Ciliate DNA barcoding

DNA barcoding is a taxonomic method that uses a short genetic marker in an organisms DNA to identify it as belonging to a particular species. A desirable gene region for DNA barcoding should be standardized, present in most of the taxa of interest and sequencable without species-specific PCR primers, short enough to be easily sequenced with current technology, and provide a large variation between species yet a relatively small amount of variation within a species. For animals and many other eukaryotes, the mitochondrial CO1 gene have been suggested, and verified as most suitable gene region for DNA barcoding. However this CO1 has been used rarely as DNA barcoding gene for many microbial eukaryotes such as ciliates because the universal primers for CO1 are seldom universal for these organisms. Ribosomal RNA gene often used in phylogenetic and molecular taxonomic studies because of its ubiquitous and well conserved nature in eukaryotes. In particular, the highly conservative nature of SSU rRNA gene facilitates its utility in the study of culture-independent microbial eukaryote communities using universal PCR primers. To verify which regions of rRNA gene are suitable as a diagnostic marker for species discrimination, SSU~the partial large subunit (LSU) including D1 and D2 (SSU~D2) region were sequenced from the species belonging to family Euplotidae and Loxophyllum and sequences divergencea of interest and sequencable without species-specific PCR primers, short enough to be easily sequenced with current technology, and provide a large variation between species yet a relatively small amount of variation within a species. For animals and many other eukaryotes, the mitochondrial CO1 gene have been suggested, and verified as most suitable gene region for DNA barcoding. However this CO1 has been used rarely as DNA barcoding gene for many microbial eukaryotes such as ciliates because the universal primers for CO1 are seldom universal1

First Record of Three Uronychia Species (Ciliophora: Spirotrichea: Euplotida) from Korea

Three morphospecies of the genus Uronychia, i.e. U. setigera Calkins, 1902, U. binucleata Young, 1922, and U. multicirrus Song, 1997, were collected from the coastal waters of Gumjin-ri on the East Sea and the public waterfront of Incheon on the Yellow Sea in Korea, respectively. These species are described based on live observation, protargol impregnation, silver nitrate impregnation, and their morphometrics. Diagnostic keys for these species are also provided. In addition, their small subunit ribosomal DNA sequences were compared with previously known sequences of Uronychia species. Diagnostics of three Uronychia species are as follows: U. setigera: 50−80μm long in vivo, oval-shaped, 2 macronuclear nodules (Ma), 1 spur on the left margin, 11 adoral membranelles (AM) 1, 4 AM2, 1 buccal cirrus (BC), 4 frontal cirri (FC), 3 left marginal cirri (LMC), 2 ventral cirri (VC), 5 transverse cirri (TC), 3 caudal cirri (CC), 6 dorsal kineties (DK), and approximately 23 cilia in the leftmost kinety. U. binucleata: 70−110μm long in vivo, oval to slightly rectangular shaped, 2 Ma, 1 micronucleus (Mi), 2 spurs on the posterior region, 11 AM1, 4 AM2, 1 BC, 4 FC, 3 LMC, 2 VC, 5 TC, 3 CC, 6 DK, and approximately 37 cilia in the leftmost kinety. U. multicirrus: 140−200μm long in vivo, oval to slightly rectangular shaped, ca. 7 Ma, 1 Mi, 11 AM1, 4 AM2, 1 BC, 4 FC, 3 LMC, approximately 8 VC, 5 TC, 3 CC, and 6 DK. This study presents the first record of this genus in Korea.22Nkc

A new species of the family Callianassidae (Crustacea: Decapoda: Axiidea) from vent fields at Tofua Arc in the Southwest Pacific

A population of ghost shrimp was isolated from sediments of vent fields collected by TV grab of R/V SONNE. Calliaxina sp. nov. was identified a new species of the family Callianassidae. The new species is described and illustrated on the basis of 18 specimens. The habitat of this species was determined by zinc (Zn) poor crust in low temp vent fields. Calliaxina sp. nov. is characterized by broadly triangular rostrum, pointed lateral projections on carapace, laterally compressed chelipeds (first pair of legs) and third maxillipeds with the presence of exopods as found the other nominal species belonging to the genus Calliaxina Ngoc-Ho, 2003. However, it mainly differs by asymmetrical-sized chelipeds and absent denticules on the lower marginal side of ischium and merus in shape from congeners. In addition, three mitochondrial 16S ribosomal DNA (16S rDNA) sequences were newly determined from Calliaxina sp. nov. This new species show the closest relationship with C. sakaii (Accession No. EU882905, bootstrap value 83 % in NJ) based on 16S rDNA. This study combined morphological and molecular approach helps delineate the phylogenetic relationship within the family Callianassidae that is a complex group in decapods.e basis of 18 specimens. The habitat of this species was determined by zinc (Zn) poor crust in low temp vent fields. Calliaxina sp. nov. is characterized by broadly triangular rostrum, pointed lateral projections on carapace, laterally compressed chelipeds (first pair of legs) and third maxillipeds with the presence of exopods as found the other nominal species belonging to the genus Calliaxina Ngoc-Ho, 2003. However, it mainly differs by asymmetrical-sized chelipeds and absent denticules on the lower marginal side of ischium and merus in shape from congeners. In addition, three mitochondrial 16S ribosomal DNA (16S rDNA) sequences were newly determined from Calliaxina sp. nov. This new species show the closest relationship1

Morphology and DNA barcode of benthic ciliates (Protozoa: Ciliophora)

저서성 섬모충류의 형태분류 및 DNA 바코드라는 연구 주제를 가지고 2007년부터 현재까지 남/북극과 국내 섬모충류에 대해 연구를 실시하였으며 결과를 요약하면 다음과 같다: i) 신속 1속, 신종 9종, 국내미기록종 10종 발굴 ii) 차세대 염기서열 결정 기술(NGS: pyrosequencing)에 적합한 섬모충류 특이 마커 개발 iii) 하모류(Hypotrichia) 미토콘드리아 DNA 바코드 개발(mtCO1). 섬모충류는 물이 존재하는 거의 대부분의 환경에 서식하며 전세계적으로 약 8,000종이 알려져 있다. 이들의 다양성은 40,000여종에 달할 것으로 추정되고 있지만 국내의 경우 200여종만이 알려져 있을 뿐이다. 채집된 섬모충류의 형태, 형태발생, 18S~28S 리보솜 DNA(약 3,000 bp), pyrosequencing 데이터 분석 결과는 이들의 다양성이 상당히 높다는 것을 뒷받침하고 있으며, 형태적 수렴진화에 의한 분류체계의 문제점을 나타내고 있다. 또한, 새로 개발된 하모류의 미토콘드리아 CO1 마커는 리보좀 유전자를 이용한 종동정과 계통분석에서의 단점을 보완할 수 있을 것으로 판단되며, 차후 계통지리분포 연구에 활용할 수 있을 것으로 사료된다.(NGS: pyrosequencing)에 적합한 섬모충류 특이 마커 개발 iii) 하모류(Hypotrichia) 미토콘드리아 DNA 바코드 개발(mtCO1). 섬모충류는 물이 존재하는 거의 대부분의 환경에 서식하며 전세계적으로 약 8,000종이 알려져 있다. 이들의 다양성은 40,000여종에 달할 것으로 추정되고 있지만 국내의 경우 200여종만이 알려져 있을 뿐이다. 채집된 섬모충류의 형태, 형태발생, 18S~28S 리보솜 DNA(약 3,000 bp), pyrosequencing 데이터 분석 결과는 이들의 다양성이 상당히 높다는 것을 뒷받침하고 있으며, 형태적 수렴진화에 의한 분류체계의 문제점을 나타내고 있다. 또한, 새로 개발된 하모류의 미토콘드리아 CO1 마커는 리보좀 유전자를 이용한 종동정과 계통분석에서의 단점을 보완할 수 있을 것으로 판단되며, 차후 계통지리분포 연구에 활용할 수 있을 것으로 사료된다.2

New candidate genus in Oxytrichidae (Protozoa: Ciliophora)

Two new oxtrytrichids were discovered from brackish water from South Korea. Based on morphology, ontogenesis, and molecular analyses, we confirm that these species belong to new genus. The new candidate genus has similar morphology to Cyrtohymena (i.e., typical 18-cirri, Cyrtohymena undulating membranes pattern), however, the candidate genus can be distinguished mainly by decreased/lacking of caudal cirri and lacking of dorsal kinety fragmentation. On gene tress of 18S rDNA sequences, the new two species were clustered together and showed a sister relationship with Cyrtohymena-Notohymena-Paraurostyla. In addition, our results support that the oral apparatus of Cyrtohymena might be evolved two times or existed in last common ancestor, and more taxon-samplings of DNA sequences are necessary to reconstruct a robust phylogenetic tree for better understanding of their relationships.hymena (i.e., typical 18-cirri, Cyrtohymena undulating membranes pattern), however, the candidate genus can be distinguished mainly by decreased/lacking of caudal cirri and lacking of dorsal kinety fragmentation. On gene tress of 18S rDNA sequences, the new two species were clustered together and showed a sister relationship with Cyrtohymena-Notohymena-Paraurostyla. In addition, our results support that the oral apparatus of Cyrtohymena might be evolved two times or existed in last common ancestor, and more taxon-samplings of DNA sequences are necessary to reconstruct a robust phylogenetic tree for better understanding of their relationships.2

Discrimination of chum salmon stocks by stock-specific PCR based on single nucleotide polymorphisms

The chum salmon (Oncorhynchus keta) is an anadromous fish distributed all around the North Pacific. Artificial production and release of the juveniles are being made by Korea, Japan, Russia, Canada and the United States for enrichment of the stocks. Since the released salmons migrate through EEZ of the neighboring countries and intermix in the open ocean, it becomes important to set up some criteria and methods discriminating each stock in order to clarify each nation's right of harvest of the salmon resources. Here, we report a few stock-specific single nucleotide polymorphisms (SNPs) in the COIII-ND3-ND4L region of the mitochondrial DNA and present a simple method of stock separation by polymerase chain reaction(PCR) utilizing the SNPs. A total of 141 chum salmons from Korea, Japan, Canada, and the United States were analyzed and 744 nucleotide sequences of the COIII-ND3-ND4L region were compared each other in this study. Several SNPs were observed in the region, some of which were stock-specific (e.g., positions 57, 70, 246, 303, 307, 534, 591). Based on these SNPs, 20-25 nucleotide long stock-specific PCR primers were designed so that a single PCR with these primers could discriminate salmon stocks among Korea, Japan, Canada and the United States. The primers have the most 3'end nucleotide identical to the SNPs and the second to the 3'end being a mismatch to any of the sequences. With such primer design, a PCR with SF0 (a primer of a conserved sequence) and SR1 (a primer with Korea stock-specific SNP) amplified DNA only from the Korea salmons. This study suggests that SNPs in the COIII-ND3-ND4L region would be useful genetic markers for stock separation and PCR with the modified primers utilizing the SNPs provide a simple method for such purpose.2

Morphology and DNA barcode of benthic ciliates (Protozoa: Ciliophora)

저서성 섬모충류의 형태분류 및 DNA 바코드라는 연구 주제를 가지고 2007년부터 현재까지 남/북극과 국내 섬모충류에 대해 연구를 실시하였으며 결과를 요약하면 다음과 같다: i) 신속 1속, 신종 9종, 국내미기록종 10종 발굴 ii) 차세대 염기서열 결정 기술(NGS: pyrosequencing)에 적합한 섬모충류 특이 마커 개발 iii) 하모류(Hypotrichia) 미토콘드리아 DNA 바코드 개발(mtCO1). 섬모충류는 물이 존재하는 거의 대부분의 환경에 서식하며 전세계적으로 약 8,000종이 알려져 있다. 이들의 다양성은 40,000여종에 달할 것으로 추정되고 있지만 국내의 경우 200여종만이 알려져 있을 뿐이다. 채집된 섬모충류의 형태, 형태발생, 18S~28S 리보솜 DNA(약 3,000 bp), pyrosequencing 데이터 분석 결과는 이들의 다양성이 상당히 높다는 것을 뒷받침하고 있으며, 형태적 수렴진화에 의한 분류체계의 문제점을 나타내고 있다. 또한, 새로 개발된 하모류의 미토콘드리아 CO1 마커는 리보좀 유전자를 이용한 종동정과 계통분석에서의 단점을 보완할 수 있을 것으로 판단되며, 차후 계통지리분포 연구에 활용할 수 있을 것으로 사료된다.(NGS: pyrosequencing)에 적합한 섬모충류 특이 마커 개발 iii) 하모류(Hypotrichia) 미토콘드리아 DNA 바코드 개발(mtCO1). 섬모충류는 물이 존재하는 거의 대부분의 환경에 서식하며 전세계적으로 약 8,000종이 알려져 있다. 이들의 다양성은 40,000여종에 달할 것으로 추정되고 있지만 국내의 경우 200여종만이 알려져 있을 뿐이다. 채집된 섬모충류의 형태, 형태발생, 18S~28S 리보솜 DNA(약 3,000 bp), pyrosequencing 데이터 분석 결과는 이들의 다양성이 상당히 높다는 것을 뒷받침하고 있으며, 형태적 수렴진화에 의한 분류체계의 문제점을 나타내고 있다. 또한, 새로 개발된 하모류의 미토콘드리아 CO1 마커는 리보좀 유전자를 이용한 종동정과 계통분석에서의 단점을 보완할 수 있을 것으로 판단되며, 차후 계통지리분포 연구에 활용할 수 있을 것으로 사료된다.2

Discrimination of chum salmon stocks by stock-specific PCR based on single nucleotide polymorphisms

The chum salmon (Oncorhynchus keta) is an anadromous fish distributed all around the North Pacific. Artificial production and release of the juveniles are being made by Korea, Japan, Russia, Canada and the United States for enrichment of the stocks. Since the released salmons migrate through EEZ of the neighboring countries and intermix in the open ocean, it becomes important to set up some criteria and methods discriminating each stock in order to clarify each nation's right of harvest of the salmon resources. Here, we report a few stock-specific single nucleotide polymorphisms (SNPs) in the COIII-ND3-ND4L region of the mitochondrial DNA and present a simple method of stock separation by polymerase chain reaction(PCR) utilizing the SNPs. A total of 141 chum salmons from Korea, Japan, Canada, and the United States were analyzed and 744 nucleotide sequences of the COIII-ND3-ND4L region were compared each other in this study. Several SNPs were observed in the region, some of which were stock-specific (e.g., positions 57, 70, 246, 303, 307, 534, 591). Based on these SNPs, 20-25 nucleotide long stock-specific PCR primers were designed so that a single PCR with these primers could discriminate salmon stocks among Korea, Japan, Canada and the United States. The primers have the most 3'end nucleotide identical to the SNPs and the second to the 3'end being a mismatch to any of the sequences. With such primer design, a PCR with SF0 (a primer of a conserved sequence) and SR1 (a primer with Korea stock-specific SNP) amplified DNA only from the Korea salmons. This study suggests that SNPs in the COIII-ND3-ND4L region would be useful genetic markers for stock separation and PCR with the modified primers utilizing the SNPs provide a simple method for such purpose.2

DNA barcode of spirotrich ciliates (Protozoa: Ciliophora) with emphasis on the high resolution among closely related species

The mitochondrial cytochrome c oxidase subunit I (COI) gene has been utilized for DNA barcoding to discriminate species. The CO1 barcode was also applied for ciliates and showed high resolution. However, previous primer pairs are not enough to uncover the class Spirotrichea of which one of species-rich groups in Ciliophora. Here we designed new primer pair for ciliate barcoding, especially for the class Spirotrichea. To design new primers, we sequenced mitochondrial genomes of two pseudokeronopsids by using next generation sequencing technique (HiSeq™ 2000). Based on the newly obtained CO1 gene fragments of the pseudokeronopsids, we designed new primer pair for spirotrichean ciliates and successfully sequenced CO1 barcode of 69 ciliate populations composed of 47 species. The CO1 barcode showed great potential to discriminate closely related species. We suggest reconsideration of Euplotes crassus as a distinct species, which was considered as a junior synonym of E. vannus, and found some candidates of cryptic species in Caudiholosticha sylvatica, Diophrys scutum, Euplotes crassus. Additionally, among the successfully sequenced 47 species, common species/genera were included and their barcodes will support to prove or reinforce biogeographical distribution pattern. to uncover the class Spirotrichea of which one of species-rich groups in Ciliophora. Here we designed new primer pair for ciliate barcoding, especially for the class Spirotrichea. To design new primers, we sequenced mitochondrial genomes of two pseudokeronopsids by using next generation sequencing technique (HiSeq™ 2000). Based on the newly obtained CO1 gene fragments of the pseudokeronopsids, we designed new primer pair for spirotrichean ciliates and successfully sequenced CO1 barcode of 69 ciliate populations composed of 47 species. The CO1 barcode showed great potential to discriminate closely related species. We suggest reconsideration of Euplotes crassus as a distinct species, which was considered as a junior synonym of E. vannus, and found some candidates of cryptic species in Caudiholosticha sylvatica, Diophrys scutum, Euplotes crassus. Additionally, among the successfully sequenced 47 species, common species/genera were included and their barcodes will support to prove or reinforce biogeographical distribution pattern.1

Genetic Identification of Korean Polychaetes collected from Chuja and Jeju island, using the DNA Barcode

A total of 63 individuals belonging to 33 species in 8 order of polychaetes collected from Chuja and Jeju island were sequenced for a 658bp partial region of the mitochondrial cytochrome c oxidase subunit I(COI) gene, the typical molecular marker called DNA barcode. The species include Nicomache minor, Maldane pigmentata, Praxillella affinis of the order Capitellida, Dorvillea annulata, Protodorvillea kefersteini, Marphysa bellii, Lumbrineris japonica, Lumbrineris latreilli, Marphysa mortenseni, Diopatra bilobata of the order Eunicida, Pherusa parmata of the order Flabelligerida, Armandia lanceolata, Scalibregma inflatum of the order Opheliida, Glycera chirori, Glycera onomichiensis, Micropodarke dubia, Nephtys oligobranchia, Nephtys ciliata, Paralacydonia paradoxa, Eteone longa, Sigambra tentaculata, Syllis spongiphila, Trypanosyllis taeniaformis, Aglaophamus sinensis, Inermonephtys japonica, Anaitides koreana of the order Phllodocida, Owenia fusiformis, Chone teres of the order Sabellida, Laonice cirrata of the order Spionida, Ampharete arctica, Amphicteis gunneri, Melinna cristata, Terebellides horikoshii of the order Terebellida. Average intraspecific and interspecific distances based on Kimura two parameter model(K2P) were 3.5% and 34.9% respectively. The Neighbor-joining tree for all 63 sequences revealed the phylogenetic relationship among the species. The result supports the role of the COI gene as useful momarker called DNA barcode. The species include Nicomache minor, Maldane pigmentata, Praxillella affinis of the order Capitellida, Dorvillea annulata, Protodorvillea kefersteini, Marphysa bellii, Lumbrineris japonica, Lumbrineris latreilli, Marphysa mortenseni, Diopatra bilobata of the order Eunicida, Pherusa parmata of the order Flabelligerida, Armandia lanceolata, Scalibregma inflatum of the order Opheliida, Glycera chirori, Glycera onomichiensis, Micropodarke dubia, Nephtys oligobranchia, Nephtys ciliata, Paralacydonia paradoxa, Eteone longa, Sigambra tentaculata, Syllis spongiphila, Trypanosyllis taeniaformis, Aglaophamus sinensis, Inermonephtys japonica, Anaitides koreana of the order Phllodocida, Owenia fusiformis, Chone teres of the order Sabellida, Laonice cirrata of the order Spionida, Ampharete arctica, Amphicteis gunneri, Melinna cristata, Terebellides horikoshii of the order Terebellida. Average intraspecific and interspecific distances based on Kimura two parameter model(K2P) were 3.5% and 34.9% respectively. The Neighbor-joining tree for all 63 sequences revealed the phylogenetic relationship among the species. The result supports the role of the COI gene as useful mo2