Molecular Taxonomy of Cupped Oysters (Crassostrea, Saccostrea, and Striostrea) in Thailand Based on COI, 16S, and 18S rDNA Polymorphism

Genetic diversity of oysters Crassostrea belcheri (Sowerby, 1871), C. iredalei (Faustino, 1932), Saccostrea cucullata (Born, 1778), S. forskali (Gmelin, 1791), and Striostrea (Parastriostrea) mytiloides (Lamarck, 1819) (Ostreoida, Mollusca) was analyzed by polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP) of 16S ribosomal DNA with AcsI, AluI, DdeI, DraI, RsaI, and TaqI, 18S ribosomal DNA with HinfI, and cytochrome oxidase subunit I with AcsI, DdeI and MboI. A total of 54 composite haplotypes were observed. Species-diagnostic markers were specifically found in C. belcheri, C. iredalei, and S. cucullata, but not in S. forskali and Striostrea mytiloides, which shared common composite haplotypes. Neighbor-joining trees constructed from genetic distances between pairs of composite haplotypes and species indicated large genetic differences between Crassostrea and Saccostrea (including Striostrea mytiloides), but closer relationships were observed within each genus. Four groups of unidentified oysters (Crassostrea sp. and Saccostrea sp. groups 1, 2, and 3) were also genetically analyzed. Fixed RFLP markers were found in Crassostrea sp. and Saccostrea sp. group 2, but not in Saccostrea sp. groups 1 and 3. Phylogenetic and genetic heterogeneity analyses indicated that Crassostrea sp. and Saccostrea sp. group 2 should be considered as newly unidentified oyster species in Thailand.     

Molecular Genetic Identification Tools for Three Commercially Cultured Oysters (<Emphasis Type="Italic">Crassostrea belcheri</Emphasis>, <Emphasis Type="Italic">Crassostrea iredalei</Emphasis>, and <Emphasis Type="Italic">Saccostrea cucullata</Emphasis>) in Thailand

Abstract Molecular genetic keys for identification of 3 commercially cultured oysters (Crassostrea belcheri, Crassostrea iredalei, and Saccostrea cucullata) in Thailand were developed based on restriction analysis of 18S ribosomal DNA and cytochrome oxidase subunit I (COI). Digestion of the amplified 18S rDNA with Hinf I unambiguously differentiated Crassostrea oysters from Saccostrea oysters and Striostrea (Parastriostrea) mytiloides. In addition, species-specific restriction fragment length polymorphism patterns of C. belcheri, C. iredalei, and S. cucullata were consistently observed when the gel-eluted COI was digested with Mbo I and Dde I. Thirty composite haplotypes were observed across all individuals. Species-specific composite haplotypes were found in C. belcheri (AAAA and AAAB), C. iredalei (AABC and AABU), and S. cucullata (BBCD and BBCE), respectively. The most common composite haplotype of COI in C. belcheri (AAAA), C. iredalei (AABC), and S. cucullata (BBCD) was amplified, cloned, and sequenced. Detection of C. belcheri and C. iredalei based on polymerase chain reaction was further developed using more specific primers (HCO2198 and R372) followed by digestion of a 372-bp product with Mbo I.     

Development of Species-Specific Markers of the Tropical Oyster (Crassostrea belcheri) in Thailand

Randomly amplified polymorphic DNA (RAPD) analysis was used to identify species-specific markers of 5 oyster species in Thailand: Crassostrea belcheri, Crassostrea iredalei, Saccostrea cucullata, Saccostrea forskali, and Striostrea (Parastriostrea) mytiloides. Species-specific markers were found in C. belcheri, C. iredalei, and S. cucullata but not in S. forskali and S. mytiloides. Three C. belcheri–specific RAPD fragments were cloned and sequenced. A primer set was designed from each of the recombinant clones (pPACB1, pPACB2, and pPACB3). The polymerase chain reaction products showed expected sizes of 536, 600, and 500 bp, respectively, with the sensitivity of detection approximately 30 pg of C. belcheri total DNA template. The specificity of pPACB1 was examined against 135 individuals of indigenous oyster species in Thailand and against outgroup references S. commercialis (N= 12) and Perna viridis (N= 12). Results indicated the species-specific nature of primers developed from pPACB1. This primer set can be used for broodstock selection and determination of C. belcheri larvae to assist the selective breeding program for this commercially important species. Received December 8, 1999; accepted March 16, 2000.     

Genetic variation in Malaysian oysters: taxonomic ambiguities and evidence of biological invasion

Genetic diversities in two cultured oyster species, Crassostrea iredalei (Faustino 1932) and Crassostrea belcheri (Sowerby 1871) were assessed using a 581-nucleotide fragment of the mtDNA cytochrome oxidase subunit 1 (CO1) gene. A total of 103 C. iredalei individuals and 120 C. belcheri from 12 populations were sampled along the coast of Malaysia. Trees of unique haplotype samples generated based on Neighbor-Joining (NJ) algorithm revealed that many individuals had been misidentified and did not cluster with their presumed species based on morphological identification. BLAST results of DNA sequences showed presence of previously unreported C. madrasensis in Peninsular Malaysian waters (98% maximum identity). The true identity of the Muar (Crassostrea sp.) and Semporna (Saccostrea sp.) populations were unresolved by two BLAST search and showed less than 88% identity with other species in GenBank. Repeated analysis of these two populations using 487 bp of the mitochondrial 16S gene data showed only a maximum identity less than 97%. Hence, the identity of these specimens remains unclear. Evolutionary divergences within presumed species were 0.001–0.011 and 0.034–0.313 between species. Findings from this study have important implications for aquaculture, management and monitoring of cultured populations as well as conservation of wild oyster species in Malaysia.     

Molecular phylogeny of mangrove oysters (Crassostrea) from Brazil

As a result of phenotypic plasticity, the cupped oysters (Crassostrea)are difficult to identify by means of their morphology. However,molecular DNA markers are a useful means of discriminating amongthese species. Cupped oysters are one of the most widely culturedmarine invertebrates and correct species identification is importantin aquaculture. Moreover, the molecular phylogeny of the genusCrassostrea and the subfamily Crassostreinae is still not clear.In order to identify the Brazilian cupped oysters and to clarifythe phylogenetic relationships of these species, we sequenceda fragment of mitochondrial DNA (16S rRNA gene) from 120 specimenscollected at nine different sites distributed along the Braziliancoast. The results identified two native species of oyster:Crassostrea gasar, from the Amazon to the Parnaíba delta;and Crassostrea rhizophorae, from the northeast (Fortim) tothe south of Brazil. An exotic Crassostrea species, closelyrelated to Indo-Pacific Crassostrea, was found in one locationin the north of Brazil. Crassostrea showed monophyly and theAtlantic oysters are clearly separated from the Indo-Pacificcluster. (Received 30 May 2006; accepted 12 April 2007)     

Unusual conservation of mitochondrial gene order in <Emphasis Type="Italic">Crassostrea</Emphasis>oysters: evidence for recent speciation in Asia

Background  

Oysters are morphologically plastic and hence difficult subjects for taxonomic and evolutionary studies. It is long been suspected, based on the extraordinary species diversity observed, that Asia Pacific is the epicenter of oyster speciation. To understand the species diversity and its evolutionary history, we collected five Crassostrea species from Asia and sequenced their complete mitochondrial (mt) genomes in addition to two newly released Asian oysters (C. iredalei and Saccostrea mordax) for a comprehensive analysis.     

SPERM ULTRASTRUCTURE IN THE AUSTRALIAN OYSTER SACCOSTREA COMMERCIALIS (IREDALE & ROUGHLEY) (BIVALVIA: OSTREOIDEA)

Sperm ultrastructurc is investigated in the economically importantAustralian oyster Saccostrea commercialis (Iredale & Roughley).Mature spermatozoa consist of a broad, cap-shaped acrosomalvesicle, sub-acrosomal material (axial rod embedded in a granularmatrix), a round nucleus (deeply invaginated anteriorly), twotriplet substructure centrioles surrounded by four sphericalmitochondria, and a flagellum anchored via satellite fibresto the distal centriole and plasma membrane. Spermatozoa ofS. commercialis closely resemble those of other investigatedostreids, with the exception that the anterior region of theacrosomal vesicle is transversely banded. Differences in acrosomalsubstructure correlate with the known inability of Saccostreaand Crassostrea gametes to interact. Potential exists for useof sperm morphology in the resolution of taxonomic problemswithin the Ostreoidea. (Received 23 April 1990; accepted 28 June 1990)     

Identification of molecular markers linked to the Yr15 stripe rust resistance gene of wheat originated in wild emmer wheat, Triticum dicoccoides

 The Yr15 gene of wheat confers resistance to the stripe rust pathogen Puccinia striiformis West., which is one of the most devastating diseases of wheat throughout the world. In the present study, molecular markers flanking the Yr15 gene of wheat have been identified using the near-isogenic-lines approach. RFLP screening of 76 probe-enzyme combinations revealed one polymorphic marker (Nor/TaqI) between the susceptible and the resistant lines. In addition, out of 340 RAPD primers tested, six produced polymorphic RAPD bands between the susceptible and the resistant lines. The genetic linkage of the polymorphic markers was tested on segregating F₂ population (123 plants) derived from crosses between stripe rust-susceptible Triticum durum wheat, cv D447, and a BC₃F₉ resistant line carrying Yr15 in a D447 background. A 2.8-kb fragment produced by the Nor RFLP probe and a 1420-bp PCR product generated by the RAPD primer OPB13 showed linkage, in coupling, with the Yr15 gene. Employing the standard maximum-likelihood technique it was found that the order OPB13 ₁₄₂₀ –Yr15–Nor1 on chromosome 1B appeared to be no less than 1000-times more probable than the closest alternative. The map distances between OPB13 ₁₄₂₀ –Yr15–Nor1 are 27.1 cM and 11.0 cM for the first and second intervals, respectively. The application of marker-assisted selection for the breeding of new wheat cultivars with the stripe rust resistance gene is discussed. Received: 27 February 1997/Accepted: 7 March 1997     

EST‐SSR markers from the Pacific oyster,Crassostrea gigas

Ten polymorphic microsatellite repeat markers were identified from Crassostrea gigas, expressed sequence tags (EST) deposited in public sequence database. Number of alleles per locus ranged from three to 18, expected and observed heterozygosities ranged from 0.071 to 0.738 and from 0.306 to 0.913, respectively. Marker transferability was tested on other two Crassostrea species and polymorphic products were detected at nine loci. EST‐derived simple sequence repeats provide robust, informative and potentially transferable polymorphic markers suitable for population genetic, parentage, and mapping studies of C. gigas.     

Complete mitochondrial DNA sequence of Crassostrea nippona: comparative and phylogenomic studies on seven commercial Crassostrea species

The complete mitochondrial genome of Crassostrea nippona was determined and compared with six other Crassostrea mitogenomes from GenBank in an attempt to shed light on the evolutionary relatedness within Crassostrea. The total length of the mitogenome was 20,030 bp for C. nippona, which was the largest among seven Crassostrea mitogenomes. Among six Asian oysters, the gene order of mitochondrial DNA was identical except for C. nippona with a transposition of trnG. While the American oyster C. virginica and Asian oysters showed broad differences in gene order with relocation of most tRNA genes and indels of duplicated tRNAs and rrnS, indicating the relatively distant relationships between the American oyster and Asian oysters. Different from other six Crassostrea oysters, C. nippona had two repeats of 66 bp in non-coding regions. Pairwise divergence among the seven Crassostrea oysters based on DNA sequences of 12 protein-coding genes ranged from 3.1 to 44.4% (Kimura two-parameter distance, K2P). The close relationship between C. nippona and C. hongkongensis was revealed by K2P of 18.9%. Phylogenetic analyses robustly revealed Crassostrea monophyly, with C. virginica at the basal position. The results of phylogenetic analyses strongly supported C. gigas and C. angulata had the closest relationship, with C. sikamea being the sister taxon. These findings presented here provide a better insight into the relationships within Crassostrea and will be useful for further evolution studies of oysters.     

Hermaphroditism and sex reversal in the four common oviparous species of oysters from the coast of Karachi

In the four common oviparous oysters from the coast of Karachi, Crassostrea rivularis, C. madrasensis, Saccostrea glomerata and S. cucculata, the gonad appeared at the age of 2–3 months, at a length of 0.4–0.6 cm. Sex is determined from the origin and hermaphrodites are rare, their percentage among the population increasing as growth proceeds, but is not affected by changes of the seasons. Incidence of hermaphrodites and changes in sex ratio suggest a change of sex as seen in oysters of temperate zones.     

Molecular identification, phylogeny and geographic distribution of Brazilian mangrove oysters (Crassostrea)

Oysters (Ostreidae) manifest a high degree of phenotypic plasticity, whereby morphology is of limited value for species identification and taxonomy. By using molecular data, the aim was to genetically characterize the species of Crassostrea occurring along the Brazilian coast, and phylogenetically relate these to other Crassostrea from different parts of the world. Sequencing of the partial cytochrome oxidase c subunit I gene (COI), revealed a total of three species of Crassostrea at 16 locations along the Brazilian coast. C. gasar was found from Curuçá (Pará state) to Santos (São Paulo state), and C. rhizophorae from Fortim (Ceará state) to Florianópolis (Santa Catarina state), although small individuals of the latter species were also found at Ajuruteua beach (municipality of Bragança, Pará state). An unidentified Crassostrea species was found only on Canela Island, Bragança. Crassostrea gasar and C. rhizophorae grouped with C. virginica, thereby forming a monophyletic Atlantic group, whereas Crassostrea sp. from Canela Island was shown to be more similar to Indo-Pacific oysters, and either arrived in the Atlantic Ocean before the convergence of the Isthmus of Panama or was accidentally brought to Brazil by ship.     

Intraspecific Variation in Mitogenomes of Five <Emphasis Type="Italic">Crassostrea</Emphasis> Species Provides Insight into Oyster Diversification and Speciation

A large number of Crassostrea oysters are found in Asia-Pacific. While analyses of interspecific variation have helped to establish historical relationships among these species, studies on intraspecific variation are necessary to understand their recent evolutionary history and current forces driving population biology. We resequenced 18 and analyzed 31 mitogenomes of five Crassostrea species from China: Crassostrea gigas, Crassostrea angulata, Crassostrea sikamea, Crassostrea ariakensis, and Crassostrea hongkongensis. Our analysis finds abundant insertions, deletions, and single-nucleotide polymorphisms in all species. Intraspecific variation varies greatly among species with polymorphic sites ranging from 54 to 293 and nucleotide diversity ranging from 0.00106 to 0.00683. In all measurements, C. hongkongensis that has the narrowest geographic distribution exhibits the least sequence diversity; C. ariakensis that has the widest distribution shows the highest diversity, and species with intermediate distribution show intermediate levels of diversity. Low sequence diversity in C. hongkongensis may reflect recent bottlenecks that are probably exacerbated by human transplantation. High diversity in C. ariakensis is likely due to divergence of northern and southern China populations that have been separated without gene flow. The significant differences in mitogenome diversity suggest that the five sister species of Crassostrea have experienced different evolutionary forces since their divergence. The recent divergence of two C. ariakensis populations and the C. gigas/angulata species complex provides evidence for continued diversification and speciation of Crassostrea species along China’s coast, which are shaped by unknown mechanisms in a north–south divide.     

Proteomic clues to the identification of QX disease-resistance biomarkers in selectively bred Sydney rock oysters,Saccostrea glomerata

The Sydney rock oyster, Saccostrea glomerata, is susceptible to infection by the protozoan parasite, Marteilia sydneyi, the causative agent of QX disease. M. sydneyi infection peaks during summer when QX disease can cause up to 95% mortality. The current study takes a proteomic approach using 2-dimensional electrophoresis and mass spectrometry to identify markers of QX disease resistance among Sydney rock oysters. Proteome maps were developed for QX disease-resistant and -susceptible oysters. Six proteins in those maps were clearly associated with resistance and so were characterized by mass spectrometry. Two of the proteins (p9 and p11) were homologous to superoxide dismutase-like molecules from the Pacific oyster, Crassostrea gigas, and the Eastern oyster, Crassostrea virginica. The remaining S. glomerata proteins had no obvious similarities to known molecules in sequence databases. p9 and p11 are currently being investigated as potential markers for the selective breeding of QX disease-resistant oysters.     

Kanagawa-Negative, <Emphasis Type="Italic">tdh</Emphasis>- and <Emphasis Type="Italic">trh</Emphasis>-Positive <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> Isolated from Fresh Oysters Marketed in Fortaleza,Brazil

Between October 2008 and June 2009, 15 samples of 10 live oysters each (Crassostrea rhizophorae) measuring 8.31–10.71 cm were purchased from a restaurant on the seashore of Fortaleza, Brazil. The Vibrio count ranged from 75 (estimated) to 43,500 CFU/g. Fourteen species were identified among the 56 isolated Vibrio strains, with V. parahaemolyticus as the most prevalent. Two of the 17 V. parahaemolyticus strains were urease-positive and tdh- and trh-positive on multiplex PCR, but neither produced β-hemolysis halos in Wagatsuma agar. Thus, fresh oysters served in natura in Fortaleza, Brazil, were found to contain Vibrio strains known to cause gastroenteritis in humans.     

GENETIC DETERMINANTS OF PROTANDRIC SEX IN THE PACIFIC OYSTER,CRASSOSTREA GIGAS THUNBERG

A unique feature of sex in Crassostrea oysters is the coexistence of protandric sex change, dioecy, and hermaphroditism. To determine whether such a system is genetically controlled, we analyzed sex ratios in 86 pair-mated families of the Pacific oyster, Crassostrea gigas Thunberg. The overall female ratios of one-, two-, and three-year-old oysters were 37%, 55%, and 75%, respectively, suggesting that a significant proportion of oysters matured first as males and changed to females in later years. Detailed analysis of sex ratios in factorial and nested crosses revealed significant paternal effects, which corresponded to two types of sires. No major maternal effects on sex were observed. Major genetic control of sex was further indicated by the distribution of family sex ratios in two to four apparently discreet groups. These and other data from the literature are compatible with a single-locus model of primary sex determination with a dominant male allele (M) and a protandric female allele (F), so that MF are true males and FF are protandric females that are capable of sex change. The rate of sex change of FF individuals may be influenced by secondary genes and/or environmental factors. Strong maternal and weak paternal effects on sexual maturation or time of spawning were also suggested.     

Channa nox, a new channid fish lacking a pelvic fin from Guangxi, China

 A new species of channid fish, genus Channa, is described from 7 specimens collected from the vicinity of Hepu, Guangxi Province, southern China. The new species, Channa nox, is distinguished from all other channid species by the following combination of characters: absence of pelvic fins, small rounded head (22.1%–26.8% SL), narrow interorbital width (19.6%–26.7% HL), short snout length (3.6%–5.1% SL), predorsal and prepectoral lengths (26.9%–28.4% SL and 24.8%–28.3% SL, respectively), 47–51 dorsal fin rays, 31–33 anal fin rays, 55–63 lateral line scales, 5.5–6.5 scales above lateral line, 9–13 cheek scales, 53–55 total vertebrae, 1 or 2 scale(s) on each side of lower jaw undersurface, the black upper half of body with 8–11 irregular (often anteriorly pointed V-shaped) bands or blotches, a large white-rimmed black ocellus on caudal peduncle and sparse white spots on the dark brown body and dorsal and caudal fins, as well as the shape of the hyomandibular process of the suprabranchial organs. Channa nox is sympatrically distributed with its morphologically most similar congener, C. asiatica. Received: January 18, 2001 / Revised: November 2, 2001 / Accepted: December 12, 2001     

Mating system and seed variation of <Emphasis Type="Italic">Acacia</Emphasis> hybrid (<Emphasis Type="Italic">A. mangium</Emphasis> × <Emphasis Type="Italic">A. auriculiformis</Emphasis>)

The mating system and seed variation of Acacia hybrid (A. mangium × A. auriculiformis) were studied using allozymes and random amplified polymorphic DNA (RAPD) markers, respectively. Multi-locus outcrossing rate estimations indicated that the hybrid was predominantly outcrossed (mean±s.e. t _m = 0.86±0.01). Seed variation was investigated using 35 polymorphic RAPD fragments. An analysis of molecular variance (AMOVA) revealed the highest genetic variation among seeds within a pod (66%–70%), followed by among pods within inflorescence (29%–37%), and the least variation among inflorescences within tree (<1%). In addition, two to four RAPD profiles could be detected among seeds within pod. Therefore, the results suggest that a maximum of four seeds per pod could be sampled for the establishment of a mapping population for further studies.     

Molecular Phylogenetics and Systematics of the Bivalve Family Ostreidae Based on rRNA Sequence-Structure Models and Multilocus Species Tree

The bivalve family Ostreidae has a worldwide distribution and includes species of high economic importance. Phylogenetics and systematic of oysters based on morphology have proved difficult because of their high phenotypic plasticity. In this study we explore the phylogenetic information of the DNA sequence and secondary structure of the nuclear, fast-evolving, ITS2 rRNA and the mitochondrial 16S rRNA genes from the Ostreidae and we implemented a multi-locus framework based on four loci for oyster phylogenetics and systematics. Sequence-structure rRNA models aid sequence alignment and improved accuracy and nodal support of phylogenetic trees. In agreement with previous molecular studies, our phylogenetic results indicate that none of the currently recognized subfamilies, Crassostreinae, Ostreinae, and Lophinae, is monophyletic. Single gene trees based on Maximum likelihood (ML) and Bayesian (BA) methods and on sequence-structure ML were congruent with multilocus trees based on a concatenated (ML and BA) and coalescent based (BA) approaches and consistently supported three main clades: (i) Crassostrea, (ii) Saccostrea, and (iii) an Ostreinae-Lophinae lineage. Therefore, the subfamily Crassotreinae (including Crassostrea), Saccostreinae subfam. nov. (including Saccostrea and tentatively Striostrea) and Ostreinae (including Ostreinae and Lophinae taxa) are recognized. Based on phylogenetic and biogeographical evidence the Asian species of Crassostrea from the Pacific Ocean are assigned to Magallana gen. nov., whereas an integrative taxonomic revision is required for the genera Ostrea and Dendostrea. This study pointed out the suitability of the ITS2 marker for DNA barcoding of oyster and the relevance of using sequence-structure rRNA models and features of the ITS2 folding in molecular phylogenetics and taxonomy. The multilocus approach allowed inferring a robust phylogeny of Ostreidae providing a broad molecular perspective on their systematics.     

Recently established <Emphasis Type="Italic">Crassostrea</Emphasis>-reefs versus native <Emphasis Type="Italic">Mytilus</Emphasis>-beds: differences in ecosystem engineering affects the macrofaunal communities (Wadden Sea of Lower Saxony,southern German Bight)

Since 1998 the non-indigenous Pacific oyster Crassostrea gigas (Thunberg 1793) has been invading the Wadden Sea of Lower Saxony, southern German Bight. C. gigas settles predominantly on intertidal Mytilus-beds (M. edulis L.) and subsequently create rigid reef-like structures. Both bivalve species are ecosystem engineers in sedimentary tidal flats. They provide hard substrate for sessile species, mobile organisms find refuge within the habitat matrix of dense suspension feeders, and biodeposits enrich the sediments with organic matter. The transformation of Mytilus-beds into Crassostrea-reefs gives rise to the question whether the invader may affect the native community. We investigated two parts of a changing bivalve bed in the backbarrier area of the island of Juist in March 2005. One part was still dominated by M. edulis whereas the other part was already densely colonized by C. gigas. Crassostrea-reefs compensate for the conceivable loss of Mytilus-beds in the intertidal of the Wadden Sea by replacing the ecological function of M. edulis. There was no indication of a suppression of indigenous species. This even applied to M. edulis, which persisted at the site invaded by C. gigas. The associated macrofaunal community showed increased species richness, abundance, biomass, and diversity in the Crassostrea-reef. The latter particularly favored sessile species like anthozoans, hydrozoans, and barnacles. Higher abundance and biomass for vagile epizoic species like the shore crab Carcinus maenas and the periwinkle Littorina littorea also occurred among oysters. Abundance of deposit feeding oligochaetes was enhanced by oysters as well. More opportunistic, facultative filter-feeding polychaetes occurred in the Crassostrea-reef.