Assigning the unassigned: A signature-based classification of rDNA metabarcodes reveals new deep-sea diversity.

Environmental DNA metabarcoding reveals a vast genetic diversity of marine eukaryotes. Yet, most of the metabarcoding data remain unassigned due to the paucity of reference databases. This is particularly true for the deep-sea meiofauna and eukaryotic microbiota, whose hidden diversity is largely unexplored. Here, we tackle this issue by using unique DNA signatures to classify unknown metabarcodes assigned to deep-sea foraminifera. We analyzed metabarcoding data obtained from 311 deep-sea sediment samples collected in the Clarion-Clipperton Fracture Zone, an area of potential polymetallic nodule exploitation in the Eastern Pacific Ocean. Using the signatures designed in the 37F hypervariable region of the 18S rRNA gene, we were able to classify 802 unassigned metabarcodes into 61 novel lineages, which have been placed in 27 phylogenetic clades. The comparison of new lineages with other foraminiferal datasets shows that most novel lineages are widely distributed in the deep sea. Five lineages are also present in the shallow-water datasets; however, phylogenetic analysis of these lineages separates deep-sea and shallow-water metabarcodes except in one case. While the signature-based classification does not solve the problem of gaps in reference databases, this taxonomy-free approach provides insight into the distribution and ecology of deep-sea species represented by unassigned metabarcodes, which could be useful in future applications of metabarcoding for environmental monitoring.

Taxonomy and Molecular Phylogeny of the Sea Anemone Macrodactyla (Haddon, 1898) (Cnidaria, Actiniaria), with a Description of a New Species from Singapore.

Sea anemones (Cnidaria, Actiniaria) are a successful group of marine invertebrates found in a diverse range of environments globally. In spite of their ubiquity, identities for many sea anemones remain unverified, especially those from the Indo-West Pacific region. Here, we clarify the taxonomy of the poorly known Macrodactyla aspera, a shallow-water species first described from the Torres Straits in northern Australia. We re-describe M. aspera based on new morphological and molecular data gathered from the type specimen, other museum vouchers, and from fresh material collected from Singapore. We tested the monophyly of Macrodactyla using three mitochondrial (12S, 16S and cox3) and one nuclear (28S) marker based on three congeners, recovering this genus to be polyphyletic. As a consequence, we transferred M. doreensis to the genus Heteractis, and describe a new species, Macrodactyla fautinae sp. nov. While both M. aspera and M. fautinae sp. nov. share the same arrangement and number of complete mesenteries, a similar distribution of cnidae, and are not symbiotically associated with any other biota, M. fautinae sp. nov. has perforated, lobe-like verrucae on its column, and lacks nematocyst batteries on its tentacles, unlike M. aspera. These two species also occur in similar habitats in Singapore. Finally, because M. aspera strongly resembles Dofleinia armata, the latter species flagged as a danger to public health due to its ability to inflict painful stings, we tested the relationship between these species and found them not to be closely related. However, tentacles of M. aspera, like D. armata, are densely covered with nematocyst batteries and harbour large nematocysts; we infer that M. aspera may also be capable of delivering stings that endanger public health. This study builds upon a growing number of studies that aim to ascertain identities and systematics of sea anemones historically reported from the Indo-West Pacific. Our findings will facilitate accurate species identification, which is crucial for advancing research, formulating conservation measures, and protecting public health.

Interactions between coral propagules in aquarium and field conditions.

The effects of intraspecific and interspecific interactions between three species of scleractinian coral micro-colonies, namely Lithophyllon undulatum, Turbinaria mesenterina and Platygyra sinensis were evaluated for their survivorship, tissue loss and growth in both field (in-situ) and aquarium (ex-situ) conditions over 12 weeks. Regardless of environmental conditions and interactions, L. undulatum survived better (91.7 ± 6.2%) than T. mesenterina (75.0 ± 25.0%) and P. sinensis (60.4 ± 39.5%). Similarly, L. undulatum registered the lowest tissue loss (0.5 ± 0.7%) as compared to T. mesenterina (14.3 ± 19.4%) and P. sinensis (22.0 ± 30.0%). However, P. sinensis gained more weight (3.2 ± 5.2 g) than either T. mesenterina (2.7 ± 2.4 g) or L. undulatum (0.8 ± 1.1 g). In both environments, all three species in intraspecific interaction generally had higher survivorship, lower tissue loss and better growth than those in interspecific interaction except the latter in in-situ conditions had a twofold increase in growth (5.8 ± 3.7 g) than the former in-situ conditions (2.8 ± 3.7 g). Hence, all three species are potentially suitable for transplantation and mariculture except perhaps for P. sinensis which performed poorly in ex-situ conditions. Corals can be transplanted either with different colonies of the same species or together with other coral taxa. This study demonstrated that L. undulatum should be transplanted between T. mesenterina and P. sinensis for optimal growth and survival.

Removal of larvae of two marine invasive bivalves, Mytilopsis sallei (Récluz, 1849) and Mytella strigata (Hanley, 1843), by water treatment processes.

The uptake and discharge of bivalve larvae through ballast water operations is a highly viable mechanism for transfer of shellfish. In this paper, we investigate the effects of common water treatment processes on the planktotrophic larvae of the two invasive species of shellfish, Mytilopsis sallei and Mytella strigata. The study found that common water treatment processes used in many ballast water treatment systems were effective for the removal of bivalve larvae, although later stages of larval forms required more effort to remove.

Sea anemones (Cnidaria, Actiniaria) of Singapore: redescription and taxonomy of Phymanthuspinnulatus Martens in Klunzinger, 1877.

Despite the ubiquity of sea anemones (Cnidaria: Actiniaria) in tropical ecosystems, our understanding of their biodiversity and taxonomy is limited. Here we re-establish the identity of an intertidal zooxanthellate species, Phymanthuspinnulatus Martens in Klunzinger, 1877. Originally described from a single preserved specimen in the Berlin Museum by CB Klunzinger, his brief footnote lacked crucial details to positively identify the species. Our redescription is based on more than 50 living individuals of P.pinnulatus collected from its type locality, Singapore. These were examined and compared with type materials of the species and its congeners. Specimens of P.pinnulatus differ from syntypes of species described as Phymanthuslevis Kwietniewski, 1898 from Indonesia, as well as Phymanthussansibaricus Carlgren, 1900 and Phymanthusstrandesi Carlgren, 1900, both described from East Africa. Phymanthuspinnulatus was encountered on the lower intertidal, among coral rubble and between rocky crevices. It is vibrantly coloured and has 96 marginal tentacles with branching outgrowths along each, resulting in a 'frilly' appearance. The anemone has a flat expanded oral disc, with discal tentacles that are inconspicuous and reduced, unlike syntypes of its congeners. Details of its live appearance, musculature, and cnidom are also provided for the first time. Overall, types of cnidae and capsule sizes differ from other known species of Phymanthus documented elsewhere. It is inferred that P.pinnulatus has a wide distribution that extends eastwards from Singapore, as far as Ambon and the Torres Straits. Some individuals reported as Phymanthusmuscosus Haddon and Shackleton, 1893 and Phymanthusbuitendijki Pax, 1924 are probably P.pinnulatus. This morphological analysis provides new insights into the characters used to delimit P.pinnulatus, clarifies its geographical distribution, and contributes to an ongoing revision of the genus Phymanthus.

Integrated Genomic and Metabolomic Approach to the Discovery of Potential Anti-Quorum Sensing Natural Products from Microbes Associated with Marine Samples from Singapore.

With 70% of the Earth's surface covered in water, the marine ecosystem offers immense opportunities for drug discovery and development. Due to the decreasing rate of novel natural product discovery from terrestrial sources in recent years, many researchers are beginning to look seaward for breakthroughs in new therapeutic agents. As part of an ongoing marine drug discovery programme in Singapore, an integrated approach of combining metabolomic and genomic techniques were initiated for uncovering novel anti-quorum sensing molecules from bacteria associated with subtidal samples collected in the Singapore Strait. Based on the culture-dependent method, a total of 102 marine bacteria strains were isolated and the identities of selected strains were established based on their 16S rRNA gene sequences. About 5% of the marine bacterial organic extracts showed quorum sensing inhibitory (QSI) activity in a dose-dependent manner based on the Pseudomonas aeruginosa QS reporter system. In addition, the extracts were subjected to mass spectrometry-based molecular networking and the genome of selected strains were analysed for known as well as new biosynthetic gene clusters. This study revealed that using integrated techniques, coupled with biological assays, can provide an effective and rapid prioritization of marine bacterial strains for downstream large-scale culturing for the purpose of isolation and structural elucidation of novel bioactive compounds.

Managing the risk of non-indigenous marine species transfer in Singapore using a study of vessel movement.

Shipping is recognized as a major vector for the global transfer of non-indigenous marine species (NIMS). As a major transshipment port, Singapore can minimize the risk of NIMS transfer by implementing pragmatic management strategies, such as using vessel movement information to assess the risk of NIMS transfer. Findings from vessel movement information in a major port terminal in Singapore showed that vessel residence time is short, with >92% of vessels spending seven days or less. There was little variation in vessel residence time to vessel arrival numbers, while the top three last ports of call were found to be from regional ports. Using two key features obtained from vessel movement records, 1) vessel residence time and 2) biogeographic origin of the vessels' last port of call, a simple risk assessment matrix was constructed and applied to assess the level of risk of NIMS transfer by transiting vessels.

Limpet feeding rate and the consistency of physiological response to temperature.

Thermal reaction norms are fundamental relationships for geographic comparisons of organism response to temperature. They are shaped by an organism's environmental history and provide insights into both the global patterns of thermal sensitivity and the physiological mechanisms underlying temperature response. In this study we conducted the first measure of the thermal reaction norm for feeding, comparing the radula rasping rate of two tropical and one polar limpet species. The consistency of thermal response was tested through comparisons with limpet duration tenacity. Feeding and duration tenacity of limpets are ecologically important muscular mechanisms that rely on very different aspects of muscle physiology, repeated concentric (shortening) and isometric (fixed length) contraction of muscles, respectively. In these limpets the thermal reaction norms of feeding limpets were best described by a single break point at a maximum temperature with linear declines at higher (Siphonaria atra) or lower temperatures (Nacella concinna and Cellana radiata) rather than a bell-shaped curve. The thermal reaction norms for duration tenacity were similar in the two tropical limpets. However, the rasping rate in Antarctic N. concinna increased linearly with temperature up to a maximum at 12.3 °C (maximal range 8.5-12.3 °C) when feeding stopped. In contrast, duration tenacity in N. concinna was maximal at 1.0 °C (-0.6 to 3.8 °C) and linearly decreased with increasing temperature. The thermal reaction norms of muscular activity were, therefore, inconsistent within and between species, indicating that different mechanisms likely underlie different aspects of species sensitivities to temperature.

Low global sensitivity of metabolic rate to temperature in calcified marine invertebrates.

Metabolic rate is a key component of energy budgets that scales with body size and varies with large-scale environmental geographical patterns. Here we conduct an analysis of standard metabolic rates (SMR) of marine ectotherms across a 70° latitudinal gradient in both hemispheres that spanned collection temperatures of 0-30 °C. To account for latitudinal differences in the size and skeletal composition between species, SMR was mass normalized to that of a standard-sized (223 mg) ash-free dry mass individual. SMR was measured for 17 species of calcified invertebrates (bivalves, gastropods, urchins and brachiopods), using a single consistent methodology, including 11 species whose SMR was described for the first time. SMR of 15 out of 17 species had a mass-scaling exponent between 2/3 and 1, with no greater support for a 3/4 rather than a 2/3 scaling exponent. After accounting for taxonomy and variability in parameter estimates among species using variance-weighted linear mixed effects modelling, temperature sensitivity of SMR had an activation energy (Ea) of 0.16 for both Northern and Southern Hemisphere species which was lower than predicted under the metabolic theory of ecology (Ea 0.2-1.2 eV). Northern Hemisphere species, however, had a higher SMR at each habitat temperature, but a lower mass-scaling exponent relative to SMR. Evolutionary trade-offs that may be driving differences in metabolic rate (such as metabolic cold adaptation of Northern Hemisphere species) will have important impacts on species abilities to respond to changing environments.

Integrative and comparative analysis of coiled-coil based marine snail egg cases - a model for biomimetic elastomers.

Integrative and comparative analyses of biomaterials systems offer the potential to reveal conserved elements that are essential for mechanical function. The approach also affords the opportunity to identify variation in designs at multiple length scales, enabling the delineation of a range of parameters for creating precisely tuned biomimetic materials. We investigated the molecular design and structural hierarchy of elastomeric egg capsules from the marine snail Pugilina cochlidium (family Melongenidae) and compared these data with all available published studies in order to infer the structure-property relationships of the egg case from the molecular to the macroscopic scale. While mechanical similarities had previously been observed for two other marine melongenid snails, Busycotypus canaliculatus and Busycon carica, B. canaliculatus was the only species for which detailed molecular and nanostructural data were available. Egg capsules from P. cochlidium were found to exhibit mechanical properties and shock absorbing potential that was similar to B. canaliculatus. The two species also displayed similarity in hierarchical fibril bundling and a sub-micron staggering of 100-105 nm within filaments, as shown by atomic force microscopy and small angle X-ray diffraction. In situ Raman micro spectroscopy indicated that P. cochlidium egg cases undergo a stress-induced coiled-coil to extended ß-strand structural transformation that is very similar to that of B. canaliculatus. These observations supported the view that these structural and hierarchical elements are essential for egg case function. Comparative analysis of the primary amino acid sequences and structural predictions for all known egg case proteins suggested that while the proteins all contain sequences prone to adopt α-helical structures, the predicted location of coiled-coil domains and stutter perturbations varied within and between species. Despite these differences, mixtures of denatured native egg case proteins readily re-folded in citrate-phosphate assembly buffer into α-helix rich, coiled-coil based oligomers, as determined by attenuated total reflection Fourier transform infrared spectroscopy, circular dichroism and MALDI-TOF. It is concluded that both conserved and divergent designs in marine snail egg cases offer inspiration for the engineering of biomimetic elastomeric materials with a unique capability for mechanical energy absorption.

Thermal reaction norms and the scale of temperature variation: latitudinal vulnerability of intertidal nacellid limpets to climate change.

The thermal reaction norms of 4 closely related intertidal Nacellid limpets, Antarctic (Nacella concinna), New Zealand (Cellana ornata), Australia (C. tramoserica) and Singapore (C. radiata), were compared across environments with different temperature magnitude, variability and predictability, to test their relative vulnerability to different scales of climate warming. Lethal limits were measured alongside a newly developed metric of "duration tenacity", which was tested at different temperatures to calculate the thermal reaction norm of limpet adductor muscle fatigue. Except in C. tramoserica which had a wide optimum range with two break points, duration tenacity did not follow a typical aerobic capacity curve but was best described by a single break point at an optimum temperature. Thermal reaction norms were shifted to warmer temperatures in warmer environments; the optimum temperature for tenacity (T(opt)) increased from 1.0°C (N. concinna) to 14.3°C (C. ornata) to 18.0°C (an average for the optimum range of C. tramoserica) to 27.6°C (C. radiata). The temperature limits for duration tenacity of the 4 species were most consistently correlated with both maximum sea surface temperature and summer maximum in situ habitat logger temperature. Tropical C. radiata, which lives in the least variable and most predictable environment, generally had the lowest warming tolerance and thermal safety margin (WT and TSM; respectively the thermal buffer of CT(max) and T(opt) over habitat temperature). However, the two temperate species, C. ornata and C. tramoserica, which live in a variable and seasonally unpredictable microhabitat, had the lowest TSM relative to in situ logger temperature. N. concinna which lives in the most variable, but seasonally predictable microhabitat, generally had the highest TSMs. Intertidal animals live at the highly variable interface between terrestrial and marine biomes and even small changes in the magnitude and predictability of their environment could markedly influence their future distributions.

Marine invertebrate skeleton size varies with latitude, temperature and carbonate saturation: implications for global change and ocean acidification.

There is great concern over the future effects of ocean acidification on marine organisms, especially for skeletal calcification, yet little is known of natural variation in skeleton size and composition across the globe, and this is a prerequisite for identifying factors currently controlling skeleton mass and thickness. Here, taxonomically controlled latitudinal variations in shell morphology and composition were investigated in bivalve and gastropod molluscs, brachiopods, and echinoids. Total inorganic content, a proxy for skeletal CaCO3 , decreased with latitude, decreasing seawater temperature, and decreasing seawater carbonate saturation state (for CaCO3 as calcite (Ωcal )) in all taxa. Shell mass decreased with latitude in molluscs and shell inorganic content decreased with latitude in buccinid gastropods. Shell thickness decreased with latitude in buccinid gastropods (excepting the Australian temperate buccinid) and echinoids, but not brachiopods and laternulid clams. In the latter, the polar species had the thickest shell. There was no latitudinal trend in shell thickness within brachiopods. The variation in trends in shell thickness by taxon suggests that in some circumstances ecological factors may override latitudinal trends. Latitudinal gradients may produce effects similar to those of future CO2 -driven ocean acidification on CaCO3 saturation state. Responses to latitudinal trends in temperature and saturation state may therefore be useful in informing predictions of organism responses to ocean acidification over long-term adaptive timescales.

Upper temperature limits of tropical marine ectotherms: global warming implications.

Animal physiology, ecology and evolution are affected by temperature and it is expected that community structure will be strongly influenced by global warming. This is particularly relevant in the tropics, where organisms are already living close to their upper temperature limits and hence are highly vulnerable to rising temperature. Here we present data on upper temperature limits of 34 tropical marine ectotherm species from seven phyla living in intertidal and subtidal habitats. Short term thermal tolerances and vertical distributions were correlated, i.e., upper shore animals have higher thermal tolerance than lower shore and subtidal animals; however, animals, despite their respective tidal height, were susceptible to the same temperature in the long term. When temperatures were raised by 1°C hour(-1), the upper lethal temperature range of intertidal ectotherms was 41-52°C, but this range was narrower and reduced to 37-41°C in subtidal animals. The rate of temperature change, however, affected intertidal and subtidal animals differently. In chronic heating experiments when temperature was raised weekly or monthly instead of every hour, upper temperature limits of subtidal species decreased from 40°C to 35.4°C, while the decrease was more than 10°C in high shore organisms. Hence in the long term, activity and survival of tropical marine organisms could be compromised just 2-3°C above present seawater temperatures. Differences between animals from environments that experience different levels of temperature variability suggest that the physiological mechanisms underlying thermal sensitivity may vary at different rates of warming.