Solving the Coral Species Delimitation Conundrum.

Distinguishing coral species is not only crucial for physiological, ecological, and evolutionary studies but also to enable effective management of threatened reef ecosystems. However, traditional hypotheses that delineate coral species based on morphological traits from the coral skeleton are frequently at odds with tree-based molecular approaches. Additionally, a dearth of species-level molecular markers has made species delimitation particularly challenging in species-rich coral genera, leading to the widespread assumption that interspecific hybridization might be responsible for this apparent conundrum. Here, we used three lines of evidence-morphology, breeding trials, and molecular approaches-to identify species boundaries in a group of ecologically important tabular Acropora corals. In contrast to previous studies, our morphological analysis yielded groups that were congruent with experimental crosses as well as with coalescent-based and allele sharing-based multilocus approaches to species delimitation. Our results suggest that species of the genus Acropora are reproductively isolated and independently evolving units that can be distinguished morphologically. These findings not only pave the way for a taxonomic revision of coral species but also outline an approach that can provide a solid basis to address species delimitation and provide conservation support to a wide variety of keystone organisms. [Acropora; coral reefs; hybridization; reproductive isolation; taxonomy.].

Coral larvae suppress heat stress response during the onset of symbiosis decreasing their odds of survival.

The endosymbiosis between most corals and their photosynthetic dinoflagellate partners begins early in the host life history, when corals are larvae or juvenile polyps. The capacity of coral larvae to buffer climate-induced stress while in the process of symbiont acquisition could come with physiological trade-offs that alter behaviour, development, settlement and survivorship. Here we examined the joint effects of thermal stress and symbiosis onset on colonization dynamics, survival, metamorphosis and host gene expression of Acropora digitifera larvae. We found that thermal stress decreased symbiont colonization of hosts by 50% and symbiont density by 98.5% over 2 weeks. Temperature and colonization also influenced larval survival and metamorphosis in an additive manner, where colonized larvae fared worse or prematurely metamorphosed more often than noncolonized larvae under thermal stress. Transcriptomic responses to colonization and thermal stress treatments were largely independent, while the interaction of these treatments revealed contrasting expression profiles of genes that function in the stress response, immunity, inflammation and cell cycle regulation. The combined treatment either cancelled or lowered the magnitude of expression of heat-stress responsive genes in the presence of symbionts, revealing a physiological cost to acquiring symbionts at the larval stage with elevated temperatures. In addition, host immune suppression, a hallmark of symbiosis onset under ambient temperature, turned to immune activation under heat stress. Thus, by integrating the physical environment and biotic pressures that mediate presettlement event in corals, our results suggest that colonization may hinder larval survival and recruitment under projected climate scenarios.

Unique environmental Symbiodiniaceae diversity at an isolated island in the northwestern Pacific.

Dinoflagellates in the family Symbiodiniaceae are intensively investigated as algal symbionts of corals and other invertebrates, underpinning coral reef ecosystems as primary producers. Diversity, including regional diversification, of free-living communities is less studied. In this study, an environmental Symbiodiniaceae community at an isolated island, Okinotori Island, Japan, was investigated to determine whether the community is endemic or common with other locations near continents and major ocean currents. Symbiotic algae in common corals at the island were the same type as those of the corals from other Japanese waters. In the environmental samples, genera Symbiodinium (formerly clade A), Cladocopium (clade C), Durusdinium (clade D), and clades F (including Freudenthalidium), G, and I, were identified through analysis of internal transcribed spacer region 2 of nuclear ribosomal RNA gene (ITS2) sequences. Interestingly, some sequences found were genetically different from those of previously reported genera/clades. These unknown sequences were genetically included in the Symbiodiniaceae linage, but they were differentiated from the previously known nine clades. The sequences formed a cluster in the phylogenetic tree based on 28S nrDNA. These sequences were thus considered members of a novel clade in the family (clade J). In total, 120 kinds of ITS2 sequences were produced; while 10 were identical to previously reported sequences, the majority were highly divergent. These genetically unique Symbiodiniaceae types, including novel clade J, may have evolved in isolation and reflect the environmental characteristics of the Okinotori Island.

N-Acetyl-d-Glucosamine-Binding Lectin in Acropora tenuis Attracts Specific Symbiodiniaceae Cell Culture Strains.

Many corals establish symbiosis with Symbiodiniaceae cells from surrounding environments, but very few Symbiodiniaceae cells exist in the water column. Given that the N-acetyl-d-glucosamine-binding lectin ActL attracts Symbiodiniaceae cells, we hypothesized that corals must attract Symbiodiniaceae cells using ActL to acquire them. Anti-ActL antibody inhibited acquisition of Symbiodiniaceae cells, and rearing seawater for juvenile Acropora tenuis contained ActL, suggesting that juvenile A. tenuis discharge ActL to attract these cells. Among eight Symbiodiniaceae cultured strains, ActL attracted NBRC102920 (Symbiodinium tridacnidorum) most strongly followed by CS-161 (Symbiodinium tridacnidorum), CCMP2556 (Durusdinium trenchii), and CCMP1633 (Breviolum sp.); however, it did not attract GTP-A6-Sy (Symbiodinium natans), CCMP421 (Effrenium voratum), FKM0207 (Fugacium sp.), and CS-156 (Fugacium sp.). Juvenile polyps of A. tenuis acquired limited Symbiodiniaceae cell strains, and the number of acquired Symbiodiniaceae cells in a polyp also differed from each other. The number of Symbiodiniaceae cells acquired by juvenile polyps of A. tenuis was correlated with the ActL chemotactic activity. Thus, ActL could be used to attract select Symbiodiniaceae cells and help Symbiodiniaceae cell acquisition in juvenile polyps of A. tenuis, facilitating establishment of symbiosis between A. tenuis and Symbiodiniaceae cells.

Effect of elevated temperature on fecundity and reproductive timing in the coral Acropora digitifera.

The synchrony of spawning is of paramount importance to successful coral reproduction. The precise timing of spawning is thought to be controlled by a set of interacting environmental factors, including regional wind field patterns, timing of the sunset, and sea surface temperatures (SST). Climate change is resulting in increased SST, which is causing physiological stress in corals and could also be altering spawning synchrony and timing. In this study, we examined the effect of increasing seawater temperature by 2°C for 1 month prior to the predicted spawning time on reproduction in the coral Acropora digitifera. This short period of elevated temperature caused spawning to advance by 1 day. In animals incubated at elevated temperature, egg number per egg bundle did not change, however, egg volume significantly decreased as did sperm number. Our results indicate that temperature is acting both as a proximate cue to accelerate timing and as a stressor on gametogenesis to reduce fecundity. This finding suggests that increasing SSTs could play a dramatic role in altering reproductive timing and the success of corals in an era of climate change.

Genetic diversity, paraphyly and incomplete lineage sorting of mtDNA, ITS2 and microsatellite flanking region in closely related Heliopora species (Octocorallia).

Examining genetic diversity and lineage sorting of different genes in closely related species provide useful information for phylogenetic analyses and ultimately for understanding the origins of biodiversity. In this study, we examined inter- and intraspecific genetic variation in internal transcribed spacer 2 (ITS2), partial mitochondrial gene (mtMutS), and nuclear microsatellite flanking region in two closely related octocoral species (Heliopora coerulea, HC-A and HC-B). These species were recently identified in a population genetic study using microsatellite markers. The two species have different reproductive timing, which ecologically promotes lineage sorting. In this study, we examined whether species boundaries could be detected by the commonly used nuclear ITS2 and mtMutS, as well as by possibly neutral microsatellite flanking sequences. Haplotype network analysis of microsatellite flanking region revealed that a possible ancestral haplotype was still shared between the two species, indicating on-going lineage sorting. Haplotype network analysis of ITS2 and microsatellite flanking region revealed shared haplotypes between the two lineages. The two species shared fewer ITS2 sequences than microsatellite flanking region sequences. The almost fixed point mutation at the tip of helix 3 of ITS2 was not associated with the secondary structure or compensatory base changes (CBCs). The phylogenetic tree of ITS2 showed paraphyly and that of the microsatellite flanking region indicated that lineage sorting for the two species may be incomplete. Much higher intra- and inter-individual variation of ITS2 was observed in HC-B than that in HC-A, highlighting the importance of examining ITS2 from multiple individuals to estimate genetic diversity. The mitochondrial mtMutS gene sequences from 39 individuals, including both species collected from Japan and Taiwan, showed no variation because of slow rates of mitochondrial nucleotide substitution. This study suggests caution is warranted when reciprocal monophyly in a phylogenetic tree is used as the criterion for delimiting closely related octocoral species based on ITS2 or mtMtuS sequences. Detection of boundaries between closely related species requires multi-locus analysis, such as genetic admixture analysis using multiple individuals.

Tolerance to Elevated Temperature and Ocean Acidification of the Larvae of the Solitary Corals Fungia fungites (Linnaues, 1758) and Lithophyllon repanda (Dana, 1846).

Increase in atmospheric CO2is the main driver of global climate change and is projected to elevate sea surface temperature by at least 2°C and to decrease oceanic pH by 0.3 to 0.4 units by the end of the century. These factors seriously threaten coral reef ecosystems worldwide. In Okinawa, solitary corals are an important feature of the coral community structure. While previous studies on the effects of ocean warming (OW), ocean acidification (OA) and its combination on larval survival focused on colonial coral species, the present study assessed the effect of high temperature on larvae from solitary corals. In this study, we examined the influence of OW (control = 28°C; control +3 = 31°C; control + 6 = 34°C) and OA (control, pCO2= 400 to 500 µatm; medium, pCO2= 1000 to 1300 µatm; high, pCO2= 1700 to 2200 µatm) on the larval survival of two solitary corals, Fungia fungites and Lithophyllon repanda for eight days. Results showed that F. fungites was neither affected by OW, OA, nor its combination. Similarly, survival of L. repanda was not affected by OA however it was significantly affected by temperature. Temperature tolerance varies between species; L. repanda (+3°C above ambient) has lower tolerance than F. fungites (+6°C above ambient). This observation suggests that fungiid larvae had higher tolerance to elevated temperature stress relative to other scleractinian corals. With the projected increase in OW and OA in the future, fungiidsmay retain good potential to widely disperse and successfully recruit to natal and other neighbouring reefs.

Acclimation potential of Acropora to mesophotic environment.

Mesophotic coral ecosystems may serve as a refuge for reef-building corals to survive the ongoing climate change. Distribution of coral species changes during larval dispersal. However, the acclimation potential in the early life stages of corals at different depths is unknown. This study investigated the acclimation potential of four shallow Acropora species at different depths via the transplantation of larvae and early polyps settled on tiles to 5, 10, 20, and 40 m depths. We then examined physiological parameters, such as size, survival, growth rate, and morphological characteristics. The survival and size of juveniles of A. tenuis and A. valida at 40 m depth were significantly higher than those at other depths. In contrast, A. digitifera and A. hyacinthus showed higher survival rates at shallow depths. The morphology (i.e., size of the corallites) also varied among the depths. Collectively, the shallow coral larvae and juveniles displayed substantial plasticity at depth.

Reactions of juvenile coral to three years of consecutive thermal stress.

As global temperatures continue to rise, corals are being exposed to increasing heat stress throughout their early life stages; however, the impact of this phenomenon is poorly understood. We exposed the reef-building coral Acropora tenuis juveniles to ∼26-28 °C (control) and ∼ 31 °C (heat stress) for one week per year over three consecutive years. In the first year of heat stress, >96 % of juveniles survived despite symbiotic algal densities in juvenile corals declining. In comparison, survival rates in the third year of heat stress declined to 50 %. Survival rates under natural conditions after stress also gradually decreased in the stressed groups. The rate in the reduction of survivorship was prominent in the consecutive thermally stressed groups (juveniles stressed twice in two years). Symbiotic algal density and photosynthetic activity (Fv/Fm) also declined in stressed juvenile groups. However, heat stress did not significantly affect the growth of juveniles. In the third year of heat stress, temperature negatively affected the physiology of juveniles in terms of survivorship, brightness (an indicator of bleaching), symbiotic algal density, and photosynthetic efficiency. Stress across consecutive years appeared to cause the survivorship of juvenile corals to decline, with three years of stress contributing to the severe decline of a reef. In conclusion, A. tenuis juveniles are not able to acclimatize to heat stress, with successive heat waves of <7 days in the summer potentially negatively affecting resilience.

Response of resistant larvae of the coral Acropora tenuis to future thermal stress.

Seawater temperatures are rising rapidly and severely due to climate change, negatively affecting coral reef communities. The persistence of coral populations depends on their success during the early life stages. Thermal conditioning during the larval stage can increase coral larvae's ability to tolerate high temperatures in subsequent stages. We studied the response of resistant larvae of Acropora tenuis to thermal stress to increase their thermal tolerance during the juvenile stage. Larvae were exposed to ambient (∼26 °C) and thermal stress (∼31 °C) temperatures. Then, settlement success on preconditioned tiles was determined. After 28 days at ambient temperature, the juveniles were exposed to thermal stress for 14 days, and their survival was assessed. Our results showed that thermal stress in the larval stage did not alter the thermal tolerance of juveniles, and they could not acclimate to heat stress. As a result, the summer's heat waves could potentially threaten their resilience.

Plasticity of shallow reef corals across a depth gradient.

Global warming harms coral reefs. Mesophotic coral reef ecosystems (MCEs) have been suggested to serve as refugia for shallow reefs. Information on the adaptation potential of shallow corals at MCEs is a prerequisite for understanding the refuge potential of MCEs. In this study, we investigated the photoacclimation potential of four shallow coral species transplanted at different depths over 1 year. The results showed that the corals-Pocillopora damicornis, Porites cylindrica, and Turbinaria reniformis-survived and acclimated to a wide range of light regimes at the depths of 5, 20, and 40 m. However, Acropora tenuis survived only at 5 and 20 m depth and showed significant morphological alteration at 20 m depth. Our results indicate that shallow corals have substantial plasticity with respect to depth changes. Changes in photosynthetic performance and phenotypic plasticity within these coral species may act as a buffer for depth-related changes and as modulators of evolutionary responses.

Limited acclimation of early life stages of the coral Seriatopora hystrix from mesophotic depth to shallow reefs.

Mesophotic coral ecosystems (MCEs, reefs between 30 and 150 m depth) have been hypothesized to contribute to shallow reef recovery through the recruitment of larvae. However, few studies have directly examined this. Here we used mesophotic colonies of Seriatopora hystrix, a depth generalist coral, to investigate the effect of light intensity on larval behavior and settlement through ex situ experiments. We also investigated juvenile survival, growth, and physiological acclimation in situ. Bleached larvae and a significant reduction in settlement rates were found when the mesophotic larvae were exposed to light conditions corresponding to shallow depths (5 and 10 m) ex situ. The in situ experiments showed that mesophotic juveniles survived well at 20 and 40 m, with juveniles in shaded areas surviving longer than three months at 3-5 m during a year of mass bleaching in 2016. Juvenile transplants at 20 m showed a sign of physiological acclimation, which was reflected by a significant decline in maximum quantum yield. These results suggest that light is a significant factor for successful recolonization of depth-generalist corals to shallow reefs. Further, recolonization of shallow reefs may only occur in shaded habitats or potentially through multigenerational recruitments with intermediate depths acting as stepping stones.

The early acquisition of symbiotic algae benefits larval survival and juvenile growth in the coral Acropora tenuis.

Larval dispersal and postsettlement survival of corals play significant roles in the maintenance of coral populations. Most corals acquire their symbiotic algae (Symbiodiniaceae) from the environment in each generation (horizontal transmission). For horizontal transmitters, the quick establishment of symbiosis is important for their survival, since the photosynthetic activity of symbiotic algae provides energy. However, recent studies have indicated that oxidative stress resulting from photosynthesis might also harm coral larvae. Therefore, it remains unclear whether symbionts contribute energy sources along with intrinsic lipids from eggs and assist in settlement/metamorphosis in early life stages. In the present study, we show that symbiotic algae contribute supplemental energy and are also associated with settlement. Furthermore, although juveniles acquired symbiotic algae after settlement, the acquisition of symbiotic algae in the larval stages caused higher growth (number of polyps and size) and low mortality in the juvenile stage. Our data suggest that symbiotic larvae potentially have longer dispersal periods due to their lower lipid consumption rates, which make them better able to retain buoyancy and motility, increasing the ability of symbiotic larvae to settle in favored locations compared with aposymbiotic larvae. Moreover, postsettlement juveniles may continue to benefit from symbiotic relationships formed during the larval stage. Overall, these findings reveal that the effects of symbiotic algae on Acropora tenuis coral larvae are beneficial, particularly under normal seawater temperature conditions.

Development of an automated transportable continuous system to measure the total alkalinity of seawater.

Anthropogenic CO2 emissions are contributing to global warming and ocean acidification. Rapid and accurate measurements of seawater carbonate chemistry are critical to understand current changes in the ocean and to predict future effects of such changes on marine organisms and ecosystems. Total alkalinity (AT) measurements can be used to directly determine the calcification rate, but they are time-consuming and require large sample volumes. Herein, we describe an automated and transportable flow-through system that can conduct continuous AT measurement using an ion sensitive field effect transistor (ISFET) - Ag/AgCl sensor and three different reference materials. The response time, stability, and uncertainty of our system were evaluated by comparing AT values of calibrated reference materials to those calculated by our system. Our system requires only small amounts of seawater (<10 mL) and a short time per sample (<5 min) to produce results with a relative uncertainty of less than 0.1% (approx. 2.2 µmol kg-1). This system is expected to facilitate easy and rapid in-situ measurement of AT. Continuous AT measurements would enable us to determine short-term calcification responses to changes in light or temperature and improve our understanding of the metabolic mechanisms of creatures such as corals.

Morphological stasis masks ecologically divergent coral species on tropical reefs.

Coral reefs are the epitome of species diversity, yet the number of described scleractinian coral species, the framework-builders of coral reefs, remains moderate by comparison. DNA sequencing studies are rapidly challenging this notion by exposing a wealth of undescribed diversity, but the evolutionary and ecological significance of this diversity remains largely unclear. Here, we present an annotated genome for one of the most ubiquitous corals in the Indo-Pacific (Pachyseris speciosa) and uncover, through a comprehensive genomic and phenotypic assessment, that it comprises morphologically indistinguishable but ecologically divergent lineages. Demographic modeling based on whole-genome resequencing indicated that morphological crypsis (across micro- and macromorphological traits) was due to ancient morphological stasis rather than recent divergence. Although the lineages occur sympatrically across shallow and mesophotic habitats, extensive genotyping using a rapid molecular assay revealed differentiation of their ecological distributions. Leveraging "common garden" conditions facilitated by the overlapping distributions, we assessed physiological and quantitative skeletal traits and demonstrated concurrent phenotypic differentiation. Lastly, spawning observations of genotyped colonies highlighted the potential role of temporal reproductive isolation in the limited admixture, with consistent genomic signatures in genes related to morphogenesis and reproduction. Overall, our findings demonstrate the presence of ecologically and phenotypically divergent coral species without substantial morphological differentiation and provide new leads into the potential mechanisms facilitating such divergence. More broadly, they indicate that our current taxonomic framework for reef-building corals may be scratching the surface of the ecologically relevant diversity on coral reefs, consequently limiting our ability to protect or restore this diversity effectively.

An Indo-Pacific coral spawning database.

The discovery of multi-species synchronous spawning of scleractinian corals on the Great Barrier Reef in the 1980s stimulated an extraordinary effort to document spawning times in other parts of the globe. Unfortunately, most of these data remain unpublished which limits our understanding of regional and global reproductive patterns. The Coral Spawning Database (CSD) collates much of these disparate data into a single place. The CSD includes 6178 observations (3085 of which were unpublished) of the time or day of spawning for over 300 scleractinian species in 61 genera from 101 sites in the Indo-Pacific. The goal of the CSD is to provide open access to coral spawning data to accelerate our understanding of coral reproductive biology and to provide a baseline against which to evaluate any future changes in reproductive phenology.

Early molecular responses of coral larvae to hyperthermal stress.

Most of the work on the impact of elevated temperature and light on Symbiodinium-invertebrate symbioses have focused primarily on how the photosynthetic (algal) partner is impacted. Understanding how the same stresses affect the invertebrate host, however, is in its infancy. In this study, we re-examined the direct effect of elevated temperatures on the invertebrate host exploring the early transcriptional response of aposymbiotic (without algal symbionts) coral larvae. The temperatures tested in the experimental design were 24 degrees C (ambient seawater temperature), 28 degrees C and 31 degrees C; and the sampling points were 3 and 10 h after temperature exposure. We explored relative changes in transcription using a cDNA microarray constructed for the scleractinian coral, Acropora millepora, and containing 18 142 expressed sequence tag (EST) clones/8386 unigenes. Our study identified 29 genes that were significantly up- and down-regulated when A. millepora coral larvae were exposed to elevated temperatures. Down-regulation of several key components of DNA/RNA metabolism was detected implying inhibition of general cellular processes. The down-regulation of protein synthesis, however, was not simple and random, which suggested that the stress response was a more complicated adjustment of cellular metabolism. We identified four significant outcomes during the very early hours of the transcriptional response to hyperthermal stress in coral larvae. First, the expression of heat-shock proteins increased rapidly (within 3 h) in response to hyperthermal stress. Second, a fluorescent protein homologue, DsRed-type FP, decreased its expression in response to elevated temperature reinforcing a potential role as a molecular marker for monitoring hyperthermal stress in nature. Third, the down-regulation of a coral mannose-binding C-type lectin under elevated temperature suggests that heat stress might compromise some components of the coral immune defence and therefore might bring about susceptibility to pathogenic diseases. And last, genes involved in protecting cells against oxidative stress showed little response at the early hours to heat stress, supporting the proposal that up-regulation of cnidarian host oxidative stress genes may require reactive oxygen species generated by stressed algal symbionts.

Demystifying Circalunar and Diel Rhythmicity in Acropora digitifera under Constant Dim Light.

Life on earth has evolved under constant environmental changes; in response to these changes, most organisms have developed an endogenous clock that allows them to anticipate daily and seasonal changes and adapt their biology accordingly. Light cycles synchronize biological rhythms and are controlled by an endogenous clock that is entrained by environmental cues. Light is known to play a key role in the biology of symbiotic corals as they exhibit many biological processes entrained by daily light patterns. In this study, we aimed at determining the effect of constant dim light on coral's perception of diel and monthly cycles. Our results show that under constant dim light corals display a loss of rhythmic processes and constant stimuli by light, which initiates signal transduction that results in an abnormal cell cycle, cell proliferation, and protein synthesis. The results emphasize how constant dim light can mask the biological clock of Acropora digitifera.

Repetitive sex change in the stony coral Herpolitha limax across a wide geographic range.

Sex change has been widely studied in animals and plants. However, the conditions favoring sex change, its mode and timing remain poorly known. Here, for the first time in stony corals, we report on a protandrous (youngest individuals are males) repetitive sex change exhibited by the fungiid coral Herpolitha limax across large spatial scales (the coral reefs of Japan, Jordan and Israel) and temporal scales (2004-2017). In contrast to most corals, this species is a daytime spawner (08:00-10:00 AM) that spawned at the same time/same date across all the study sites. The sporadically scattered populations of H. limax among the coral reefs of Eilat (Israel) and Aqaba (Jordan) exhibited significantly slower growth, earlier sex change, and lower percentages of reproduction and sex change in comparison to the densely aggregated populations in Okinawa (Japan). At all sites, sex ratio varied among years, but was almost always biased towards maleness. Growth rate decreased with size. We conclude that comparable to dioecious plants that display labile sexuality in response to energetic and/or environmental constraints, the repetitive sex change displayed by H. limax increases its overall fitness reinforcing the important role of reproductive plasticity in the Phylum Cnidaria in determining their evolutionary success.

Deciphering the nature of the coral-Chromera association.

Since the discovery of Chromera velia as a novel coral-associated microalga, this organism has attracted interest because of its unique evolutionary position between the photosynthetic dinoflagellates and the parasitic apicomplexans. The nature of the relationship between Chromera and its coral host is controversial. Is it a mutualism, from which both participants benefit, a parasitic relationship, or a chance association? To better understand the interaction, larvae of the common Indo-Pacific reef-building coral Acropora digitifera were experimentally infected with Chromera, and the impact on the host transcriptome was assessed at 4, 12, and 48 h post-infection using Illumina RNA-Seq technology. The transcriptomic response of the coral to Chromera was complex and implies that host immunity is strongly suppressed, and both phagosome maturation and the apoptotic machinery is modified. These responses differ markedly from those described for infection with a competent strain of the coral mutualist Symbiodinium, instead resembling those of vertebrate hosts to parasites and/or pathogens such as Mycobacterium tuberculosis. Consistent with ecological studies suggesting that the association may be accidental, the transcriptional response of A. digitifera larvae leads us to conclude that Chromera could be a coral parasite, commensal, or accidental bystander, but certainly not a beneficial mutualist.