST, Waheed Z, Affendi YA, Yeemin T, Chou LM (2014) Extraordinary diversity of reef corals in the South China Sea. Mar Biodiv 45:157-168
Huang D, Benzoni F, Fukami H, Knowlton N, Smith ND, Budd AF (2014) Taxonomic classification of the reef coral families Merulinidae, Montastraeidae, and Diploastraeidae (Cnidaria: Anthozoa: Scleractinia). Zool J Linnean Soc 171:277-355
Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:754-755
Hughes TP, Baird AH, Dinsdale EA, Moltschaniwskyj NA, Prachett MS, Tanner JE, Willis BL (2000) Supply-side ecology works both ways: the link between benthic adults, fecundity, and larval recruits. Ecology 81:2241-2249
Hughes TP, Baird AH, Bellwood DR, Card M, Connolly SR, Folke C, Grosberg R, Hoegh-Gludberg O, Jackson JBC, Kleypas J, Lough JM, Marshall P, Nystrom M, Palumbi SR, Pandolfi JM, Rosen B, Roughgarden J (2003) Climate change, human impacts and the resilience of coral reefs. Science 301:929
Hughes TP, Graham NA, Jackson JB, Mumby PJ, Steneck RS (2010) Rising to the challenge of sustaining coral reef resilience. Trends Ecol Evol 25:633-642
Johnsen H, Andersen Ø (2012) Sex dimorphic expression of five dmrt genes identified in the Atlantic cod genome. The fish-specific dmrt2b diverged from dmrt2a before the fish whole-genome duplication. Gene 505:221-223 

Kato Y, Kobayashi K, Watanabe H, Iguchi T (2011) Environmental sex determination in the branchiopod crustacean Daphnia magna: deep conservation of a doublesex gene in the sex-determining pathway. PLoS Genet 7:e1001345
Keshavmurthy S, Hsu CM, Kuo CT, Denis V, Leung JKL, Fontana S, Hsieh HJ, Tsai WS, Su WC, Chen CA (2012) Larvae development of fertilized "Pseudo-Gynodioecious" eggs suggests a sexual pattern of gynodioecy in Galaxea fascicularis (Scleractinia: Euphyllidae). Zool Stud 51:143-149
Kettlewell JR, Raymond CS, Zarkower D (2000) Temperature-dependent expression of turtle Dmrt1 prior to sexual differentiation. Genesis 26:174-178

Kim S, Kettlewell JR, Anderson RC, Bardwell VJ, Zarkower D (2003) Sexually dimorphic expression of multiple doublesex-related genes in the embryonic mouse gonad. Gene Expr Patterns 3:77-82
Kimble J, Sharrock WJ (1983) Tissue-specific synthesis of yolk proteins in Caenorhabditis elegans. Dev Biol 96:189-196
Kimura K, Hachiya T, Koganezawa M, Tazawa T, Yamamoto D (2008) Fruitless and doublesex coordinate to generate male-specific neurons that can initiate courtship. Neuron 59:759-769
Kleypas JA, McManus JW, Meñez LAB (1999) Environmental limits to coral reef development: Where do we Draw the line? Am Zool 39:146-159
Kongjandtre N, Ridgway T, Ward S, Hoegh-Guldberg O (2009) Broadcast spawning patterns of Favia species on the inshore reefs of Thailand. Coral reefs 29:227-234
Kojis BL, Quinn NJ (1982) Reproductive strategies in four species of Porites (scleractinia). Proc 4th Int Coral Reef Symp 2:145-151
Kolinski SP, Cox EF (2003) An update on modes and timing of gamete and planula release in Hawaiian scleractinian corals with implications for conservation and management. Pac Sci 57:17-27
Kondo M, Froschauer A, Kitano A, Nanda I, Hornung U, Volff JN, Asakawa S, Mitani H, Naruse K, Tanaka M, Schmid M, Shimizu N, Schartl M, Shima A (2002) Molecular cloning and characterization of DMRT genes from the medaka Oryzias latipes and the platyfish Xiphophorus maculatus. Gene 295:213-222
Krentz AD, Murphy MW, Sarver AL, Griswold MD, Bardwell V, Zarkower D (2011) DMRT1 promotes oogenesis by transcriptional activation of Stra8 in the mammalian fetal ovary. Dev Biol 356:63-70
Kuo CY, Meng PJ, Ho PH, Wang JT, Chen JP, Chiu YW, Lin HJ, Chang YC, Fan TY, Chen CA (2011) Damage to the reefs of Siangjiao Bay marine protected area of Kenting National Park, southern Taiwan during typhoon Morakot. Zool Stud 50:85
Kuo CY, Yuen YS, Meng PJ, Ho PH, Wang JT, Liu PJ, Chang YC, Dai CF, Fan TY, Lin HJ, Baird AH, Chen CA (2012) Recurrent disturbances and the degradation of hard coral communities in Taiwan. PLoS One 7:e44364
Lints R, Emmons SW (2002) Regulation of sex-specific differentiation and mating behavior in C. elegans by a new member of the DM domain transcription factor family. Genes Dev 16:2390-2402
Loya Y, Sakai K (2008) Bidirectional sex change in mushroom stony corals. Proc Biol Sci 275:2335-2343
Lu H, Huang X, Zhang L, Guo Y, Cheng H, Zhou R (2007) Multiple alternative splicing of mouse Dmrt1 during gonadal differentiation. Biochem Biophys Res Commun 352:630-634
Madin JS, Hoogenboom MO, Connolly SR, Darling ES, Falster DS, Huang D, Keith SA, Mizerek T, Pandolfi JM, Putnam HM, Baird AH (2016) A trait-based approach to advance coral reef science Trends Ecol Evol 31:419-428
Mallatt J, Craig CW, Yoder MJ (2010) Nearly complete rRNA genes assembled from across the metazoan animals: effects of more taxa, a structure-based alignment, and paired-sites evolutionary models on phylogeny recon- struction. Mol Phylogenet Evol 55:1-17
Matson CK, Murphy MW, Griswold MD, Yoshida S, Bardwell VJ, Zarkower D (2010) The mammalian doublesex homolog DMRT1 is a transcriptional gatekeeper that controls the mitosis versus meiosis decision in male germ cells. Dev Cell 19:612-624 

Maynard JA, Anthony KRN, Marshall PA, Masiri I (2008) Major bleaching events can lead to increased thermal tolerance in corals. Mar Biol 155:173-182
Mezaki T, Tohru H, Fumihito I, Shuu N, Yoko N, Mai M, Motoyuki T (2007) Spawning patterns of high latitude scleractinian corals from 2002 to 2006 at Nishidomari, Otsuki, Kochi, Japan. Kuroshio Biosphere 3:33-47
Miller SW, Hayward DC, Bunch TA, Miller DJ, Ball EE, Bardwell VJ, Zarkower D, Brower DL (2003) A DM domain protein from a coral, Acropora millepora, homologous to proteins important for sex determination. Evol Dev 5:251-258
Moniot B, Berta P, Scherer G, Sudbeck P, Poulat F (2000) Male specific expression suggests role of DMRT1 in human sex determination. Mech Dev 91:323-325
Muramatsu D, Silveira FL (2008) Gametogenesis in Madracis decactis Lyman, 1859 (Cnidaria, Scleractinia) from Ilha Grande Bay (Rio de Janeiro), Southeastern Brazil. Braz J Oceanorgr 56:297-305
Nozawa Y, Tokeshi M, Nojima S (2006) Reproduction and recruitment of scleractinian corals in a high-latitude coral community, Amakusa, southwestern Japan. 149:1047-1058
Nozawa Y, Tokeshi M, Nojima S (2008) Structure and dynamics of a high-latitude scleractinian coral community in Amakusa, southwestern Japan. Mar Ecol Prog Ser 358:151-160
Nozawa Y (2012) Annual variation in the timing of coral spawning in a high-latitude environment: Influence of temperature. Biol Bull 222:192-202
Nylander JAA, Wilgenbusch JC, Warren DL, Swofford DL (2008) AWTY (are we there yet?): a system for graphical exploration of MCMC convergence in Bayesian phylogenetics. Bioinformatics 24:581-583
Ottolenghi C, Veitia R, Quintana-murci L, Torchard D, Scapoli L, Souleyreau-Therville N, Beckmann J, Fellous M, Mcelreavey K (2000) The region on 9p associated with 46, XY sex reversal contains several transcripts expressed in the urogenital system and a novel doublesex- related domain. Genomics 64:170-178 

Ottolenghi C, Fellous M, Barbieri M, McElreavey K (2002) Novel paralogy relations among human chromosomes support a link between the phylogeny of doublesex-related genes and the evolution of sex determination. Genomics 79:333-343
Õunap K, Uibo O, Zordania R, Kiho L, Ilus T, Õiglane-Shlik E, Bartsch O (2004) Three patients with 9p deletions including DMRT1 and DMRT2: a girl with XY complement, bilateral ovotestes, and extreme growth retardation, and two XX females with normal pubertal development. Am J Med Genet 130A:415-423
Palmer CV, Traylor-Knowles NG, Willis BL, Bythell JC (2011) Corals use similar immune cells and wound-healing processes as those of higher organisms. PLoS One 6:e23992
Palumbi SR (2003) Population genetics, demographic connectivity, and the design of marine reserves. Ecol Appl 13:S146-S158
Pandolfi JM, Connolly SR, Marshall DJ, Cohen AL (2011) Projecting coral reef futures under global warming and ocean acidification. Science 333:418-422
Parlier D, Moers V, Van Campenhout C, Preillon J, Leclère l, Saulnier A, Sirakov M, Busengdal H, Kricha S, Marine JC, Rentzsch F, Bellefroid EJ (2013) The Xenopus doublesex-related gene Dmrt5 is required for olfactory placode neurogenesis. Dev Biol 373:39-52
Penland L, Kloulechad J, Idip D, van Woesik R (2004) Coral spawning in the western Pacific Ocean is related to solar insolation: evidence of multiple spawning events in Palau. Coral Reefs 23:133-140
Postlethwait JH, Laugé G, Handler AM (1980) Yolk protein synthesis in ovariectomized and genetically agametic [X87] Drosophila melanogaster. Gen Comp Endocr 40:385-390
Rambaut A. Sequence alignment editor, v2.0a11. Oxford, UK: University of Oxford; 1996. Available at: http://tree.bio.ed.ac.uk/ software/seal/. Accessed January 12, 2016
Rambaut A. FigTree v1.3.1: tree figure drawing tool, 2009. Available at: http://treebioedacuk/software/figtree/ Accessed April 20, 2012.
Raymond CS, Kettlewell JR, Hirsch B, Bardwell VJ, Zarkower D (1999a) Expression of Dmrt1 in the genital ridge of mouse and chicken embryos suggests a role in vertebrate sexual development. Dev Biol 215:208-220
Raymond CS, Parker ED, Kettlewell JR, Brown LG, Page DC, Kusz K, Jaruzelska J, Reinberg Y, Flejter WL, Bardwell VJ, Hirsch B, Zarkower D (1999b) A region of human chromosome 9p required for testis development contains two genes related to known sexual regulators. Hum Mol Genet 8:989-996

Raymond CS, Murphy MW, O’Sullivan MG, Bardwell VJ, Zarkower D (2000) Dmrt1, a gene related to worm and fly sexual regulators, is required for mammalian testis differentiation. Genes Dev 14:2587-2595
Ribas-Deulofeu L, Denis V, De Palmas S, Kuo CY, Hsieh HJ, Chen CA (2016) Structure of benthic communities along the Taiwan latitudinal gradient. PLoS One 11:e0160601
Richmond RH, Hunter CL (1990) Reproduction and recruitment of corals: comparisons among the Caribbean, the Tropical Pacific and the Red Sea. Mar Ecol Prog Ser 60:185-203
Rideout EJ, Dornan AJ, Neville MC, Eadie S, Goodwin SF (2010) Control of sexual differentiation and behavior by the doublesex gene in Drosophila melanogaster. Nat Neurosci 13:458-466
Rinkevich B, Loya Y (1979) Reproduction of the Red Sea coral Stylophora pistillata II. Synchronization in breeding and seasonality of planulae shedding. Mar Ecol Prog Ser 1:145-152
Rosser NL, Gilmour JP (2008) New insights into patterns of coral spawning on western Australian reefs. Coral Reefs 27:345-349
Ryan JF, Pang K, Schnitzler CE, Nguyen AD, Moreland RT, Simons DK, Koch BJ, Francis WR, Havlak P (2013) The genome of the ctenophore Mnemiopsisleidyi and its implications for cell type evolution. Science 342:1336-1344 

Shen MM, Hodgkin J (1988) Mab-3, a gene required for sex-specific yolk protein expression and a male-specific lineage in C. elegans. Cell 54:1019-1031
Shetty S, Kirby P, Zarkower D, Graves JAM (2002) DMRT1 in a ratite bird: evidence for a role in sex determination and discovery of a putative regulatory element Cytogenet Genome Res 99:245-251
Shikina S, Chen CJ, Liou JY, Shao ZF, Chung YJ, Lee YH, Chang CF (2012) Germ cell development in the scleractinian coral Euphyllia ancora (Cnidaria, Anthozoa). PLoS One 7:e41569
Shikina S, Chen CJ, Chung YJ, Shao ZF, Liou JY, Tseng HP, Lee YH, Chang CF (2013) Yolk formation in a stony coral Euphyllia ancora (Cnidaria, Anthozoa): insight into the evolution of vitellogenesis in nonbilaterian animals. Endocrinology 154:3447-3459
Shikina S, Chung YJ, Wang HM, Chiu YL, Shao ZF, Lee YH, Chang CF (2015a) Localization of early germ cells in a stony coral, Euphyllia ancora: potential implications for a germline stem cell system in coral gametogenesis. Coral Reefs 34:639-653 

Shikina S, Chiu YL, Lee YH, Chang CF (2015b) From somatic cells to oocytes: a novel yolk protein produced by ovarian somatic cells in a stony coral, Euphyllia ancora. Biol Reprod 93:1-10
Shikina S, Chiu YL, Chung YJ, Chen CJ, Lee YH, Chang CF (2016a) Oocytes express an endogenous red fluorescent protein in a stony coral, Euphyllia ancora: a potential involvement in coral oogenesis. Sci Rep 6:25868
Shikina S, Chang CF (2016b) Sexual reproductionin stony corals and insight into the evolution of oogenesis in Cnidaria. In: Goffredo S, Dubinsky Z (eds) The Cnidaria, Past, Present and Future. Springer, Switzerland, pp249-268
Smith CA, Hurley TM, McClive PJ, Sinclair AH (2002) Restricted expression of DMRT3 in chicken and mouse embryos. Mech Develop 119:73-76
Smith CA, Roeszler KN, Ohnesorg T, Cummins DM, Farlie PG, Doran TJ, Sinclair AH (2009) The avian Z-linked gene DMRT1 is required for male sex determination in the chicken. Nature 461:267-271 

Soong K (1991) Sexual reproductive patterns of shallow-water reef corals in Panama. Bull Mar Sci 49:832-846
Soong K, Chen CA, Chang JC (1999) A very large poritid colony at Green Island, Taiwan. Coral reefs 18:42
Suzuki G (2012) Simultaneous spawning of Pocillopora and Goniopora corals in the morning time. Galaxea 14:115-116
Szmant AM (1986) Reproduction ecology of Caribbean reef corals. Coral Reefs 5:43-54
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731-2739
Tomascik T, Sander F (1987) Effect of eutrophication on reef building corals III. Reproduction of the reef building coral Porites porites. Mar Biol 94:77-94
Traylor-Knowles NG, Kane EG, Sombatsaphay V, Finnerty JR, Reitzel AM (2015) Sex-specific and developmental expression of Dmrt genes in the starlet sea anemone, Nematostella vectensis. Evodevo 6:13 

Treml EA, Halpin PN, Urban DL, Praton LF (2008) Modeling population connectivity by ocean currents, a graph-theoretic approach for marine conservation. Landscape Ecol 23:19-23
Tribble GW, Randall RH (1986) A description of the high-latitude shallow water coral communities of Miyake-jima, Japan. 4:151-159
Vargas-Ángel B, Colley SB, Hoke SM, Thomas JD (2005) The reproductive seasonality and gametogenic cycle of Acropora cervicornis off Broward County, Florida, USA. Coral Reefs 25:110-122
Veith A, Klattig J, Dettai A, Schmidt C, Englert C, Volff J (2006) Male-biased expression of X-chromosomal DM domain-less Dmrt8 genes in the mouse. Genomics 88:185-195
Veron JEN (1986) Corals of Australia and the Indo-Pacific. Angus and Robertson, Sydney
Wallace CC (1985) Seasonal peaks and annual fluctuations in recruitment of juvenile scleractinian corals. Mar Ecol Prog Ser 21:289-298
Warner RR (1975) The adaptive significance of sequential hermaphroditism in animals. Amer Nat 965:61-82
Wang J, Miao L, Li M, Guo X, Pan N, Chen Y (2014) Cloning the Dmrt1 and DmrtA2 genes of Ayu (Plecoglossus altivelis) and their expression in adult, larval, and embryonic stages. Zool Res 35:1-9
Weil E, Vargas WL (2010) Comparative aspects of sexual reproduction in the Caribbean coral genus Diploria (Scleractinia: Faviidae). Mar Biol 157:413-426
Wen A, You F, Tan X, Sun P, Ni J, Zhang Y, Xu D, Wu Z, Xu Y, Zhang P (2009) Expression pattern of dmrt4 from olive flounder (Paralichthys olivaceus) in adult gonads and during embryogenesis. Fish Physiol 
Biochem 35:421-433
Wexler JR, Plachetzki DC, Kopp A (2014) Pan-metazoan phylogeny of the DMRT gene family: a framework for functional studies. Dev Genes Evol 224:175-181
Willis BL, Babcock RC, Harrison PL, Oliver JK, Wallace CC (1985) Patterns in the mass spawning of corals on the Great Barrier Reef from 1981 to 1984. Proc 5th Int Coral Reef Symp, Tahiti, French Polynesia. pp.343-348
Willis BL, Oliver JK (1988) Inter-reef dispersal of coral larvae following the annual mass spawning on the Great Barrier Reef. Proc 6th Int Coral Reef Symp 2:853-859
Wilson JR, Harrison PL. (2003) Spawning patterns of scleractinian corals at the Solitary Islands-a high latitude coral community in eastern Australia. Mar Ecol Prog Ser 260:115-123
Winkler C, Hornung U, Kondo M, Neuner C, Duschl J, Shima A, Schartl 
M (2004) Developmentally regulated and non-sex-specific expression of autosomal dmrt genes in embryos of the Medaka fish (Oryzias latipes). Mech Develop 121:997-1005 

van Woesik R (2009) Calm before the spawn: global coral spawning patterns are explained by regional wind fields. Proc. R. Soc. B 277:715-722
Wu GC, Chiu PC, Lin CJ, Lyu YS, Lan DS, Chang CF (2012) Testicular dmrt1 is involved in the sexual fate of the ovotestis in the protandrous Black Porgy. Biol Reprod 86:1-11 

Yamano H, Hori K, Yamauchi M, Yamagawa O, Ohmura A (2001) Highest-latitude coral reef at Iki Island, Japan. Coral Reefs 20:9-12
Yoshimoto S, Okada E, Umemoto H, Tamura K, Uno Y, Nishida-Umehra C, Matsuda Y, Takamatsu N, Shiba T, Ito MA (2008) W-linked DM- domain gene, DM-W, participates in primary ovary development in Xenopus laevis. Proc Natl Acad Sci USA 105:2469-2474
Yoshimoto S, Ikeda N, Izutsu Y, Shiba T, Takamatsu N, Ito M (2010) Opposite roles of DMRT1 and its W-linked paralogue, DM-W, in sexual dimorphism of Xenopus laevis: implications of a ZZ/ZW-type sex-determining system. Development 137:2519-2526