Phylogeography of Brazilian scleractinian corals

Orientador: Vera Nisaka SolferiniTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Recifes de corais são importantes ecossistemas para a biodiversidade marinha, entretanto, a sua história evolutiva tem sido pouco estudada. Os recifes brasileiros são considerados marginais devido a distância do centro de diversidade caribenho e às condições ambientais não ideais em que se desenvolvem. Estudos sugerem que a sua formação atual é recente uma vez que, no Último Máximo Glacial, o nível do mar teria descido 120 m e a plataforma continental onde ocorrem hoje estaria exposta. Hipóteses sobre a formação da sua diversidade atual incluem colonizações recentes a partir do Caribe ou dispersão a partir de refúgios climáticos em montanhas submersas próximas ao banco de Abrolhos. Neste trabalho, nós associamos abordagens filogeográficas à análises morfológicas e modelagens paleoclimáticas para estudar processos históricos responsáveis pela distribuição atual da diversidade de dois grupos de corais na costa do Brasil, o gênero Siderastrea e a espécie endêmica Mussismilia braziliensis. No primeiro capítulo, `Phylogeography of the genus Siderastrea (Anthozoa, Scleractinia) in Southwest Atlantic: insights about the historical formation of coral biodiversity in marginal reefs¿, os resultados indicaram que as duas espécies do gênero encontradas no Brasil são muito similares às congêneres S. radians e S. siderea e que a endêmica S. stellata pode ser uma variação morfológica de S. siderea, o que mostrou a necessidade de uma revisão taxonômica do grupo. As análises intraespecíficas de diversidade genética, estrutura e demografia não corroboram as hipóteses de colonização recente e indicam que as espécies parecem ter mantido a sua amplitude latitudinal de ocorrência atual ao longo do tempo. No segundo capítulo, `Paleoclimatic distribution and phylogeography of Mussismilia braziliensis, and endemic coral of Brazilian coast¿, tanto as análises filogeográficas quanto as simulações paleoclimáticas indicaram que a M. braziliensis também parece ter mantido a sua distribuição latitudinal ao longo das últimas variações no nível do mar. Os resultados para ambos os grupos sugerem que a costa do Brasil pode ter sido relativamente estável ambientalmente no tempo geológico em relação à outras regiões no Atlântico. Este trabalho traz importantes informações sobre os grupos estudados e sobre a biogeografia histórica dos atuais recifes de corais brasileirosAbstract: Coral reefs are important ecosystems for marine biodiversity, however, their evolutionary history has been poorly studied. Brazilian coral reefs are considered marginal due to the distance from the Caribbean center of diversity and due to the suboptimal environmental conditions. The formation of their current reefs structures is very recent once the continental shelf where they occurs today were exposed in the Last Glacial Maximum. Some studies suggest colonization from the Caribe or dispersal from refuges in submerged mountain near the Abrolhos bank. Here, we associate phylogeographic approaches to morphological analysis and paleoclimatic modeling to study historical processes responsible for the current distribution of the diversity of two groups of Brazilian corals, the genus Siderastrea and the endemic species Mussismilia braziliensis. In the first chapter, entitled `Phylogeography of the genus Siderastrea (Anthozoa, Scleractinia) in Southwest Atlantic: insights about the historical formation of coral biodiversity in marginal reefs', the results indicated that the two Brazilian species are strictly similar to the Caribbean congeners S. radians and S. siderea and that the endemic S. stellata seems to be a morphological variation of S. siderea, which showed the need for a taxonomic revision of the group. Genetic diversity, structure, and demography into both species do not corroborate the hypotheses of recent colonizations and indicate that the species have maintained their current latitudinal range of occurrence along geological time. In the second chapter, entitled `Paleoclimatic distribution and phylogeography of Mussismilia braziliensis, an endemic coral of Brazilian reefs¿, both, phylogeographic analyzes and paleoclimatic simulations indicated that M. braziliensis also appears to have maintained its current latitudinal distribution over last sea level variation. The results for both studies suggest that the Brazilian coast remained environmentally stable when compared to other regions in the Atlantic along the geological time. This work presents important information on the general knowledge of the studied corals and on the historical biogeography of the current Brazilian coral reefsDoutoradoEcologiaDoutora em EcologiaCAPE

Gorgonian Responses to Environmental Change on Coral Reefs in SE Sulawesi, Indonesia

Gorgonian corals (Cnidaria: Anthozoa: Octocorallia) are conspicuous, diverse and often dominant components of benthic marine environments. Intra- & interspecific morphological variability in gorgonians are influenced by environmental factors such as light, sedimentation and flow rates. Yet, little is known about the responses of gorgonian taxa to environmental parameters particularly in Indonesia, despite their high regional abundance and diversity. With a burgeoning human population and subsequent marine resource exploitation, reefs throughout the Indonesian archipelago are under rapid decline and often destroyed. Conservation surveys are however, underway with a tendency to overlook gorgonian taxa primarily due to unresolved taxonomic assignment leading to difficulties in field identification. The aims of this study were to: 1) characterise gorgonian diversity and ecology across a gradient of habitat quality within the Wakatobi Marine National Park (WMNP), SE Sulawesi, Indonesia, 2) assess morphological and genetic variability between morphotypes of the ubiquitous zooxanthellate isidid Isis hippuris Linnaeus 1758 from healthy and degraded reefs, 3) determine if I. hippuris morphotypes are environmentally induced (plastic) or genetically derived through reciprocal transplant experiments (RTEs) between contrasting reefs and thus, 4) identify mechanisms of plasticity capacity or divergence through phenotypic trait integration in response to environmental change. Ecological surveys revealed considerable gorgonian diversity with a total of 197 species and morphotypes from 42 genera, and 12 families within the suborders Calcaxonia and Holaxonia and the group Scleraxonia, with current estimates of over 21 new species and 28 new species records for the region. Gorgonian abundance and diversity increased with reef health and bathymetry. However, a clear loss of gorgonian diversity existed with increased sedimentation and reduced light due to anthropogenic disturbance. In particular, two distinct I. hippuris morphotypes were highly abundant between environmental clines: short-branched multi/planar colonies on healthy reefs, and long-branched bushy colonies on degraded reefs. Comparative morphological and molecular analyses using ITS2 sequence and predicted secondary structure, further corroborated haplotype differences relative to morphotypes between environments. However, unsatisfactory assignment of I. hippuris morphotypes to previously described alternatives (Isis reticulata Nutting 1910, Isis minorbrachyblasta Zou, Huang & Wang 1991) questions the validity to such taxonomic assignments. Phylogenetic analyses also confirm that the polyphyletic nature of the Isididae lies in its type species I. hippuris, being unrelated to the rest of its family members. A one-year RTE revealed three key results, that: 1) reduced survivorship of healthy reef morphotypes on degraded reefs implied the onset of lineage segregation through immigrant inviability, 2) prominent phenotypic traits were at the morphological and bio-optical levels revealing high phenotypic plasticity in healthy clones, and relative insensitivity to environmental change in degraded reef morphotypes, indicative of local adaptation leading to incipient ecological divergence, and 3) photoacclimation at the bio-optical level was not attributed to endosymbiont diversity or shuffling, with all test colonies possessing a novel clade D1a Symbiodinium. While it is clear that gorgonian taxa within the WMNP are of exceptional diversity and abundance, responses to environmental perturbation highlight three pertinent, testable ideas. Firstly, increased species richness specifically with depth in azooxanthellate taxa, invite tests of deep-reef refugia previously established through geological change. Secondly, ecological assessment targets research on informative taxa for focused systematics and mechanisms of phenotypic divergence. Thirdly, exploring intrinsic and extrinsic interactions that define the host-symbiont relationship and differential biological success using physiological and next generation sequencing approaches. These objectives would provide considerable insight into the evolutionary processes to environmental change, accelerated by anthropogenic encroachment. Taken together, this work signifies that gorgonian corals within the WMNP are of foremost diversity and concern, exhibiting informative ecological and mechanistic responses to environmental perturbation. This evidence elicits tests of deep-reef refugia, priority systematics, mechanisms of ecological divergence and physiological assessment. Such tests inevitably expand our understanding of the intrinsic and extrinsic associations of gorgonian taxa to environmental change from an historical and predictive perspective yielding benefits to conservation assessment and management

Speciation, evolution and phylogeny of some shallow-water octocorals (Cnidaria: Anthozoa)

Shallow-water octocorals are among the most abundant macro-benthic organisms inhabiting tropical subtropical and temperate communities. In spite of being worldwide distributed and highly diverse, the systematics of many octocorals remains controversial and the understanding of the processes who led to their diversification is largely unexplored. This study includes five chapters, each dealing with different aspects of the systematics, phylogeny and evolution of six soft coral genera such as Lobophytum, Sarcophyton, Paramuricea, Leptogorgia, Muricea and Pacifigorgia. One of the main goals of the study was to explore, through the use of molecular methods, the genetic variation within species notoriously difficult to identify. Besides the use of standard molecular methods for phylogenetic reconstruction and species delimitation, the effectiveness of Next Generation Sequencing (NGS) technologies was tested for mitogenomic and genotyping analyses. In the first chapter the use of single-locus markers (e.g. COI, mtMutS and 28S rDNA) was investigated and different automated species delimitation methods (e.g. ABGD, bPTP) were employed to assess species richness among soft coral genera from Western Australia. The methods used appeared suitable for preliminary and rapid diversity assessments especially in the presence of species-rich genera such as Lobophytum and Sarcophyton where morphological identification is particularly difficult and time consuming. In the second chapter, along with the sequencing of complete mitogenomes of Mediterranean Paramuricea species (P. clavata and P. macrospina), the biogeography of the genus was investigated. The results revealed nucleotide and genome size polymorphisms, while the biogeographic predictions suggested that the Mediterranean species have resulted from independent speciation events, explaining in part the high phylogenetic divergence detected. In the third chapter, the sequencing of complete mitogenomes of five Leptogorgia species from different geographic areas (eastern Pacific, eastern Atlantic and Mediterranean) was followed by phylogenetic reconstructions based on an extended mtMutS dataset. The phylogenetic tree recovered Leptogorgia polyphyletic with a clear segregation between the eastern Pacific and eastern Atlantic forms. A time calibrated phylogeny provided insights into the evolution of the genus. In chapter four, using NGS approaches, the complete mitochondrial genome of two eastern Pacific Muricea species (M. crassa and M. purpurea) has been sequenced. The recovery of complete mitogenomes allowed to evaluate the presence of variable and informative regions and to infer a more robust phylogeny. Overall, the results showed high nucleotide diversity in the intergenic spacers, making these regions new potential molecular markers for species-level identifications. In the last chapter a genome-wide Single Nucleotide Polymorphisms (SNPs) and a Bayes Factor Delimitation method were used to infer the genetic relationships within species of the genus Pacifigorgia. The data obtained showed incongruence between molecular and morphological investigations suggesting the possibility of alternative taxonomic assignments for these species. This study provides information on the evolution and speciation of ecologically important soft corals, which distribution range from the littoral and sublittoral zones of the Mediterranean to the tropical and subtropical reefs of Western Australia (WA) and eastern Pacific (EP). The use of mitochondrial markers such as MutS allowed to shed some light on the biogeography and evolutionary history of widespread gorgonians with special emphasis on the Mediterranean endemics and the Atlantic species. Concerning the Western Australia, the obtained results will support the management and conservation of under-investigated marine biodiversity hotspots and potentially species-rich localities such as the Kimberley. In terms of species delimitation, the application of genome-wide SNPs and the use of NGS technologies showed a higher resolution when compared with the traditional methods based on DNA barcoding and single-locus phylogenies. The data generated have been used to clarify the systematics of the species investigated and will be considered as a baseline for future studies on population genetics with a closer look on the adaptive processes

Speciation, evolution and phylogeny of some shallow-water octocorals (Cnidaria: Anthozoa)

Shallow-water octocorals are among the most abundant macro-benthic organisms inhabiting tropical subtropical and temperate communities. In spite of being worldwide distributed and highly diverse, the systematics of many octocorals remains controversial and the understanding of the processes who led to their diversification is largely unexplored. This study includes five chapters, each dealing with different aspects of the systematics, phylogeny and evolution of six soft coral genera such as Lobophytum, Sarcophyton, Paramuricea, Leptogorgia, Muricea and Pacifigorgia. One of the main goals of the study was to explore, through the use of molecular methods, the genetic variation within species notoriously difficult to identify. Besides the use of standard molecular methods for phylogenetic reconstruction and species delimitation, the effectiveness of Next Generation Sequencing (NGS) technologies was tested for mitogenomic and genotyping analyses. In the first chapter the use of single-locus markers (e.g. COI, mtMutS and 28S rDNA) was investigated and different automated species delimitation methods (e.g. ABGD, bPTP) were employed to assess species richness among soft coral genera from Western Australia. The methods used appeared suitable for preliminary and rapid diversity assessments especially in the presence of species-rich genera such as Lobophytum and Sarcophyton where morphological identification is particularly difficult and time consuming. In the second chapter, along with the sequencing of complete mitogenomes of Mediterranean Paramuricea species (P. clavata and P. macrospina), the biogeography of the genus was investigated. The results revealed nucleotide and genome size polymorphisms, while the biogeographic predictions suggested that the Mediterranean species have resulted from independent speciation events, explaining in part the high phylogenetic divergence detected. In the third chapter, the sequencing of complete mitogenomes of five Leptogorgia species from different geographic areas (eastern Pacific, eastern Atlantic and Mediterranean) was followed by phylogenetic reconstructions based on an extended mtMutS dataset. The phylogenetic tree recovered Leptogorgia polyphyletic with a clear segregation between the eastern Pacific and eastern Atlantic forms. A time calibrated phylogeny provided insights into the evolution of the genus. In chapter four, using NGS approaches, the complete mitochondrial genome of two eastern Pacific Muricea species (M. crassa and M. purpurea) has been sequenced. The recovery of complete mitogenomes allowed to evaluate the presence of variable and informative regions and to infer a more robust phylogeny. Overall, the results showed high nucleotide diversity in the intergenic spacers, making these regions new potential molecular markers for species-level identifications. In the last chapter a genome-wide Single Nucleotide Polymorphisms (SNPs) and a Bayes Factor Delimitation method were used to infer the genetic relationships within species of the genus Pacifigorgia. The data obtained showed incongruence between molecular and morphological investigations suggesting the possibility of alternative taxonomic assignments for these species. This study provides information on the evolution and speciation of ecologically important soft corals, which distribution range from the littoral and sublittoral zones of the Mediterranean to the tropical and subtropical reefs of Western Australia (WA) and eastern Pacific (EP). The use of mitochondrial markers such as MutS allowed to shed some light on the biogeography and evolutionary history of widespread gorgonians with special emphasis on the Mediterranean endemics and the Atlantic species. Concerning the Western Australia, the obtained results will support the management and conservation of under-investigated marine biodiversity hotspots and potentially species-rich localities such as the Kimberley. In terms of species delimitation, the application of genome-wide SNPs and the use of NGS technologies showed a higher resolution when compared with the traditional methods based on DNA barcoding and single-locus phylogenies. The data generated have been used to clarify the systematics of the species investigated and will be considered as a baseline for future studies on population genetics with a closer look on the adaptive processes

Exploiting whole-genome sequencing to understand the evolution of corals and their symbionts

Jia Zhang's research undertook an in-depth study of Acropora coral population genomics using whole-genome sequencing data. Her focus was the genetic diversity and evolution of these corals in the Great Barrier Reef and Kimberley, northwestern Australia. The study's findings enhance our understanding of coral speciation and provide projections on coral adaptability to future climate changes

11th International Coral Reef Symposium Abstracts

https://nsuworks.nova.edu/occ_icrs/1001/thumbnail.jp

11th International Coral Reef Symposium Proceedings

A defining theme of the 11th International Coral Reef Symposium was that the news for coral reef ecosystems are far from encouraging. Climate change happens now much faster than in an ice-age transition, and coral reefs continue to suffer fever-high temperatures as well as sour ocean conditions. Corals may be falling behind, and there appears to be no special silver bullet remedy. Nevertheless, there are hopeful signs that we should not despair. Reef ecosystems respond vigorously to protective measures and alleviation of stress. For concerned scientists, managers, conservationists, stakeholders, students, and citizens, there is a great role to play in continuing to report on the extreme threat that climate change represents to earth’s natural systems. Urgent action is needed to reduce CO2 emissions. In the interim, we can and must buy time for coral reefs through increased protection from sewage, sediment, pollutants, overfishing, development, and other stressors, all of which we know can damage coral health. The time to act is now. The canary in the coral-coal mine is dead, but we still have time to save the miners. We need effective management rooted in solid interdisciplinary science and coupled with stakeholder buy in, working at local, regional, and international scales alongside global efforts to give reefs a chance.https://nsuworks.nova.edu/occ_icrs/1000/thumbnail.jp

공생성 와편모류 종들에 대한 분류, 혼합영양성 및 종속 영양성 와편모류들의 광합성유전자 전사체 분석, 적조 유발 와편모류의 정량화를 위한 RT-PCR 방법의 개선

학위논문 (박사)-- 서울대학교 대학원 : 지구환경과학부 해양학 전공, 2017. 2. 정해진.Dinoflagellates are ubiquitous protists and live in marine environments as diverse forms. Although they often cause red tides or harmful algal blooms, they also play such important roles in marine food webs as prey, predator, endosymbiont, and parasite. However, in studying taxonomy, genetics and quantification of some dinoflagellate species, there have been some difficulties and problems in identifying species, quantifying their abundances, and understanding their molecular genetic characteristics. To solve the difficulties in exploring the taxonomy, the trophic mode associated molecular genetic characteristics, and improved qPCR based quantification of small dinoflagellates and mixotrophic protists, this study focused on 1) discovering and distinguishing symbiotic species by molecular and morphological method to set up their morphological standard, 2) comparing the genetic status with the trophic mode of dinoflagellates by analyzing the expressed genes through transcriptome analysis, and 3) developing a fast and accurate method to detect red-tide organisms. Among the dinoflagellate species, the genus Symbiodinium forms symbiosis with a broad diversities of host species such as corals, jellyfish, sea anemones or giant clams. Especially they provide organic photosynthases to host species, mostly coral, and in return, corals provide other nutrients to symbiodinium. Thus, they are crucial components for survivals of corals and for building tropical reef ecosystems. However, despite their ecological and economic importance in marine ecosystems, the taxonomy of Symbiodinium remains limited due to their small size and fragile surface, which has been causing the difficulties in identification of these species for several decade. Especially, type species of genus Symbiodinium have been described to have incomplete morphology, although the morphological and molecular characteristics of type species plays as a overall standard for species belonging to the genus. These incomplete morphological standards have ended with multiple synonyms for a single species. Thus, here, I completed the morphological characterization of Symbiodinium microadriaticum, the type species, and also established a new Symbiodinium species which is symbiotic to the giant clam (Tridanidae), to name it as Symbiodinium tridacnidorum sp. nov.. Furthermore, I clarified the unreported morphological characteristics of two clade B Symbiodinium, Symbiodinium minutum and Symbiodinium psygmorphilum, to complete their plate formula. The dinoflagellate are known to have three different trophic modesautotrophic, heterotrophic and mixotrophic. Due to these characteristics, dinoflagellates plays diverse roles in marine food webs as prey and predators. However, the difference in molecular genetic characteristics between mixotrophic and heterotrophic dinoflagellates have not been reported so far. Thus, molecular genetic characteristics of mixotrophic and heterotrophic dinoflagellates should be compared to trace evolutionary routes of trophic modes in dinoflagellates. I explored the transcriptome of two closely related dinoflagllates which have high similarity in their morphology, ribosomal DNA, and edible prey species, but have different trophic modes in order to discover any parallellism between the gene expression and the trophic modes. When I compared the expressed gene sets of the mixotrophic dinoflagellate Paragymnodinium shiwhaense and the heterotrophic dinoflagllate Gyrodiniellum shiwhaense by analyzing their transcriptome, the expressed genes exhibited huge dissimilarities, although there were many similar expressed genes, especially in the gene groups related to photosynthesis. Furthermore, based on this comparison analysis, I proved that the mixotrophic dinoflagllate may have more genes that play diverse functional characteristics in genetic level than the heterotropic dinoflagellates because the former must conduct both photosynthesis and phagotrophy in contrast to the latter performing phagotrophy only. Every summer, Korea suffers from harmful algal blooms (HABs) caused by red tide dinoflagellate Cochlodinium polykrikoides. Thus, it is important to detect and quantifying their abundances accurately. However, quantifying the harmful dinoflagellate Cochlodinium polykrikoides in natural samples is not easy due to similarity in morphologies between Cochlodinium polykrikoides and closely related species. Furthermore, qPCR method for C. polykrikoides is also not easy due to potential variations in DNA contents among individual cells. Here, I also developed new species-specific primers and probe for detecting all the 3 ribotypes of ichthyotoxic dinoflagellate Cochlodinium polykrikoides, and comparatively evaluated the efficiencies of the 4 different preparation methods used to determine standard curves. Furthermore, to confirm the accuracy of result, the abundance of C. polykrikoides in the > 500 samples collected from the coastal South Sea of Korea, in 2014 and 2015, were independently determined using all the 4 methods. Standard curves constructed by by extraction of DNA from each of the serially diluted cultures with different concentrations of cultured C. polykrikoides were most accurate followed by the standard curve obtained by extracting DNA from each of serially diluted field sample with different concentrations of C. polykrikoides. Thus, this study provided more accurately modified methods to detect dinoflagellate species from natural sea water samples. The result of this thesis provided complete morphological standard and eventually provided the basis of overall understanding of taxonomy of symbiotic dinoflagellate Symbiodinium species. Furthermore, the result of comparative transcriptome analyses of dinoflagellates possessing different trophic modes obtained in this thesis will provide better understanding of genetic influence to the trophic mode of dinoflagellate species. In addition, the improved qPCR methods and a newly developed set of specific primer and probe set resolving the two recently discovered 2 new ribotypes of Cochlodinium polykrikoides will facilitate simple and automatic estimation of C. polykrikoides abundance.Chapter 1. Overall Introduction 1 Chapter 2. Report of new symbiotic dinoflagellate Symbiodinium tridacnidorum sp. nov., and a revised morphological description of S. microadriaticum Freudenthal, emended Trench et Blank 7 2.1. Introduction 7 2.2. Materials and methods 11 2.2.1. Cultures and growth conditions of Symbiodinium spp. 11 2.2.2. Morphological analysis of Symbiodinium using Scanning electron microscopy (SEM) 13 2.2.3. Ultrastructure analysis of Symbiodinium using Transmission electron microscopy (TEM) 14 2.2.4. Nucleic acid extraction, PCR amplification, sequencing, and phylogenetic analyses 18 2.3. Results 20 2.3.1. Taxonomic descriptions 20 2.3.2. Morphology of Symbiodinium microadriaticum 22 2.3.3. Morphology of Symbiodinium tridacnidorum 29 2.3.4. Phylogenetic delineation of Clade A Symbiodinium 38 2.4. Discussion 44 2.4.1. Morphological comparisons among Clade A Symbiodinium 44 2.4.2. The genetic, ecological, and geographical attributes of described species in Symbiodinium Clade A 49 Chapter 3. Morphological characterization of the two new Symbiotic dinoflagellate Symbiodinium minutum and S. psygmophilum belonging to clade B 53 3.1. Introduction 53 3.2. Materials and methods 56 3.2.1. Collection and culture of Symbiodinium spp. 56 3.2.2. Morphological analysis of Symbiodinium spp. 56 3.3. Results 60 3.3.1. Morphology of Symbiodinium minutum 60 3.3.2. Morphology of Symbiodinium psygmophilum 66 3.4. Discussion 71 Chapter 4. Comparative de novo transcriptome analysis of the mixotrophic dinoflagellate Paragymnodinium shiwhaense and the heterotrophic dinoflagellate Gyrodinium shiwhaense. 73 4.1. Introduction 73 4.2. Materials and methods 76 4.2.1. Sample preparations and cell harvest 76 4.2.2. RNA isolation and sequencing 78 4.2.3. Identification of plastid-derived genes and bioinformatics 78 4.2.4. Phylogenetic analysis of photosynthesis related gene and ribosomal DNA. 79 4.2.5. Gene-specific primer-probe set design, DNA extraction and RT-PCR amplification. 79 4.3. Results 82 4.3.1. Sequence analysis and assembly 82 4.3.2. Functional classification by eggNOG and Gene Ontology 85 4.3.3. Photosynthesis and carbon fixation related genes classified by KEGG in G. shiwhaense and P. shiwhaense. 86 4.3.4. Calvin cycle related gene expression in G. shiwhaense and P. shiwhaense 92 4.4. Discussion 98 Chapter 5. Real-time PCR based quantification of the red tide dinoflagellate Cochlodinium polykrikoides in South Sea of Korea in 2014 122 5.1. Introduction 122 5.2. Material and Methods 125 5.2.1. Sample collection for standard curve generation and field sample test 125 5.2.2. Four different sample preparations utilized to determine standard curves 128 5.2.3. PCR amplification, sequencing, and phylogenetic analysis. 132 5.2.4. Design of TaqMan probe and primer set for detection. 133 5.2.5. Determination of threshold cycle and standard curve conduction 134 5.2.6. Determination of Cochlodinium polykrikoides cell abundance in field samples using the 4 different standard curves 135 5.3. Results 136 5.4. Discussion 139 Chapter 6. Overall Discussion 145 Reference 150 Abstract (in Korean) 176Docto