Multispecies environmental DNA metabarcoding sheds light on annual coral spawning events.

Synchronous multispecific coral spawning generally occurs annually and forms an integral part of the coral life cycle. Apart from spawning times and species participation, however, much else remains unknown. Here, we applied environmental DNA (eDNA) metabarcoding to study two tropical reef sites of contrasting coral cover before, during and after coral spawning. Using coral-ITS2 and vertebrate-12S markers, we evaluated eDNA as an alternative monitoring tool by assessing its capabilities in detecting spawning species and tracking relative abundances of coral and fish eDNA. Over 3 years, elevated eDNA coral signals during the event (proportional read increase of up to five-fold) were observed, detecting a total of 38 coral and 133 fish species with all but one of the coral species visually observed to be spawning. This is also the first demonstration that eDNA metabarcoding can be used to infer the diurnal partitioning of night- and day-time spawning, spawning in coral species overlooked by visual surveys, and the associated changes in fish trophic structures as an indicator of spawning events. Our study paves the way for applied quantitative eDNA metabarcoding approaches to better study ephemeral and important biological events.

Resting Stage of Plankton Diversity from Singapore Coastal Water: Implications for Harmful Algae Blooms and Coastal Management.

Resting strategies of planktonic organisms are important for the ecological processes of coastal waters and their impacts should be taken into consideration in management of water bodies used by multiple industries. We combined different approaches to evaluate the importance of resting stages in Singapore coastal waters. We used molecular approaches to improve the knowledge on Singapore biodiversity, we sampled and extracted cysts from sediments to evaluate the density of resting stages in Johor Strait, and we compared systematically information on Singapore planktonic biodiversity to existing published information on resting stages from these reported organisms. This is the first study evaluating the importance of resting stages in Singapore waters. Above 120 species reported in Singapore are known to produce resting stages though no previous work has ever been done to evaluate the importance of these strategies in these waters. The results from the resting stage survey confirmed 0.66 to 5.34 cyst g-1 dry weight sediment were present in the Johor Strait suggesting that cysts may be flushed by tidal currents into and out of the strait regularly. This also suggest that the blooms occurring in Singapore are likely due to secondary growth of Harmful Algae Bloom species in the water rather than from direct germination of cysts from sediment. Finally, we discuss the importance of these resting eggs for three main national industries in Singapore (shipping, marine aquaculture and provision of drinking water through seawater desalination). We argue that this study will serve as a baseline for some of the future management of Singapore waters.

Low genetic diversity and predation threaten a rediscovered marine sponge.

Discovered in 1819 in the tropical waters off Singapore, the magnificent Neptune's cup sponge Cliona patera (Hardwicke, 1820) was harvested for museums and collectors until it was presumed extinct worldwide for over a century since 1907. Recently in 2011, seven living individuals were rediscovered in Singapore with six relocated to a marine protected area in an effort to better monitor and protect the population, as well as to enhance external fertilisation success. To determine genetic diversity within the population, we sequenced the complete mitochondrial genomes and nuclear ribosomal DNA of these six individuals and found extremely limited variability in their genes. The low genetic diversity of this rediscovered population is confirmed by comparisons with close relatives of C. patera and could compromise the population's ability to recover from environmental and anthropogenic pressures associated with the highly urbanised coastlines of Singapore. This lack of resilience is compounded by severe predation which has been shrinking sponge sizes by up to 5.6% every month. Recovery of this highly endangered population may require ex situ approaches and crossbreeding with other populations, which are also rare.

Assessing the Diversity and Biomedical Potential of Microbes Associated With the Neptune's Cup Sponge, Cliona patera.

Marine sponges are known to host a complex microbial consortium that is essential to the health and resilience of these benthic invertebrates. These sponge-associated microbes are also an important source of therapeutic agents. The Neptune's Cup sponge, Cliona patera, once believed to be extinct, was rediscovered off the southern coast of Singapore in 2011. The chance discovery of this sponge presented an opportunity to characterize the prokaryotic community of C. patera. Sponge tissue samples were collected from the inner cup, outer cup and stem of C. patera for 16S rRNA amplicon sequencing. C. patera hosted 5,222 distinct OTUs, spanning 26 bacterial phyla, and 74 bacterial classes. The bacterial phylum Proteobacteria, particularly classes Gammaproteobacteria and Alphaproteobacteria, dominated the sponge microbiome. Interestingly, the prokaryotic community structure differed significantly between the cup and stem of C. patera, suggesting that within C. patera there are distinct microenvironments. Moreover, the cup of C. patera had lower diversity and evenness as compared to the stem. Quorum sensing inhibitory (QSI) activities of selected sponge-associated marine bacteria were evaluated and their organic extracts profiled using the MS-based molecular networking platform. Of the 110 distinct marine bacterial strains isolated from sponge samples using culture-dependent methods, about 30% showed quorum sensing inhibitory activity. Preliminary identification of selected QSI active bacterial strains revealed that they belong mostly to classes Alphaproteobacteria and Bacilli. Annotation of the MS/MS molecular networkings of these QSI active organic extracts revealed diverse classes of natural products, including aromatic polyketides, siderophores, pyrrolidine derivatives, indole alkaloids, diketopiperazines, and pyrone derivatives. Moreover, potential novel compounds were detected in several strains as revealed by unique molecular families present in the molecular networks. Further research is required to determine the temporal stability of the microbiome of the host sponge, as well as mining of associated bacteria for novel QS inhibitors.

The different fates of two Asian horseshoe crab species with different dispersal abilities.

Impending anthropogenic climate change will severely impact coastal organisms at unprecedented speed. Knowledge on organisms' evolutionary responses to past sea-level fluctuations and estimation of their evolutionary potential is therefore indispensable in efforts to mitigate the effects of future climate change. We sampled tens of thousands of genomic markers of ~300 individuals in two of the four extant horseshoe crab species across the complex archipelagic Singapore Straits. Carcinoscorpius rotundicauda Latreille, a less mobile mangrove species, has finer population structure and lower genetic diversity compared with the dispersive deep-sea Tachypleus gigas Müller. Even though the source populations of both species during the last glacial maximum exhibited comparable effective population sizes, the less dispersive C. rotundicauda seems to lose genetic diversity much more quickly because of population fragmentation. Contra previous studies' results, we predict that the more commonly sighted C. rotundicauda faces a more uncertain conservation plight, with a continuing loss in evolutionary potential and higher vulnerability to future climate change. Our study provides important genomic baseline data for the redirection of conservation measures in the face of climate change and can be used as a blueprint for assessment and mitigation of the adverse effects of impending sea-level rise in other systems.

From marine park to future genomic observatory? Enhancing marine biodiversity assessments using a biocode approach.

Few tropical marine sites have been thoroughly characterised for their animal species, even though they constitute the largest proportion of multicellular diversity. A number of focused biodiversity sampling programmes have amassed immense collections to address this shortfall, but obstacles remain due to the lack of identification tools and large proportion of undescribed species globally. These problems can be partially addressed with DNA barcodes ("biocodes"), which have the potential to facilitate the estimation of species diversity and identify animals to named species via barcode databases. Here, we present the first results of what is intended to be a sustained, systematic study of the marine fauna of Singapore's first marine park, reporting more than 365 animal species, determined based on DNA barcodes and/or morphology represented by 931 specimens (367 zooplankton, 564 macrofauna including 36 fish). Due to the lack of morphological and molecular identification tools, only a small proportion could be identified to species solely based on either morphology (24.5%) or barcodes (24.6%). Estimation of species numbers for some taxa was difficult because of the lack of sufficiently clear barcoding gaps. The specimens were imaged and added to "Biodiversity of Singapore" (http://singapore.biodiversity.online), which now contains images for > 13,000 species occurring in the country.

Effects of eutrophication on the planktonic food web dynamics of marine coastal ecosystems: The case study of two tropical inlets.

We studied the plankton dynamics of two semi-enclosed marine coastal inlets of the north of Jurong Island separated by a causeway (SW Singapore; May 2012-April 2013). The west side of the causeway (west station) has residence times of ca. one year and is markedly eutrophic. The east side (east station) has residence times of one month and presents lower nutrient concentrations throughout the year. The higher nutrient concentrations at the west station did not translate into significantly higher concentrations of chlorophyll a, with the exception of some peaks at the end of the South West Monsoon. Microzooplankton were more abundant at the west station. The west station exhibited more variable abundances of copepods during the year than did the east station, which showed a more stable pattern and higher diversity. Despite the higher nutrient concentrations at the west station (never limiting phytoplankton growth), the instantaneous phytoplankton growth rates there were generally lower than at the east station. The phytoplankton communities at the west station were top-down controlled, largely by microzooplankton grazing, whereas those of the east station alternated between top-down and bottom-up control, with mesozooplankton being the major grazers. Overall, the trophic transfer efficiency from nutrients to mesozooplankton in the eutrophic west station was less efficient than in the east station, but this was mostly because a poor use of inorganic nutrients by phytoplankton rather than an inefficient trophic transfer of carbon. Some hypotheses explaining this result are discussed.

Differential Response of Coral Assemblages to Thermal Stress Underscores the Complexity in Predicting Bleaching Susceptibility.

Coral bleaching events have been predicted to occur more frequently in the coming decades with global warming. The susceptibility of corals to bleaching during thermal stress episodes is dependent on many factors and an understanding of these underlying drivers is crucial for conservation management. In 2013, a mild bleaching episode ensued in response to elevated sea temperature on the sediment-burdened reefs in Singapore. Surveys of seven sites highlighted variable bleaching susceptibility among coral genera-Pachyseris and Podabacia were the most impacted (31% of colonies of both genera bleached). The most susceptible genera such as Acropora and Pocillopora, which were expected to bleach, did not. Susceptibility varied between less than 6% and more than 11% of the corals bleached, at four and three sites respectively. Analysis of four of the most bleached genera revealed that a statistical model that included a combination of the factors (genus, colony size and site) provided a better explanation of the observed bleaching patterns than any single factor alone. This underscored the complexity in predicting the coral susceptibility to future thermal stress events and the importance of monitoring coral bleaching episodes to facilitate more effective management of coral reefs under climate change.