Genome of the sea anemone Exaiptasia pallida and transcriptome profiles during tentacle regeneration.

Cnidarians including sea anemones, corals, hydra, and jellyfishes are a group of animals well known for their regeneration capacity. However, how non-coding RNAs such as microRNAs (also known as miRNAs) contribute to cnidarian tissue regeneration is poorly understood. Here, we sequenced and assembled the genome of the sea anemone Exaiptasia pallida collected in Hong Kong waters. The assembled genome size of E. pallida is 229.21 Mb with a scaffold N50 of 10.58 Mb and BUSCO completeness of 91.1%, representing a significantly improved genome assembly of this species. The organization of ANTP-class homeobox genes in this anthozoan further supported the previous findings in jellyfishes, where most of these genes are mainly located on three scaffolds. Tentacles of E. pallida were excised, and both mRNA and miRNA were sequenced at 9 time points (0 h, 6 h, 12 h, 18 h, 1 day, 2, 3, 6, and 8 days) from regenerating tentacles. In addition to the Wnt signaling pathway and homeobox genes that are shown to be likely involved in tissue regeneration as in other cnidarians, we have shown that GLWamide neuropeptides, and for the first time sesquiterpenoid pathway genes could potentially be involved in the late phase of cnidarian tissue regeneration. The established sea anemone model will be useful for further investigation of biology and evolution in, and the effect of climate change on this important group of animals.

Coastal Transient Niches Shape the Microdiversity Pattern of a Bacterioplankton Population with Reduced Genomes.

Globally dominant marine bacterioplankton lineages are often limited in metabolic versatility, owing to their extensive genome reductions, and thus cannot take advantage of transient nutrient patches. It is therefore perplexing how the nutrient-poor bulk seawater sustains the pelagic streamlined lineages, each containing numerous populations. Here, we sequenced the genomes of 33 isolates of the recently discovered CHUG lineage (~2.6 Mbp), which have some of the smallest genomes in the globally abundant Roseobacter group (commonly over 4 Mbp). These genome-reduced bacteria were isolated from a transient habitat: seawater surrounding the brown alga, Sargassum hemiphyllum. Population genomic analyses showed that: (i) these isolates, despite sharing identical 16S rRNA genes, were differentiated into several genetically isolated populations through successive speciation events; (ii) only the first speciation event led to the genetic separation of both core and accessory genomes; and (iii) populations resulting from this event are differentiated at many loci involved in carbon utilization and oxygen respiration, corroborated by BiOLOG phenotype microarray assays and oxygen uptake kinetics experiments, respectively. These differentiated traits match well with the dynamic nature of the macroalgal seawater, in which the quantity and quality of carbon sources and the concentration of oxygen likely vary spatially and temporally, though other habitats, like fresh organic aggregates, cannot be ruled out. Our study implies that transient habitats in the overall nutrient-poor ocean can shape the microdiversity and population structure of genome-reduced bacterioplankton lineages. IMPORTANCE Prokaryotic species, defined with operational thresholds, such as 95% of the whole-genome average nucleotide identity (ANI) or 98.7% similarity of the 16S rRNA gene sequences, commonly contain extensive fine-grained diversity in both the core genome and the accessory genome. However, the ways in which this genomic microdiversity and its associated phenotypic microdiversity are organized and structured is poorly understood, which disconnects microbial diversity and ecosystem functioning. Population genomic approaches that allow this question to be addressed are commonly applied to cultured species because linkages between different loci are necessary but are missing from metagenome-assembled genomes. In the past, these approaches were only applied to easily cultivable bacteria and archaea, which, nevertheless, are often not representative of natural communities. Here, we focus on the recently discovered cluster, CHUG, which are representative in marine bacterioplankton communities and possess some of the smallest genomes in the globally dominant marine Roseobacter group. Despite being over 95% ANI and identical in the 16S rRNA gene, the 33 CHUG genomes we analyzed have undergone multiple speciation events, with the first split event predominantly structuring the genomic diversity. The observed pattern of genomic microdiversity correlates with CHUG members' differential utilization of carbon sources and differential ability to explore low-oxygen niches. The available data are consistent with the idea that brown algae may be home to CHUG, though other habitats, such as fresh organic aggregates, are also possible.

Population differentiation of Rhodobacteraceae along with coral compartments.

Coral mucus, tissue, and skeleton harbor compositionally different microbiota, but how these coral compartments shape the microbial evolution remains unexplored. Here, we sampled bacteria inhabiting a prevalent coral species Platygyra acuta and sequenced genomes of 234 isolates comprising two populations in Rhodobacteraceae, an alphaproteobacterial lineage representing a significant but variable proportion (5-50%) of the coral microbiota. The Ruegeria population (20 genomes) contains three clades represented by eight, six, and six isolates predominantly sampled from the skeleton (outgroup), mucus (clade-M), and skeleton (clade-S), respectively. The clade-M possesses functions involved in the utilization of coral osmolytes abundant in the mucus (e.g., methylamines, DMSP, taurine, and L-proline), whereas the clade-S uniquely harbors traits that may promote adaptation to the low-energy and diurnally anoxic skeleton (e.g., sulfur oxidation and swimming motility). These between-clade genetic differences were largely supported by physiological assays. Expanded analyses by including genomes of 24 related isolates (including seven new genomes) from other marine environments suggest that clade-M and clade-S may have diversified in non-coral habitats, but they also consolidated a key role of distinct coral compartments in diversifying many of the above-mentioned traits. The unassigned Rhodobacteraceae population (214 genomes) varies only at a few dozen nucleotide sites across the whole genomes, but the number of between-compartment migration events predicted by the Slatkin-Maddison test supported that dispersal limitation between coral compartments is another key mechanism diversifying microbial populations. Collectively, our results suggest that different coral compartments represent ecologically distinct and microgeographically separate habitats that drive the evolution of the coral microbiota.

Hong Kong's subtropical scleractinian coral communities: Baseline, environmental drivers and management implications.

We surveyed 41 sites to provide an updated baseline of Hong Kong coral communities. Five community types were identified, among them the most common one inhabited oceanic waters and dominated by both massive and upward-plating corals. The 41 sites had 2.1-⁠79% coral cover; among them 21 in the eastern waters had >40% coral cover. Corals in several sites showed signs of external bioerosion or bleaching-induced damage. Sites in the southern waters had low coral cover. Both coral cover and generic richness correlated negatively with several water quality parameters including total inorganic nitrogen concentration and turbidity, indicating the development of Hong Kong's coral communities is constrained by water quality parameters. Management actions are proposed to reduce bioerosion, and to monitor sites affected by bleaching.

Fish and crustacean biodiversity in an outer maritime estuary of the Pearl River Delta revealed by environmental DNA.

Understanding the faunal community structure in the estuary would be crucial in assessing the health of the ecosystem. The poor visibility in the estuarine area due to the outflow from the Pearl River hinders the conventional visual census in assessing the megafaunal biodiversity. In this study, the fish and crustacean biodiversity of Hong Kong's western waters, i.e. the outer maritime estuary of the PRD, were studied through the metabarcoding of environmental DNA (eDNA). eDNA from the seawater and sediment samples was extracted from five sites in the region. After testing the performance of two genetic markers, amplicons of the cytochrome oxidase I, amplified by polymerase chain reaction, were subjected to Illumina high-throughput sequencing (MiSeq) analysis. A total of 22 fish species from 17 families and 34 crustacean species from 27 families were identified by blasting the sequences against the NCBI GenBank database, demonstrating segregation between samples from different sites. This study provides insight on the detail distribution of fish assembly in PRD, when compared with a previous eDNA study in the inner brackish PRD.

Evolutionary biogeography of the reef-building coral genus Galaxea across the Indo-Pacific ocean.

Stony corals (Scleractinia) form the basis for some of the most diverse ecosytems on Earth, but we have much to learn about their evolutionary history and systematic relationships. In order to improve our understanding of species in corals we here investigated phylogenetic relationships between morphologically defined species and genetic lineages in the genus Galaxea (Euphyllidae) using a combined phylogenomic and phylogeographic approach. Previous studies revealed the nominal species G. fascicularis included three genetically well-differentiated lineages (L, S & L+) in the western Pacific, but their distribution and relationship to other species in the genus was unknown. Based on genomic (RAD-seq) and mitochondrial sequence data (non-coding region between cytb and ND2) we investigated whether the morphological taxa represent genetically coherent entities and what is the phylogenetic relationship and spatial distribution of the three lineages of G. fascicularis throughout the observed species range. Using the RAD-seq data, we find that the genus Galaxea is monophyletic and contains three distinct clades: an Indo-Pacific, a Pacific, and a small clade restricted to the Chagos Archipelago. The three lineages of G. fascicularis were associated with different RAD-seq clades, with the 'L' lineage showing some morphological distinction from the other two lineages (larger more asymmetrical polyps). In addition to these, three more genetic lineages in G. fascicularis may be distinguished - a Chagossian, an Ogasawaran, and one from the Indian-Red Sea. Among nominal taxa for which we have multiple samples, G. horrescens was the only monophyletic species. The mitochondrial non-coding region is highly conserved apart of the length polymorphism used to define L, S & L+ lineages and lacks the power to distinguish morphological and genetic groups resolved with genomic RAD-sequencing. The polyphyletic nature of most species warrants a careful examination of the accepted taxonomy of this group with voucher collections and their comparison to type specimens to resolve species boundaries. Further insight to the speciation process in corals will require international cooperation for the sharing of specimens to facilitate scientific discovery.

Genome Size, rDNA Copy, and qPCR Assays for Symbiodiniaceae.

Symbiodiniaceae community structure in corals is crucial for understanding the plasticity of different holobionts under environmental stress. While this relies on molecular analyses, accuracy of molecular quantification, as influenced by DNA extraction efficiency and rDNA copy number variations in particular, has rarely been systematically investigated. Here, we report the development of a set of genus-specific qPCR assays. First, a protocol for efficient DNA isolation and accurate measurements of genome size and rDNA copy number was established. Second, seven newly designed genus-specific ITS2 primer sets were validated using computational and empirical analyses and qPCR assays were developed. We find that while the genome size ranges between 1.75 ± 0.21 and 4.5 ± 0.96 Gbp, rDNA copy number shows over 10-fold variation among Symbiodiniaceae species. Our protocol produced standard curves with high efficiencies (89.8-99.3%; R 2 ≥ 0.999) and tight Cq values over different PCR conditions, illustrating high specificity and sensitivity of the qPCR assays. Tested on mock communities of mixed culture species, our qPCR results agreed well with microscopic counts and facilitated calibration of metabarcoding data. To test the applicability of our protocol for field samples, we analyzed three different Hong Kong coral samples. Six Symbiodiniaceae genera were detected in Acropora valida, Oulastrea crispata, and Platygyra acuta, with Breviolum, Effrenium, Fugacium, and Gerakladium sp. being reported for the first time. Our results suggest that aggressively disrupting cells to ensure thorough cell lysis, estimating cell loss and DNA loss, and validating qPCR assays are critical for success. The number of species examined here is limited, but the primers are potentially applicable to most species in respective genera, and the protocol and the approach to develop it provide a base and template toward a standardized procedure for quantitatively characterizing Symbiodiniaceae communities in corals.

Metabolic performance and thermal and salinity tolerance of the coral Platygyra carnosa in Hong Kong waters.

Stress-tolerant coral species, such as Platygyra spp., are considered to be well adapted to survive in marginal reefs, but their physiological response to short term exposure to abnormally high temperature and lowered salinity remains poorly understood. Using non-invasive techniques to quantitatively assess the health of Platygyra carnosa (e.g. respiration, photosynthesis, biocalcification and whiteness), we identified the plasticity of its energetics and physiological limits. Although these indicators suggest that it can survive to increasing temperature (25-32 °C), its overall energetics were seriously diminished at temperatures >30 °C. In contrast, it was well adapted to hyposaline waters (31-21 psu) but with reduced biocalcification, indicating short term adaptation for expected future changes in salinity driven by increased amounts and intensities of precipitation. Our findings provide useful insights to the effect of these climate drivers on P. carnosa metabolism and thus better forecast changes in their health status under future climate change scenarios.

Localized bleaching and quick recovery in Hong Kong's coral communities.

During the summer of 2017 we visited 33 sites with substantial coral cover across Hong Kong waters. At six sites where coral bleaching was observed, video-transect surveys were conducted, which revealed 18.7% to 56.1% bleached colonies and 5.9% to 57.6% bleached coral covered area per site. Mainly affected were three plate-like and three massive coral species. Water quality parameters were analyzed, which indicated that hyposalinity might have triggered the bleaching event. Tagging and follow-up field observation revealed a pattern of recovery depending on coral growth form, with high recovery rates (>93%) at five sites dominated by massive and submassive corals, but moderate (70%) at a site dominated by plate-like corals. Our study shows that the corals of Hong Kong exhibit differential susceptibility to bleaching and ability to recover, therefore it is imperative to establish a long-term monitoring programme to detect the changes in community structure over time.

Contrasting patterns of genetic structure and phylogeography in the marine agarophytes Gelidiophycus divaricatus and G. freshwateri (Gelidiales, Rhodophyta) from East Asia.

The evolutionary and population demographic history of marine red algae in East Asia is poorly understood. Here, we reconstructed the phylogeographies of two upper intertidal species endemic to East Asia, Gelidiophycus divaricatus and G. freshwateri. Phylogenetic and phylogeographic inferences of 393 mitochondrial cox1, 128 plastid rbcL, and 342 nuclear ITS2 sequences were complemented with ecological niche models. Gelidiophycus divaricatus, a southern species adapted to warm water, is characterized by a high genetic diversity and a strong geographical population structure, characteristic of stable population sizes and sudden reduction to recent expansion. In contrast, G. freshwateri, a northern species adapted to cold temperate conditions, is genetically relatively homogeneous with a shallow population structure resulting from steady population growth and recent equilibrium. The overlap zone of the two species roughly matches summer and winter isotherms, indicating that surface seawater temperature is a key feature influencing species range. Unidirectional genetic introgression was detected at two sites on Jeju Island where G. divaricatus was rare while G. freshwateri was common, suggesting the occurrence of asymmetric natural hybrids, a rarely reported event for rhodophytes. Our results illustrate that Quaternary climate oscillations have left strong imprints on the current day genetic structure and highlight the importance of seawater temperature and sea level change in driving speciation in upper intertidal seaweed species.

Resistance to temperature stress and Drupella corallivory may promote the dominance of Platygyra acuta in the marginal coral communities in Hong Kong.

The wide fluctuation in seawater temperature (from 14 °C in winter to 31 °C in summer) has been suggested as one of the factors affecting the diversity of marginal coral communities like those in Hong Kong. We proposed in a previous study that branching corals like Acropora valida were more susceptible to low temperature stress during, and weakened corals became more vulnerable to corallivorous attack by the snails Drupella spp. In the following spring. Acropora spp., however, are not the most common species found in Hong Kong. In the present study, we examined comparable temperature effects, both low and elevated, on Platygyra acuta, one of the most dominant coral species in Hong Kong. Platygyra acuta fragments were exposed to six temperature levels ranging from 14 °C to 32 °C for 7 days before they were exposed to prey-choice experiments with Drupella. Results from these experiments indicated that P. acuta fragments were generally tolerant to temperature changes within the range tested. In contrast to those observed for A. valida, they were not found to be attractive to the subsequent Drupella corallivory. The greater tolerance of P. acuta to both these environmental and biological stresses may have contributed to its dominance in Hong Kong coral communities.

Urban coral reefs: Degradation and resilience of hard coral assemblages in coastal cities of East and Southeast Asia.

Given predicted increases in urbanization in tropical and subtropical regions, understanding the processes shaping urban coral reefs may be essential for anticipating future conservation challenges. We used a case study approach to identify unifying patterns of urban coral reefs and clarify the effects of urbanization on hard coral assemblages. Data were compiled from 11 cities throughout East and Southeast Asia, with particular focus on Singapore, Jakarta, Hong Kong, and Naha (Okinawa). Our review highlights several key characteristics of urban coral reefs, including "reef compression" (a decline in bathymetric range with increasing turbidity and decreasing water clarity over time and relative to shore), dominance by domed coral growth forms and low reef complexity, variable city-specific inshore-offshore gradients, early declines in coral cover with recent fluctuating periods of acute impacts and rapid recovery, and colonization of urban infrastructure by hard corals. We present hypotheses for urban reef community dynamics and discuss potential of ecological engineering for corals in urban areas.

A 30-year monitoring of changes in coral community structure following anthropogenic disturbances in Tolo Harbour and Channel, Hong Kong.

Coral communities in Tolo Harbour and Channel, northeastern Hong Kong, suffered from tremendous degradations in 1980s due to excessive sewage pollutions. This study examined changes in coral community structures over the last 30 years including period before, at the height of and after implementation of abatement measures of pollution impacts. Signs of coral degradations finally stopped in inner harbour and some corals started to reappear, likely due to sewage export scheme since 1998. Yet, the coral cover remained very low (<2%) in 2012. Natural recovery is limited by very low coral recruitment success other than that of Oulastrea crispata. The outer coral communities, which suffered least in 1980s, continued to decline, possibly due to new biological disturbances like sea urchin predation and bioerosion. This long-term study clearly revealed how coral communities could so easily be destroyed and yet natural recovery could be so difficult and unlikely.

Exploring coral microbiome assemblages in the South China Sea.

Coral reefs are significant ecosystems. The ecological success of coral reefs relies on not only coral-algal symbiosis but also coral-microbial partnership. However, microbiome assemblages in the South China Sea corals remain largely unexplored. Here, we compared the microbiome assemblages of reef-building corals Galaxea (G. fascicularis) and Montipora (M. venosa, M. peltiformis, M. monasteriata) collected from five different locations in the South China Sea using massively-parallel sequencing of 16S rRNA gene and multivariate analysis. The results indicated that microbiome assemblages for each coral species were unique regardless of location and were different from the corresponding seawater. Host type appeared to drive the coral microbiome assemblages rather than location and seawater. Network analysis was employed to explore coral microbiome co-occurrence patterns, which revealed 61 and 80 co-occurring microbial species assembling the Galaxea and Montipora microbiomes, respectively. Most of these co-occurring microbial species were commonly found in corals and were inferred to play potential roles in host nutrient metabolism; carbon, nitrogen, sulfur cycles; host detoxification; and climate change. These findings suggest that the co-occurring microbial species explored might be essential to maintain the critical coral-microbial partnership. The present study provides new insights into coral microbiome assemblages in the South China Sea.

Recruitment failure of scleractinian corals in a subtropical marginal environment: Three-year monitoring in a Hong Kong marine park.

Coral recruitment was monitored for three years, using terracotta tiles, in two core coral areas in Tung Ping Chau Marine Park where coral coverage is one of the richest in Hong Kong. Results revealed an extremely low recruitment success of corals, with only 10 recruits recorded on 384 tile pairs, equivalent to 0.19 recruits m-2yr-1 throughout the study period. Recruits of Platygyra acuta pre-seeded on tiles that were subsequently grown in situ experienced very high mortality following settlement, with average mortality of 80% within half a month and <0.1% survival through the first four months of deployment. Data suggested that low recruitment of corals may be due to the lack of arrival of competent coral larvae, low larval settlement; and/or low post-settlement survival of recruits due to high sedimentation or intense competition for space with fouling organisms, e.g. algae, oysters, barnacles and bryozoans, which may negatively affect coral recruitment.

High tolerance to temperature and salinity change should enable scleractinian coral Platygyra acuta from marginal environments to persist under future climate change.

With projected changes in the marine environment under global climate change, the effects of single stressors on corals have been relatively well studied. However, more focus should be placed on the interactive effects of multiple stressors if their impacts upon corals are to be assessed more realistically. Elevation of sea surface temperature is projected under global climate change, and future increases in precipitation extremes related to the monsoon are also expected. Thus, the lowering of salinity could become a more common phenomenon and its impact on corals could be significant as extreme precipitation usually occurs during the coral spawning season. Here, we investigated the interactive effects of temperature [24, 27 (ambient), 30, 32°C] and salinity [33 psu (ambient), 30, 26, 22, 18, 14 psu] on larval settlement, post-settlement survival and early growth of the dominant coral Platygyra acuta from Hong Kong, a marginal environment for coral growth. The results indicate that elevated temperatures (+3°C and +5°C above ambient) did not have any significant effects on larval settlement success and post-settlement survival for up to 56 days of prolonged exposure. Such thermal tolerance was markedly higher than that reported in the literature for other coral species. Moreover, there was a positive effect of these elevated temperatures in reducing the negative effects of lowered salinity (26 psu) on settlement success. The enhanced settlement success brought about by elevated temperatures, together with the high post-settlement survival recorded up to 44 and 8 days of exposure under +3°C and +5°C ambient respectively, resulted in the overall positive effects of elevated temperatures on recruitment success. These results suggest that projected elevation in temperature over the next century should not pose any major problem for the recruitment success of P. acuta. The combined effects of higher temperatures and lowered salinity (26 psu) could even be beneficial. Therefore, corals that are currently present in marginal environments like Hong Kong, as exemplified by the dominant P. acuta, are likely to persist in a warmer and intermittently less saline, future ocean.

Homogeneous population of the brown alga Sargassum polycystum in Southeast Asia: possible role of recent expansion and asexual propagation.

Southeast Asia has been known as one of the biodiversity hotspots in the world. Repeated glacial cycles during Pleistocene were believed to cause isolation of marine taxa in refugia, resulting in diversification among lineages. Recently, ocean current was also found to be another factor affecting gene flow by restricting larval dispersal in animals. Macroalgae are unique in having mode of reproduction that differs from that of animals. Our study on the phylogeographical pattern of the brown macroalga Sargassum polycystum using nuclear Internal Transcribed Spacer 2 (ITS2), plastidal RuBisCO spacer (Rub spacer) and mitochondrial cytochrome oxidase subunit-III (Cox3) as molecular markers revealed genetic homogeneity across 27 sites in Southeast Asia and western Pacific, in sharp contrast to that revealed from most animal studies. Our data suggested that S. polycystum persisted in single refugium during Pleistocene in a panmixia pattern. Expansion occurred more recently after the Last Glacial Maximum and recolonization of the newly flooded Sunda Shelf could have involved asexual propagation of the species. High dispersal ability through floating fronds carrying developing germlings may also contribute to the low genetic diversity of the species.

Phylogeography of the marine macroalga Sargassum hemiphyllum (Phaeophyceae, Heterokontophyta) in northwestern Pacific.

Sargassum hemiphyllum is commonly found in Japan and Korea, with a variety, var. chinense, that is found distributed in the southern Chinese coast. We previously reported distinct genetic differentiation between the two taxa based on the PCR-RFLP data of plastid RubiscoL-S spacer. The present study aims at elucidating the phylogeographic pattern of S. hemiphyllum based on more markers in the nuclear and extranuclear genomes, with a view to reveal the occurrence of hybridization. The two allopatrically distributed taxa were found to be genetically distinct in nuclear ITS2, plastidial Rubisco (Rbc) and mitochondrial TrnW_I (Trn) spacers. Their divergence was postulated to be attributable to the vicariant event which resulted from the isolation of the Sea of Japan during the late Miocene (6.58-11.25 Mya). Divergence within both S. hemiphyllum and the chinense variety was observed based on Trn spacer, while the divergence in S. hemiphyllum was further confirmed in Rbc spacer. This divergence appears to correspond to the separation of the Japanese populations between the Sea of Japan and the Pacific that occurred around 0.92-2.88 Mya (the early Pleistocene). The presence of an ITS2 clone resembling var. chinense sequences in a Japanese population of S. hemiphyllum (JpNS) raises the possibility of the introgression of var. chinense individuals into S. hemiphyllum population. Compared to that between S. hemiphyllum and the chinense variety, hybridization among the Japanese and Korean populations of S. hemiphyllum is highly probable as all these individuals share a pool of nuclear ITS2 sequences, possibly attributable to incomplete concerted evolution of ITS2.