Recurrent disease outbreak in a warm temperate marginal coral community.

Coral diseases contribute to the rapid degradation of coral reefs on a global scale. Although widespread in tropical and subtropical reefs, disease outbreaks have not been described in warm temperate areas. Here, we report the outbreak of a new coral disease in a warm temperate marginal coral community in Japan. Outbreaks of the disease have been observed during the summer and autumn months since 2014. It affects the coral species Porites heronensis and was tentatively named "White Mat Syndrome" (WMS) as it consists of a white microbial mat dominated by Thiothrix sp., a sulfide oxidizing bacteria. Outbreaks followed high seasonal temperatures and were associated with the macroalga Gelidium elegans, which acts as a pathogen reservoir. With ocean warming and the anticipated increase in novel coral-algae interactions as some coral species shift poleward, WMS and emerging diseases could hinder the role of temperate areas as a future coral refuge.

Bacterial Dynamics in the Accessory Nidamental Gland of Sepioteuthis lessoniana throughout Maturation.

The accessory nidamental gland (ANG) is part of the reproduction organ in the majority of female cephalopods, including the bigfin reef squid Sepioteuthis lessoniana, an economically important fishery product. Microbes in Alphaproteobacteria, Gammaproteobacteria, and Verrucomicrobia have been suggested to play a role in the maturation of the S. lessoniana ANG and are responsible for its color. However, the bacterial composition and dynamics of the different maturation stages of the ANG remain unclear. In the present study, we surveyed ANG-associated bacterial dynamics in wild-caught S. lessoniana at various developmental stages in different populations over 3 years. The results obtained showed that the ANG bacterial community shifted gradually and decreased in diversity throughout maturation. Verrucomicrobia occupied the ANG during the early stages in large numbers, and was replaced by Bacteroidia, Alphaproteobacteria, and Gammaproteobacteria in the later stages. Flavobacteriales and Alphaproteobacteria both appeared to contribute to pigmentation, while Bacteroidia, Alphaproteobacteria, and Gammaproteobacteria may be involved in enriching the heme biosynthesis pathway in the ANG with the maturation of S. lessoniana. The present results provide an open question of whether S. lessoniana actively selects the bacterial community in the ANG to adjust to its surrounding environment.

Demographic census confirms a stable population of the critically-endangered caryophyllid coral Polycyathus chaishanensis (Scleractinia; Caryophyllidae) in the Datan Algal Reef, Taiwan.

Polycyathus chaishanensis is a symbiotic caryophyllid coral described from a single population in a tidal pool off Chaishan, Kaohsiung, Taiwan. Due to its rarity, P. chaishanensis was declared a critically-endangered species under the Taiwan Wildlife Protection Act. In May 2017, a P. chaishanensis colony was discovered in the intertidal area of the Datan Algal Reef, Taoyuan, Taiwan. To determine whether this is a stable population in the algal reef, a demographic census-including data on occurrence, distribution, and colony size-was carried out in the algal reef in southern Taoyuan. Intertidal censuses and sediment collections were conducted at five different sections-Baiyu, Datan G1, Datan G2, Yongxing, and Yongan algal reefs-during the monthly spring low tide from July 2018 to January 2019. In total, 84 colonies-23 in Datan G1 and 61 in Datan G2-were recorded from a tidal range of - 160 to - 250 cm, according to the Taiwan Vertical Datum 2001 compiled by the Central Weather Bureau. No P. chaishanensis was found in Baiyu, Yongxing, or Yongan. The P. chaishanensis colony sizes ranged from 2.55 to 81.5 cm in diameter, with the larger P. chaishanensis present in the lower intertidal zone. Sediment was extremely high, with monthly site averages ranging from 3,818.26 to 29,166.88 mg cm-2 day-1, and there was a significant difference between sites and months, both of which affected the distribution of P. chaishanensis in the algal reef. Our study confirms the existence of a second population of P. chaishanensis in Taiwan, highlighting the importance of the Datan Algal Reef for the survival and protection of this critically-endangered caryophyllid coral and why it is so urgent that the reef should be conserved.

Development of Novel Polymorphic Microsatellite Markers in Catch Bowl Coral, Isopora palifera (Scleractinia; Acroporidae) Using Next-generation Sequencing.

Sung-Yin Yang, Wai-Ling Fong, Wenhua Savanna Chow, Chia-Min Hsu, Chia-Ling Carynn Chan, Shashank Keshavmurthy, and Chaolun Allen Chen (2018) Catch bowl coral, Isopora palifera, is a shallow- water scleractinian species distributed in the Indo-West Pacific region, and has been studied for its reproduction, symbiont diversity, and population genetics. In order to develop microsatellite markers to reveal the genetic connectivity of I. palifera in the Kenting reefs, southern Taiwan, we applied a stepwise approach including Illumina sequencing, primer screening, and validation. DNA sequences of each 6,363,035 read pairs were assembled with high coverage and sequencing depth, and 1,173,835 potential SSRs were identified. A set of 60,986 primers were designed and tested, and six novel microsatellite markers with three type motifs, including 3 di- and 3 tetra- repeats, were successfully isolated. The ranges in number of alleles per locus and observed and expected heterozygosities were 3-5, 0.444-0.538, and 0.375-0.565, respectively. Application of these loci to the genetic diversity of an I. palifera population that experienced bleaching events in the Kenting reef between 1998 and 2015 showed a signature admixture of three clusters without temporal variation. These loci are useful for studying population genetics in the genus Isopora. Our results suggest that next-generation sequencing technology is convenient and cost-effective and can be utilized to isolate microsatellites in other reef-building corals.

A preliminary survey of zoantharian endosymbionts shows high genetic variation over small geographic scales on Okinawa-jima Island, Japan.

Symbiotic dinoflagellates (genus Symbiodinium) shape the responses of their host reef organisms to environmental variability and climate change. To date, the biogeography of Symbiodinium has been investigated primarily through phylogenetic analyses of the ribosomal internal transcribed spacer 2 region. Although the marker can approximate species-level diversity, recent work has demonstrated that faster-evolving genes can resolve otherwise hidden species and population lineages, and that this diversity is often distributed over much finer geographical and environmental scales than previously recognized. Here, we use the noncoding region of the chloroplast psbA gene (psbAncr) to examine genetic diversity among clade C Symbiodinium associating with the common reef zoantharian Palythoa tuberculosa on Okinawa-jima Island, Japan. We identify four closely related Symbiodinium psbAncr lineages including one common generalist and two potential specialists that appear to be associated with particular microhabitats. The sea surface temperature differences that distinguish these habitats are smaller than those usually investigated, suggesting that future biogeographic surveys of Symbiodinium should incorporate fine scale environmental information as well as fine scale molecular data to accurately determine species diversity and their distributions.

Profiling soil microbial communities with next-generation sequencing: the influence of DNA kit selection and technician technical expertise.

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil® DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin® Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA; P < 0.006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

Diversity of Microbial Communities and Quantitative Chemodiversity in Layers of Marine Sediment Cores from a Causeway (Kaichu-Doro) in Okinawa Island, Japan.

Microbial community diversity and chemodiversity were investigated in marine sediments adjacent to the Okinawan "Kaichu-Doro" Causeway, which was constructed 46 years ago to connect a group of four islands (Henza-jima, Miyagi-jima, Ikei-jima, Hamahiga-jima) to the Okinawan main island. This causeway was not built on pilings, but by land reclamation; hence, it now acts as a long, thin peninsula. The construction of this causeway was previously shown to have influenced the surrounding marine ecosystem, causing ecosystem fragmentation and loss of water circulation. In this study, we collected sediment cores (n = 10) from five paired sites in 1 m water depths. Each pair of sites consisted of one site each on the immediate north and south sides of the causeway. Originally the members of each pair were much closer to each other (<150 m) than to other pairs, but now the members of each pair are isolated by the causeway. Each core was 60-80 cm long and was divided into 15-cm layers. We examined the vertical diversity of microbial communities and chemical compounds to determine the correlation between chemodiversity and microbial communities among marine sediment cores and layers. Principal coordinate analyses (PCoA) of detected compounds and of bacterial and archaeal operational taxonomic units (OTUs) revealed that the north and south sides of the causeway are relatively isolated, with each side having unique microbial OTUs. Additionally, some bacterial families (e.g., Acidaminobacteraceae, Rhizobiaceae, and Xanthomonadaceae) were found only on the south side of Kaichu-Doro. Interestingly, we found that the relative abundance of OTUs for some microbial families increased from top to bottom, but this was reversed in some other families. We conclude that the causeway has altered microbial community composition and metabolite profiles in marine sediments.

A citizen science approach to monitoring bleaching in the zoantharian Palythoa tuberculosa.

Coral reef bleaching events are expected to become more frequent and severe in the near future as climate changes. The zoantharian Palythoa tuberculosa bleaches earlier than many scleractinian corals and may serve as an indicator species. Basic monitoring of such species could help to detect and even anticipate bleaching events, especially in areas where more sophisticated approaches that rely on buoy or satellite measurements of sea surface temperature are unavailable or too coarse. One simple and inexpensive monitoring method involves training volunteers to record observations of host color as a proxy for symbiosis quality. Here, we trained university students to take the 'color fingerprint' of a reef by assessing the color of multiple randomly selected colonies of P. tuberculosa at one time point in Okinawa Island, Japan. We tested the reliability of the students' color scores and whether they matched expectations based on previous monthly monitoring of tagged colonies at the same locations. We also measured three traditional metrics of symbiosis quality for comparison: symbiont morphological condition, cell density, and chlorophyll a content. We found that P. tuberculosa color score, although highly correlated among observers, provided little predictive power for the other variables. This was likely due to inherent variation in colony color among generally healthy zoantharians in midwinter, as well as low sample size and brief training owing to the course structure. Despite certain limitations of P. tuberculosa as a focal organism, the citizen science approach to color monitoring has promise, and we outline steps that could improve similar efforts in the future.

Effects of causeway construction on environment and biota of subtropical tidal flats in Okinawa, Japan.

Okinawa, Japan is known for its high marine biodiversity, yet little work has been performed on examining impacts of numerous large-scale coastal development projects on its marine ecosystems. Here, we examine apparent impacts of the construction of the Kaichu-Doro causeway, which was built over 40 years ago. The causeway is a 4.75 km long embankment that divides a large tidal flat and has only two points of water exchange along its entire length. We employed quadrats, transects, sampling, visual surveys, and microbial community analyses combined with environmental, water quality data, and 1m cores, at five stations of two paired sites each (one on each side of Kaichu-Doro) to investigate how the environment and biota have changed since the Kaichu-Doro was built. Results indicate reduction in water flow, and site S1 was particularly heavily impacted by poor water quality, with low diversity and disturbed biotic communities.

Can resistant coral-Symbiodinium associations enable coral communities to survive climate change? A study of a site exposed to long-term hot water input.

Climate change has led to a decline in the health of corals and coral reefs around the world. Studies have shown that, while some corals can cope with natural and anthropogenic stressors either through resistance mechanisms of coral hosts or through sustainable relationships with Symbiodinium clades or types, many coral species cannot. Here, we show that the corals present in a reef in southern Taiwan, and exposed to long-term elevated seawater temperatures due to the presence of a nuclear power plant outlet (NPP OL), are unique in terms of species and associated Symbiodinium types. At shallow depths (<3 m), eleven coral genera elsewhere in Kenting predominantly found with Symbiodinium types C1 and C3 (stress sensitive) were instead hosting Symbiodinium type D1a (stress tolerant) or a mixture of Symbiodinium type C1/C3/C21a/C15 and Symbiodinium type D1a. Of the 16 coral genera that dominate the local reefs, two that are apparently unable to associate with Symbiodinium type D1a are not present at NPP OL at depths of <3 m. Two other genera present at NPP OL and other locations host a specific type of Symbiodinium type C15. These data imply that coral assemblages may have the capacity to maintain their presence at the generic level against long-term disturbances such as elevated seawater temperatures by acclimatization through successful association with a stress-tolerant Symbiodinium over time. However, at the community level it comes at the cost of some coral genera being lost, suggesting that species unable to associate with a stress-tolerant Symbiodinium are likely to become extinct locally and unfavorable shifts in coral communities are likely to occur under the impact of climate change.

DNA barcoding reveals the coral "laboratory-rat", Stylophora pistillata encompasses multiple identities.

Stylophora pistillata is a widely used coral "lab-rat" species with highly variable morphology and a broad biogeographic range (Red Sea to western central Pacific). Here we show, by analysing Cytochorme Oxidase I sequences, from 241 samples across this range, that this taxon in fact comprises four deeply divergent clades corresponding to the Pacific-Western Australia, Chagos-Madagascar-South Africa, Gulf of Aden-Zanzibar-Madagascar, and Red Sea-Persian/Arabian Gulf-Kenya. On the basis of the fossil record of Stylophora, these four clades diverged from one another 51.5-29.6 Mya, i.e., long before the closure of the Tethyan connection between the tropical Indo-West Pacific and Atlantic in the early Miocene (16-24 Mya) and should be recognised as four distinct species. These findings have implications for comparative ecological and/or physiological studies carried out using Stylophora pistillata as a model species, and highlight the fact that phenotypic plasticity, thought to be common in scleractinian corals, can mask significant genetic variation.

Diversity and distribution of symbiodinium associated with seven common coral species in the Chagos Archipelago, central Indian Ocean.

The Chagos Archipelago designated as a no-take marine protected area in 2010, lying about 500 km south of the Maldives in the Indian Ocean, has a high conservation priority, particularly because of its fast recovery from the ocean-wide massive coral mortality following the 1998 coral bleaching event. The aims of this study were to examine Symbiodinium diversity and distribution associated with scleractinian corals in five atolls of the Chagos Archipelago, spread over 10,000 km(2). Symbiodinium clade diversity in 262 samples of seven common coral species, Acropora muricata, Isopora palifera, Pocillopora damicornis, P. verrucosa, P. eydouxi, Seriatopora hystrix, and Stylophora pistillata were determined using PCR-SSCP of the ribosomal internal transcribed spacer 1 (ITS1), PCR-DDGE of ITS2, and phylogenetic analyses. The results indicated that Symbiodinium in clade C were the dominant symbiont group in the seven coral species. Our analysis revealed types of Symbiodinium clade C specific to coral species. Types C1 and C3 (with C3z and C3i variants) were dominant in Acroporidae and C1 and C1c were the dominant types in Pocilloporidae. We also found 2 novel ITS2 types in S. hystrix and 1 novel ITS2 type of Symbiodinium in A. muricata. Some colonies of A. muricata and I. palifera were also associated with Symbiodinium A1. These results suggest that corals in the Chagos Archipelago host different assemblages of Symbiodinium types then their conspecifics from other locations in the Indian Ocean; and that future research will show whether these patterns in Symbiodinium genotypes may be due to local adaptation to specific conditions in the Chagos.