Genetic diversity and divergence among native and translocated populations of the golden flathead goby Glossogobius aureus (Gobiiformes: Gobiidae) in Philippine lakes.

The golden flathead goby Glossogobius aureus is a native species in the Philippines, Australia, Japan, Taiwan, and many other countries in Asia. In the Philippines, it is an important food fish as it is commonly caught in major lakes. In this study, a total of 307 specimens morphologically identified as G. aureus were sampled from nine major lakes in the Philippines and were sequenced for their mitochondrial cytochrome b (cyt b) gene. Two hundred sixty of the 307 cyt b sequences had sequence similarities of ≥ 99% with G. aureus reference sequence in GenBank, while the remaining 47 (all from Lake Lanao) had sequence similarities of only 95% and were thus designated as Glossogobius cf. aureus and treated as a separate population. The sequences were then analyzed to examine the pattern of genetic diversity, relatedness, divergence, and demographic history among native and translocated populations of the species. Twenty-nine haplotypes were recovered, of which four haplotypes were shared among three to seven populations. Only one haplotype each was found in the native population in Lake Buhi and translocated population in Lake Paoay. Low haplotype and low nucleotide diversities were found for the populations in Laguna de Bay, Lanao, Bato, Buhi, Paoay, and Sebu lakes, which indicate founder event for the introduced populations in Lanao, Paoay, and Sebu lakes and recent genetic bottleneck for the native populations in Laguna de Bay, Bato, and Buhi. In contrast, high haplotype but low nucleotide diversities were found for the native populations of Taal, Naujan, and Buluan lakes, signifying a recent bottleneck followed by population expansion. Pairwise FST values showed generally large (FST = 0.168-0.249) to very large (FST = 0.302-1.000) genetic divergence between populations except between Laguna de Bay and Lake Bato, Laguna de Bay and Lake Buhi, and Lake Bato and Lake Buhi populations, which showed nonsignificant genetic differentiation. Lake Buluan and Lake Sebu populations showed moderate genetic differentiation (FST = 0.098). Neutrality tests showed significant negative Tajima's D and Fu's FS values only for the population from Laguna de Bay, which suggests that the population is undergoing expansion. These results are important for establishing scientifically sound strategies for effective conservation and sustainable exploitation of G. aureus in the Philippines.

DNA barcoding cannot discriminate between Sardinella tawilis and S. hualiensis (Clupeiformes: Clupeidae).

Sardinella tawilis, the only known freshwater sardinella in the world, is endemic to Taal Lake, Philippines. Previous studies found the Taiwan sardinella, S. hualiensis, to be morphologically very similar to S. tawilis and identified it as the marine sister species of S. tawilis. In this study, DNA barcoding using the mitochondrial cytochrome c oxidase I (COI) gene was carried out to analyze species demarcation in the Sardinella genus, focusing primarily on the relationship between S. tawilis and S. hualiensis. The neighbour-joining (NJ) tree that was constructed using Kimura 2-parameter (K2P) model showed a single clade for the two species with 100% bootstrap support. K2P interspecific genetic divergence ranged from 0% to 0.522%, which is clearly below the suggested 3-3.5% cutoff for species discrimination. Recombination activating gene 1 (RAG1), mitochondrial control region (CR), cytochrome b, 16S rRNA, and S7 markers were used to further validate the results. Sardinella tawilis and S. hualiensis clustered together with a bootstrap support of 99-100% in each of the NJ trees. Low interspecific genetic distances between S. tawilis and S. hualiensis for all the markers except CR could be attributed to incipient allopatric speciation.

Genetic diversity and patterns of demographic expansion in natural populations of milkfish, Chanos chanos (Forsskål, 1775), in the Philippines.

The milkfish, Chanos chanos (Forsskål, 1775) is an important aquaculture species produced in the tropical Pacific region. Milkfish aquaculture is heavily reliant on seedstock; thus, there is a need to assess the genetic diversity of wild populations to identify areas that could be sources of high quality seedstock and broodstock. In this study, 631 milkfish specimens from 11 Philippine and, for comparison, two Indonesian wild populations were analysed for their genetic diversities using mitochondrial control region and cytochrome b. High genetic diversity indices were found for Sarangani, Claveria, and Camarines Sur populations in the Philippines. Philippine samples had either comparable or higher genetic diversity values than Indonesian samples from Medan and Sulawesi. Based on neutrality tests and mismatch distribution analysis, Philippine samples exhibited population expansion. Such pattern was not observed among Indonesian specimens. Genetic structure among populations was weak based on analysis of molecular variance (AMOVA). Correlation between genetic distance and geographic distance was not significant. Differentiation among marine biogeographic regions was small based on hierarchical AMOVA. High gene flow due to natural dispersal and influx of haplotypes brought about by importation may have contributed to the patterns observed, but diversification events within the Philippines were still evident.

Determining species identity from confiscated pangolin remains using DNA barcoding.

Illegal wildlife trade is one of the key threats to biodiversity. A requisite in combating illegal wildlife trade is through effective and efficient identification of confiscated wildlife or wildlife remains. This can be done through DNA barcoding. In this study, DNA barcoding was employed on several cases of poaching in the Philippines involving 85 unidentified pangolin remains. Of these, 73 specimens confiscated from Palawan were identified as the Palawan endemic Manis culionensis, but no deep divergences were observed, suggesting that the samples originated from a single locality. The other 12 individuals, which were part of a large haul of pangolin carcasses recovered from a foreign fishing vessel that ran aground in Tubattaha Reefs, Philippines, were identified as the Malayan Pangolin, M. javanica. They split into two groups with 3.3% mean genetic distance, suggesting at least two geographic origins.

DNA barcoding of feral tilapias in Philippine lakes.

Tilapia (Oreochromis mossambicus) was first introduced to the Philippines in 1950 for aquaculture. Since then, other species of tilapia have been introduced to the country and some of them (mainly Oreochromis niloticus) have become established in lakes and other water bodies. In this study, DNA barcoding using the mitochondrial cytochrome c oxidase subunit I (COI) gene was done to assess the reliability of morphological identification and the degree of introgression among feral tilapias (Oreochromis spp.) in seven major Philippine lakes, namely Laguna de Bay, Lake Lanao, Taal Lake, Lake Mainit, Lake Naujan, Lake Bato, and Lake Buhi. Specimens were also collected from a private hatchery in Sual, Pangasinan to serve as reference. Morphological traits, Nucleotide BLAST (BLASTn), and Translated BLAST (BLASTx) analyses were used to classify the specimens. A Neighbor-Joining tree was constructed using the Kimura 2-Parameter method, incorporating 66 COI sequences generated from the study and 20 additional reference sequences obtained from GenBank. Three Oreochromis clusters were obtained and were classified as the O. niloticus group, O. mossambicus group, and O. aureus group, with bootstrap support values of 99%, 74%, and 99%, respectively. The mean K2P genetic distances within each group were 0.008%, 0.959%, and 0.086%, respectively. The clustering of COI sequences generated from this study corresponded with the results of the BLASTn analysis. Oreochromis hybrids were also found in all the lakes. The study highlights the usefulness of DNA barcoding for molecular identification and detection of introgressed individuals, with potential applications in management of feral stocks.

DNA barcoding of commercially important catfishes in the Philippines.

Many species of catfish are important resources for human consumption, for sport fishing and for use in aquarium industry. In the Philippines, some species are cultivated and some are caught in the wild for food and a few introduced species have become invasive. In this study, DNA barcoding using the mitochondrial cytochrome c oxidase I (COI) gene was done on commercially and economically important Philippine catfishes. A total of 75 specimens belonging to 11 species and 5 families were DNA barcoded. The genetic distances were computed and Neighbor-Joining (NJ) trees were constructed based on the Kimura 2-Parameter (K2P) method. The average K2P distances within species, genus, family and order were 0.2, 8.2, 12.7 and 21.9%, respectively. COI sequences clustered according to their species designation for 7 of the 11 catfishes. DNA barcoding was not able to discriminate between Arius dispar and A. manillensis and between Pterygoplichthys disjunctivus and P. pardalis. The morphological characters that are used to distinguish between these species do not complement molecular identification through DNA barcoding. DNA barcoding also showed that Clarias batrachus from the Philippines is different from the species found in India and Thailand, which supports earlier suggestions based on morphology that those found in India should be designated as C. magur and those in mainland Southeast Asia as C. aff. batrachus "Indochina". This study has shown that DNA barcoding can be used for species delineation and for tagging some species for further taxonomic investigation, which has implications on proper management and conservation strategies.

High gene flow in reef fishes and its implications for ad-hoc no-take marine reserves.

Integration of genetic connectivity information in effective marine reserve (MR) design is important in sustaining marine biodiversity. Here, genetic connectivity based on mitochondrial DNA (mtDNA) of three reef fish species, namely Epinephelus merra (n = 67; 32 from Bolinao, 14 from Alaminos, and 21 from Masinloc), Parupeneus multifasciatus (n = 23; 12 from Bolinao and 11 from Masinloc), and Odonus niger (n = 35; 21 from Mabini and 14 from Tingloy), sampled across western Luzon, Philippines, was inferred by assessing their genetic diversity, population genetic structure, and historical demography. The results show high haplotype and nucleotide diversity in the three species. Tests for population structure indicate high gene flow and no spatial genetic structuring for the three species. Mismatch analyses suggest unimodal distribution for E. merra and P. multifasciatus, but bimodal distribution for O. niger. Even with differences in mismatch distributions, all the three species exhibit low raggedness index indicating demographic population expansion. The bimodal distribution of O. niger could be attributed to the mixing of two isolated populations. High gene flow between sampling locations implies genetic exchanges and connectivity between many small MRs and fishing grounds in western Luzon, Philippines, at a scale similar to our study. This research is among the first few to elucidate the high genetic connectivity of reef fish communities across the Philippines (here western Luzon), but it also calls for more support (i.e. government and academia) for genetic research that aims to (1) understand the maintenance of megadiversity of the country and (2) search for effective biodiversity conservation options for the coral reefs.

Genetic diversity analysis of Arius manillensis (Siluriformes: Ariidae) using the mitochondrial control region.

Arius manillensis is a Philippine endemic species and is an economically important fishery resource in Laguna de Bay, the largest lake in the country. Drastic reduction in population sizes of A. manillensis has been recorded in the past, which may have resulted in genetic bottleneck. In this study, the genetic diversity and population structure of A. manillensis in Laguna de Bay were assessed using the mitochondrial DNA control region. Specimens were obtained from three localities along Laguna de Bay, namely Binangonan (n = 27), Tanay (n = 29), and Calamba (n = 30). Of the 86 DNA sequences generated, 22 distinct haplotypes were observed. There were four unique haplotypes for Binangonan, six for Calamba, and five for Tanay. There were two haplotypes common to the three sites. The maximum likelihood tree and median-joining network showed little geographic separation among the haplotypes. Chi-square test showed no significant differentiation in A. manillensis from the three sites. The overall computed F(ST) was 0.0144, indicating small genetic differentiation in A. manillensis from the three localities sampled. Likewise, analysis of molecular variance showed a greater percentage of variation within population (98.62%) than variation among populations (1.38%; P = 0.21). Total haplotype diversity and nucleotide diversity among the specimens from the three sites were 0.775 and 0.013, respectively. The high haplotype diversity coupled with low nucleotide diversity observed in this study confirms that genetic bottleneck occurred in A. manillensis which was followed by population expansion. This is also supported by the non-significant values for both Tajima's D and Fu's F. Furthermore, multimodal mismatch distribution plots were generated, which is consistent with the model of spatial range expansion followed by demographic expansion.

DNA barcoding of fishes of Laguna de Bay, Philippines.

Laguna de Bay, the largest lake in the Philippines, is an important part of the country's fisheries industry. It is also home to a number of endemic fishes including Gobiopterus lacustris (Herre 1927) of family Gobiidae, Leiopotherapon plumbeus (Kner 1864) of family Terapontidae, Zenarchopterus philippinus (Peters 1868) of family Hemiramphidae and Arius manillensis Valenciennes 1840 of family Ariidae. Over the years, a steady decline has been observed in the abundance and diversity of native fishes in the lake due to anthropogenic disturbances. In this study, a total of 71 specimens of 18 different species belonging to 18 genera, 16 families, and seven orders were DNA barcoded using the mitochondrial cytochrome c oxidase subunit I (COI) gene. All of the fish species were discriminated by their COI sequences and one endemic species G. lacustris, showing deep genetic divergence, was highlighted for further taxonomic investigation. Average Kimura 2-parameter genetic distances within species, family, and order were 1.33%, 18.91%, and 24.22%, respectively. These values show that COI divergence increases as taxa become less exclusive. All of the COI sequences obtained were grouped together according to their species designation in the Neighbor-joining tree that was constructed. This study demonstrated that DNA barcoding has great potential as a tool for fast and accurate species identification and also for highlighting species that warrant further taxonomic investigation.

DNA barcoding of the ichthyofauna of Taal Lake, Philippines.

This study represents the first molecular survey of the ichthyofauna of Taal Lake and the first DNA barcoding attempt in Philippine fishes. Taal Lake, the third largest lake in the Philippines, is considered a very important fisheries resource and is home to the world's only freshwater sardine, Sardinella tawilis. However, overexploitation and introduction of exotic fishes have caused a massive decline in the diversity of native species as well as in overall productivity of the lake. In this study, 118 individuals of 23 native, endemic and introduced fishes of Taal Lake were barcoded using the partial DNA sequence of the mitochondrial cytochrome c oxidase subunit I (COI) gene. These species belong to 21 genera, 17 families and 9 orders. Divergence of sequences within and between species was determined using Kimura 2-parameter (K2P) distance model, and a neighbour-joining tree was generated with 1000 bootstrap replications using the K2P model. All COI sequences for each of the 23 species were clearly discriminated among genera. The average within species, within genus, within family and within order percent genetic divergence was 0.60%, 11.07%, 17.67% and 24.08%, respectively. Our results provide evidence that COI DNA barcodes are effective for the rapid and accurate identification of fishes and for identifying certain species that need further taxonomic investigation.

DNA barcodes of Philippine accipitrids.

DNA barcoding is a molecular method that rapidly identifies an individual to a known taxon or its closest relative based on a 650-bp fragment of the cytochrome c oxidase subunit I (COI). In this study, DNA barcodes of members of the family Accipitridae, including Haliastur indus (brahminy kite), Haliaeetus leucogaster (white-bellied sea eagle), Ichthyophaga ichthyaetus (grey-headed fish eagle), Spilornis holospilus (crested serpent-eagle), Spizaetus philippensis (Philippine hawk-eagle), and Pithecophaga jefferyi (Philippine eagle), are reported for the first time. All individuals sampled are kept at the Philippine Eagle Center in Davao City, Philippines. Basic local alignment search tool results demonstrated that the COI sequences for these species were unique. The COI gene trees constructed using the maximum-likelihood and neighbour-joining (NJ) methods supported the monophyly of the booted eagles of the Aquilinae and the sea eagles of the Haliaeetinae but not the kites of the Milvinae.

Microarray analysis of gene expression in eastern oyster (Crassostrea virginica) reveals a novel combination of antimicrobial and oxidative stress host responses after dermo (Perkinsus marinus) challenge.

Dermo disease, caused by Perkinsus marinus, is one of the most severe diseases of eastern oysters, Crassostrea virginica. It causes serious mortalities in both wild and aquacultured oysters. Using existing expressed sequence tag (EST) resources, we developed a 12K in situ oligonucleotide microarray and used it for the analysis of gene expression profiles of oysters during the interactions between P. marinus and its oyster host. Significant gene expression regulation was found at day 30 post-challenge in the eastern oyster. Putative identities of the differentially expressed genes revealed a set of genes involved in several processes including putative antimicrobial defenses, pathogen recognition and uptake, anti-oxidation and apoptosis. Consistent with results obtained from previous, smaller-scale experiments, expression profiles revealed a large set of genes likely involved in an active mitigating response to oxidative stress and apoptosis induced by P. marinus. Additionally, a unique galectin from C. virginica, CvGal, which serves as a preferential receptor for P. marinus trophozoites, was found to be significantly down-regulated in gill tissue of oysters with both light and heavy infection, suggesting an attempt to control parasite uptake and proliferation in the later stages of infection. Potential histone-derived antimicrobial responses to P. marinus were also revealed in the gene expression profiles.

Assembly of 500,000 inter-specific catfish expressed sequence tags and large scale gene-associated marker development for whole genome association studies.

BACKGROUND: Through the Community Sequencing Program, a catfish EST sequencing project was carried out through a collaboration between the catfish research community and the Department of Energy's Joint Genome Institute. Prior to this project, only a limited EST resource from catfish was available for the purpose of SNP identification. RESULTS: A total of 438,321 quality ESTs were generated from 8 channel catfish (Ictalurus punctatus) and 4 blue catfish (Ictalurus furcatus) libraries, bringing the number of catfish ESTs to nearly 500,000. Assembly of all catfish ESTs resulted in 45,306 contigs and 66,272 singletons. Over 35% of the unique sequences had significant similarities to known genes, allowing the identification of 14,776 unique genes in catfish. Over 300,000 putative SNPs have been identified, of which approximately 48,000 are high-quality SNPs identified from contigs with at least four sequences and the minor allele presence of at least two sequences in the contig. The EST resource should be valuable for identification of microsatellites, genome annotation, large-scale expression analysis, and comparative genome analysis. CONCLUSIONS: This project generated a large EST resource for catfish that captured the majority of the catfish transcriptome. The parallel analysis of ESTs from two closely related Ictalurid catfishes should also provide powerful means for the evaluation of ancient and recent gene duplications, and for the development of high-density microarrays in catfish. The inter- and intra-specific SNPs identified from all catfish EST dataset assembly will greatly benefit the catfish introgression breeding program and whole genome association studies.

Generation and analysis of ESTs from the eastern oyster, Crassostrea virginica Gmelin and identification of microsatellite and SNP markers.

BACKGROUND: The eastern oyster, Crassostrea virginica (Gmelin 1791), is an economically important species cultured in many areas in North America. It is also ecologically important because of the impact of its filter feeding behaviour on water quality. Populations of C. virginica have been threatened by overfishing, habitat degradation, and diseases. Through genome research, strategies are being developed to reverse its population decline. However, large-scale expressed sequence tag (EST) resources have been lacking for this species. Efficient generation of EST resources from this species has been hindered by a high redundancy of transcripts. The objectives of this study were to construct a normalized cDNA library for efficient EST analysis, to generate thousands of ESTs, and to analyze the ESTs for microsatellites and potential single nucleotide polymorphisms (SNPs). RESULTS: A normalized and subtracted C. virginica cDNA library was constructed from pooled RNA isolated from hemocytes, mantle, gill, gonad and digestive tract, muscle, and a whole juvenile oyster. A total of 6,528 clones were sequenced from this library generating 5,542 high-quality EST sequences. Cluster analysis indicated the presence of 635 contigs and 4,053 singletons, generating a total of 4,688 unique sequences. About 46% (2,174) of the unique ESTs had significant hits (E-value