Gut content metabarcoding of specialized feeders is not a replacement for environmental DNA assays of seawater in reef environments.

In tropical marine ecosystems, the coral-based diet of benthic-feeding reef fishes provides a window into the composition and health of coral reefs. In this study, for the first time, we compare multi-assay metabarcoding sequences of environmental DNA (eDNA) isolated from seawater and partially digested gut items from an obligate corallivore butterflyfish (Chaetodon lunulatus) resident to coral reef sites in the South China Sea. We specifically tested the proportional and statistical overlap of the different approaches (seawater vs gut content metabarcoding) in characterizing eukaryotic community composition on coral reefs. Based on 18S and ITS2 sequence data, which differed in their taxonomic sensitivity, we found that gut content detections were only partially representative of the eukaryotic communities detected in the seawater based on low levels of taxonomic overlap (3 to 21%) and significant differences between the sampling approaches. Overall, our results indicate that dietary metabarcoding of specialized feeders can be complimentary to, but is no replacement for, more comprehensive environmental DNA assays of reef environments that might include the processing of different substrates (seawater, sediment, plankton) or traditional observational surveys. These molecular assays, in tandem, might be best suited to highly productive but cryptic oceanic environments (kelp forests, seagrass meadows) that contain an abundance of organisms that are often small, epiphytic, symbiotic, or cryptic.

Genetic stock structure of the silky shark Carcharhinus falciformis in the Indo-Pacific Ocean.

The silky shark, Carcharhinus falciformis, is a cosmopolitan species commonly caught as a bycatch for longline fisheries. However, the genetic stock structure for the Indo-Pacific Ocean is not well-defined yet. Here, we used eight microsatellite loci to examine the genetic stock structure and effective population size of 307 silky sharks across 5 Indo-Pacific sampling locations. A major genetic break was found between Aceh and the remaining locations (FST = 0.0505-0.0828, p = 0.001). The Indian Ocean displayed a slightly lower effective population estimate (Ne) compared to the Pacific Ocean, potentially due to the higher fishing pressure in the Indian Ocean region. The lowest Ne was found in the Aceh population (Ne = 2.3), suggesting it might be a small and endemic population. These findings offer valuable information for the conservation and management of the silky shark. We suggest that the population around Aceh waters constitutes a distinct stock and should be managed independently. Further investigations into migratory and movement patterns are needed to define the boundaries of different stocks, ensuring effective management the silky shark across the Indo-Pacific region.

Anti-Inflammatory Halogenated Monoterpenes from the Red Alga Portieria hornemannii.

The chemical investigation of a red alga Portieria hornemannii enabled the identification of three new halogenated monoterpenes (1-3) along with two previously identified metabolites (4 and 5). Their structures were determined by spectroscopic analysis and also by utilizing single-crystal diffraction analysis and quantum chemical calculation, as well as by comparison with literature data. Further corrections for dichloro and dibromo carbons using the sorted training set (STS) method were established in this study to significantly improve the accuracy in GIAO 13C NMR calculation of compounds 1-3. To discover the potential bioactive metabolites from P. hornemannii, the anti-inflammatory activities of all compounds were examined. Compounds 1 and 3-5 showed significant anti-inflammatory activity to inhibit the production of pro-inflammatory cytokines in the LPS-stimulated mature dendritic cells.

Genetic structure and relatedness of juvenile sicklefin lemon shark (Negaprion acutidens) at Dongsha Island.

Negaprion acutidens (sicklefin lemon shark) is distributed in the Indo-Pacific and in close association with coral reefs. Under the protection of the Dongsha Atoll National Park, a small but well-established juvenile population of N. acutidens inhabiting coastal areas of Dongsha Island was recently observed to display site fidelity by using acoustic telemetry. This study was designed to reveal the fine scale genetic structure and relatedness within and among 5 juvenile shark cohorts inhabiting 3 sampling sites at Dongsha Island. A total 188 juveniles were caught and sampled between 2016 and 2017, and genotyped with twelve loci. They were assigned to 5 year cohorts (2013-2017) based on the body length and date they were caught, also assigned to 3 sites based on where they were caught. Among five cohorts, the percentage of unrelated pairs within a cohort is more than 62% in average, suggesting a potential high mortality during their early life stage. The results of Fst and assignment testing showed that there was no significant genetic structure between sites and cohorts indicating that there was no fine scale genetic structure, even though the juveniles possessed strong site fidelity. A small effective population size (Ne) was detected (Ne = 86.7) which indicates the presence of a potentially isolated and vulnerable population at Dongsha. These results provide the genetic diversity as a baseline for future management and conservation of N. acutidens in the South China Sea.

Johnius sasakii, a new species of croaker (Perciformes: Sciaenidae) with a key to Johnius from East Malaysia, Borneo.

A new sciaenid fish, Johnius sasakii sp. nov. from the East Malaysian coastal waters of Sabah and Sarawak, Borneo is described herein. Johnius sasakii sp. nov. can be separated from the close congeners, Johnius heterolepis and Johnius carouna by having less gill rakers on the lower limb of the first gill arch (mode number 9 vs. 10 for J. heterolepis; 11 for J. carouna). It can be distinguished from J. heterolepis with the higher modal number of gill rakers on the upper limb of first gill arch (5 vs. 4), shorter anal-fin base length (9% vs. 10% of SL) and first anal-fin ray length (1012% vs. 1213% of SL) respectively. Furthermore, J. sasakii sp. nov. can be further differentiated from J. carouna by its shorter second anal-fin spine length 710% SL (usually 810%) vs. 1114% SL (usually 1112%) and slightly deeper body depth 2529% SL (usually 2628%) vs. 2328% SL (usually 2426%). Meanwhile, J. sasakii sp. nov. can be easily differentiated from J. macrorhynus by a present of molariform teeth on inner rows of jaws and stumpy gill rakers. Johnius (Johnius) sasakii sp. nov. can be clearly distinguished from species of the subgenus J. (Johnieops) by the lack of enlarged outer row teeth on upper jaw. A Kimura 2-parameter genetic distance comparison of 450 bp sequences of mitochondrial 16S rRNA and 488 bp nuclear gene S7 showed that J. sasakii sp. nov. are highly differentiated from J. heterolepis (16S: 10.4%, S7: 5.8%), J. carouna (16S:19.3%, S7:8.4%); and J. macrorhynus (16S: 16.7%, S7: 8.1%) respectively. The study highlights that the genetic approach from mtDNA and nDNA can contribute to the confirmation of taxonomic status of sympatric species in genus Johnius.

Dongsha Atoll is an important stepping-stone that promotes regional genetic connectivity in the South China Sea.

BACKGROUND: Understanding region-wide patterns of larval connectivity and gene flow is crucial for managing and conserving marine biodiversity. Dongsha Atoll National Park (DANP), located in the northern South China Sea (SCS), was established in 2007 to study and conserve this diverse and remote coral atoll. However, the role of Dongsha Atoll in connectivity throughout the SCS is seldom studied. In this study, we aim to evaluate the role of DANP in conserving regional marine biodiversity. METHODS: In total, 206 samples across nine marine species were collected and sequenced from Dongsha Atoll, and these data were combined with available sequence data from each of these nine species archived in the Genomic Observatories Metadatabase (GEOME). Together, these data provide the most extensive population genetic analysis of a single marine protected area. We evaluate metapopulation structure for each species by using a coalescent sampler, selecting among panmixia, stepping-stone, and island models of connectivity in a likelihood-based framework. We then completed a heuristic graph theoretical analysis based on maximum dispersal distance to get a sense of Dongsha's centrality within the SCS. RESULTS: Our dataset yielded 111 unique haplotypes across all taxa at DANP, 58% of which were not sampled elsewhere. Analysis of metapopulation structure showed that five out of nine species have strong regional connectivity across the SCS such that their gene pools are effectively panmictic (mean pelagic larval duration (PLD) = 78 days, sd = 60 days); while four species have stepping-stone metapopulation structure, indicating that larvae are exchanged primarily between nearby populations (mean PLD = 37 days, sd = 15 days). For all but one species, Dongsha was ranked within the top 15 out of 115 large reefs in the South China Sea for betweenness centrality. Thus, for most species, Dongsha Atoll provides an essential link for maintaining stepping-stone gene flow across the SCS. CONCLUSIONS: This multispecies study provides the most comprehensive examination of the role of Dongsha Atoll in marine connectivity in the South China Sea to date. Combining new and existing population genetic data for nine coral reef species in the region with a graph theoretical analysis, this study provides evidence that Dongsha Atoll is an important hub for sustaining connectivity for the majority of coral-reef species in the region.

Accessing Multiple Paternity in the Shortfin Mako Shark (Isurus oxyrinchus).

Multiple paternity has been demonstrated in a variety of sharks with different reproductive modes (i.e., viviparous, ovoviviparous, adelphophagy, oviparous), although the number of sires per litter varies considerably among species. To date, such analyses have focused mainly on coastal and nearshore shark species due to the difficulty in sampling oceanic sharks. In the present study, we observed multiple paternity in the oceanic shark Isurus oxyrinchus from seven polymorphic microsatellite loci and three litters collected from Nanfangao Fishing Port. Paternity tests showed that an average of 4.6 sires were assigned to each litter of I. oxyrinchus using COLONY software, and that the average number of sires dropped to 2.5 when using GERUD. These findings suggest that multiple paternity could be a common reproductive strategy used by the shortfin mako shark, and that this mating system should be integrated into a demographic model to make more accurate population projections and risk analyses in the future.

Genetic identification and hybridization in the seagrass genus Halophila (Hydrocharitaceae) in Sri Lankan waters.

Seagrasses, as marine angiosperms, play important roles in coastal ecosystems. With increasing anthropogenic impacts, they are facing dramatic declines on a global scale. Halophila is well-known as a complex taxonomic challenge mainly due to high morphological plasticity. By using only a morphological approach, the genus could be over-split or similar species could be erroneously lumped, thus masking its true biodiversity. In the present study, we incorporated genetic identification with morphological examination to reveal the identity of Halophila plants in southern and northwestern Sri Lankan waters. The nuclear ribosomal internal transcribed spacer (ITS) region and chloroplast ribulose-bisphosphate carboxylase gene (rbcL) were used to identify plants collected from the Gulf of Mannar, Puttalam Lagoon, and Matara, Sri Lanka. Based on genetic identification, H. major (Zoll.) Miquel is reported for the first time from Sri Lanka, which might have been misidentified as H. ovalis in previous literature based on morphology alone. We also observed a first hybridization case of Halophila cross between H. ovalis and H. major. Two potential cryptic species were found, herein designated Halophila sp. 1 (allied to H. minor) and Halophila sp. 2 (closely related to H. decipiens). In order to clarify taxonomic ambiguity caused by morphological plasticity and the low resolution of genetic markers, further comparative phylogenomic approaches might be needed to solve species boundary issues in this genus.

Integrating phylogeographic and ecological niche approaches to delimitating cryptic lineages in the blue-green damselfish (Chromis viridis).

Species delimitation is challenging in sibling species/cryptic lineages because of the absence of clear diagnostic traits. However, integration of different approaches such as phylogeography and ecological niche comparison offers one potential approach to tease apart recently diverged lineages. In this study, we estimate the ecological niche divergence among lineages in Chromis viridis in a broad-scale phylogeographic framework to test whether the combination of these two approaches can effectively distinguish recently diverged lineages. Results from Cytb and Rag2 analyses identified two cryptic lineages (C. viridis A and C. viridis B) that diverged ∼3 Myr ago. Estimates of ecological niche divergence with 11 environmental parameters across the broad geographic range of these lineages showed overlapping ecological niches and niche conservatism. However, regardless of the incongruence between genetic and ecological niche divergence, the substantial genetic divergence between the two clades of C. viridis in both mtDNA and nuclear loci strong suggest that they are cryptic taxa.

Development and characterization of nine polymorphic microsatellite markers for the deep-sea scleractinian coral Deltocyathus magnificus using paired-end Illumina shotgun sequencing.

Deltocyathus magnificus is an azooxanthellae deep-sea scleractinian coral species that is distributed in the Indo-Pacific region. In order to investigate its genetic structure and connectivity, polymorphic microsatellite markers were isolated and tested on 37 individuals collected from the South China Sea. Twenty loci were selected and primers synthesized for further PCR testing. Nine of 20 were amplified successfully with polymorphism markers. The number of alleles detected in each locus ranged from 2 to 13. The expected and observed heterozygosities of these loci were 0.053-0.819 and 0.054-0.946, respectively. These nine novel polymorphic loci were used to reveal the genetic connectivity pattern among depths and geographic locations in the South China Sea.

Genetic diversity and connectivity of the megamouth shark (Megachasma pelagios).

The megamouth shark (Megachasma pelagios) was described as a new species in 1983. Since then, only ca. 100 individuals have been observed or caught. Its horizontal migration, dispersal, and connectivity patterns are still unknown due to its rarity. Two genetic markers were used in this study to reveal its genetic diversity and connectivity pattern. This approach provides a proxy to indirectly measure gene flow between populations. Tissues from 27 megamouth sharks caught by drift nets off the Hualien coast (eastern Taiwan) were collected from 2013 to 2015. With two additional tissue samples from megamouths caught in Baja California, Mexico, and sequences obtained from GenBank, we were able to perform the first population genetic analyses of the megamouth shark. The mtDNA cox1 gene and a microsatellite (Loc 6) were sequenced and analyzed. Our results showed that there is no genetic structure in the megamouth shark, suggesting a possible panmictic population. Based on occurrence data, we also suggest that the Kuroshio region, including the Philippines, Taiwan, and Japan, may act as a passageway for megamouth sharks to reach their feeding grounds from April to August. Our results provide insights into the dispersal and connectivity of megamouth sharks. Future studies should focus on collecting more samples and conducting satellite tagging to better understand the global migration and connectivity pattern of the megamouth shark.

Role of IL-28B genetic variants in HCV-related liver disease severity in patients with different viral genotypes.

Reports of the role of host interleukin 28B (IL-28B) genetic variants in liver disease severity in patients with chronic hepatitis C (CHC) have obtained conflicting results. The impact of IL-28B in Asian patients with different viral genotypes remains elusive.We try to elucidate the effect of IL-28B genetic variants in a large Asian cohort with different viral genotypes.The association between the IL-28B rs8099917 genotype and liver fibrosis was investigated in 1288 patients with biopsy-proven CHC.Patients with hepatitis C virus genotype 1 (HCV-1) infection comprised 59.4% of the population. The remaining 40.6% (518 patients) did not have HCV-1 infection. Of the 1084 patients with the IL-28 genotype, 85.6% (928 patients) had the TT genotype. Univariate analysis revealed that, compared to patients without advanced liver fibrosis, patients with advanced liver fibrosis (Metavir fibrosis score 3-4) had an older age, a lower platelet count, a higher α-fetoprotein level, a higher alanine aminotransferase level, a higher incidence of diabetes, and a higher frequency of rs8099917 non-TT genotype carriage.Logistic regression analysis revealed that factors significantly associated with advanced liver fibrosis included age (odds ratio [OR]/95% confidence interval [CI]: 1.023/1.009-1.037, P = .001), diabetes (OR/CI: 1.736/1.187-2.539, P = .004), α-fetoprotein (OR/CI: 1.007/1.002-1.012, P = .009), platelet count (OR/CI: 0.991/0.988-0.993, P < .001), and carriage of the rs8099917 non-TT genotype (OR/CI: 0.585/0.400-0.856, P = .006). When patients were classified by viral genotype, factors that had significant independent associations with advanced liver fibrosis in patients with HCV-1 infection included diabetes (OR/CI: 2.379/1.452-3.896, P = .001), α-fetoprotein (OR/CI: 1.023/1.012-1.035, P < .001), platelet count (OR/CI: 0.99/0.987-0.994, P < .001), and carriage of the rs8099917 non-TT genotype (OR/CI: 0.529/0.328-0.854, P = .009). In patients who had advanced liver fibrosis but not HCV-1 infection, factors that had significant independent associations with advanced liver fibrosis included age (OR/CI: 1.039/1.016-1.063, P = .001) and platelet count (OR/CI: 0.99/0.986-0.995, P < .001); additionally, IL-28B genetic variants were not associated with liver disease severity.Unfavorable IL-28B genetic variants were associated with advanced liver disease. The genetic effect is limited to patients with HCV-1 infection.

Buccal venom gland associates with increased of diversification rate in the fang blenny fish Meiacanthus (Blenniidae; Teleostei).

At the macroevolutionary level, many mechanisms have been proposed to explain explosive species diversification. Among them morphological and/or physiological novelty is considered to have a great impact on the tempo and the mode of diversification. Meiacanthus is a genus of Blenniidae possessing a unique buccal venom gland at the base of an elongated canine tooth. This unusual trait has been hypothesized to aid escape from predation and thus potentially play an important role in their pattern of diversification. Here, we produce the first time-calibrated phylogeny of Blenniidae and we test the impact of two morphological novelties on their diversification, i.e. the presence of swim bladder and buccal venom gland, using various comparative methods. We found an increase in the tempo of lineage diversification at the root of the Meiacanthus clade, associated with the evolution of the buccal venom gland, but not the swim bladder. Neither morphological novelty was associated with the pattern of size disparification in blennies. Our results support the hypothesis that the buccal venom gland has contributed to the explosive diversification of Meiacanthus, but further analyses are needed to fully understand the factors sustaining this burst of speciation.

Spatial genetic structure and body size divergence in endangered Gymnogobius isaza in ancient Lake Biwa.

Gymnogobius isaza is a freshwater goby endemic to ancient Lake Biwa, the largest lake in Japan. The species is now listed as 'Critically Endangered' in the Red Data Book of Japan. Nevertheless, it remains subject to fishing without any specific management strategies. Previous studies using mitochondrial DNA markers showed that this fish species has two cryptic lineages. However, little is known about spatial genetic structure and ecological differences across the broad lakescape. In this study, we collected fish samples at nine locations along the lakeshore during the breeding season and tested for the presence of spatial heterogeneity in the lineage's composition while measuring body size as the most fundamental biological trait. The results showed that the major lineage dominated all the sampling locations whereas the minor lineage consisted of only 11% (16/143) of samples. Furthermore, although their spatial distributions overlapped (i.e. the two lineages may be well mixed), we found it possible that the minor lineage may have a potentially narrower distribution than the major lineage. In addition, we found that the two lineages differ in body size; specifically, the minor lineage is smaller in size. From the viewpoint of genetic diversity conservation and sustainable resource use, this fish should be managed as two genetic stocks and spatial and/or body size-based fishery management is desirable, with particular attention to the minor (smaller sized) lineage.

A new family of poecilostomatoid copepods (Strepidae fam. nov.) associated with the sun coral, Tubastraea coccinea Lesson, 1829 in Taiwan.

Both sexes of Strepus elongatus gen. et sp. nov. are described based on specimens obtained from the coral host, Tubastraea coccinea Lesson, 1829, collected from shallow water reefs in Taiwan. The new species belongs to a new family of the copepod order Poecilostomatoida and displays the following morphological characteristics: (1) poecilostome type of well developed piercing mandibles, (2) absence of an oral cone, (3) highly transformed body, (4) 4-segmented antennules, (5) 2-segmented maxilliped, (6) caudal rami with only two setae, and (7) complete loss of legs 1-5 in the female, but legs 1-2 being represented by a free segment bearing two terminal setae in the male. In addition, the phylogenetic analysis based on 18S ribosomal RNA sequence data showed that S. elongatus clustered with a Sabelliphilus-Anchimolgus clade (Sabelliphilidae + Xarifiidae + Rhynchomolgidae + Anchimolgidae), and that the average genetic distance between S. elongatus gen. et sp. nov. and representatives of the six most closely related families (0.064) was higher than the average distances among those families (0.028). Therefore, based on both morphological and molecular data, a new family Strepidae fam. nov. is proposed to accommodate its only known species, S. elongatus gen. et sp. nov.

DNA barcoding of shark meats identify species composition and CITES-listed species from the markets in Taiwan.

BACKGROUND: An increasing awareness of the vulnerability of sharks to exploitation by shark finning has contributed to a growing concern about an unsustainable shark fishery. Taiwan's fleet has the 4th largest shark catch in the world, accounting for almost 6% of the global figures. Revealing the diversity of sharks consumed by Taiwanese is important in designing conservation plans. However, fins make up less than 5% of the total body weight of a shark, and their bodies are sold as filets in the market, making it difficult or impossible to identify species using morphological traits. METHODS: In the present study, we adopted a DNA barcoding technique using a 391-bp fragment of the mitochondrial cytochrome oxidase I (COI) gene to examine the diversity of shark filets and fins collected from markets and restaurants island-wide in Taiwan. RESULTS: Amongst the 548 tissue samples collected and sequenced, 20 major clusters were apparent by phylogenetic analyses, each of them containing individuals belonging to the same species (most with more than 95% bootstrap values), corresponding to 20 species of sharks. Additionally, Alopias pelagicus, Carcharhinus falciformis, Isurus oxyrinchus, and Prionace glauca consisted of 80% of the samples we collected, indicating that these species might be heavily consumed in Taiwan. Approximately 5% of the tissue samples used in this study were identified as species listed in CITES Appendix II, including two species of Sphyrna, C. longimanus and Carcharodon carcharias. CONCLUSION: DNA barcoding provides an alternative method for understanding shark species composition when species-specific data is unavailable. Considering the global population decline, stock assessments of Appendix II species and highly consumed species are needed to accomplish the ultimate goal of shark conservation.

Cloning and characterization of microsatellite loci in a gorgonian coral, Junceella juncea (Anthozoa; Octocorallia; Ellisellidae) and its application in clonal genotyping.

We attempted to isolate microsatellites from a Symbiodinium-free gorgonian octocoral, Junceella juncea, using two methods, partial genomic library screening and enrichment. Among the 3856 clones screened by the partial library method, 10 possibly positive signals were found, and 3 of them could be used to design primers and amplified consistently. In contrast, only one locus isolated by the enrichment method gave reliable amplification and was useful. The results indicate that microsatellites are rare in Junceella juncea, as reported for other cnidarians. Overall, we obtained 4 polymorphic loci to test the feasibility in investigating clonal structure of J. juncea. A total of 40 multilocus genotypes were found among 152 colonies, and the number of genotypes (clones) identified at 7 reefs ranged from 2 to 16. The results of a nonmetric multidimensional scaling analysis indicated the recruitment of J. juncea populations mainly comes from self-retention. These novel microsatellite loci will provide a useful tool to study clonal structure and population genetics for J. juncea in the future.