Inferring the distribution of octocorals Nanipora and Heliopora along the coast of the Kerama Islands, Okinawa, Japan, by eDNA metabarcoding

Inferring the distribution of octocorals Nanipora and Heliopora along the coast of the Kerama Islands, Okinawa, Japan, by eDNA metabarcoding | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Inferring the distribution of octocorals Nanipora and Heliopora along the coast of the Kerama Islands, Okinawa, Japan, by eDNA metabarcoding Noriyuki Satoh, Takeshi Noda, Kanako Hisata, Yuki Yoshioka, Tomofumi Nagata, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7776767/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Mar, 2026 Read the published version in Coral Reefs → Version 1 posted 10 You are reading this latest preprint version Abstract Two zooxanthellate octocorals, Heliopora coerulea and Nanipora kamurai (family Helioporidae, order Scleralcyonacea, class Octocorallia), produce aragonite calcium carbonate skeletons similar to those of scleractinian hexacorals and contribute to the formation of complex coral reef ecosystems. H. coerulea is known to occur along the coasts of the Kerama Islands, Okinawa, Japan and N. kamurai was originally described from the Kerama Islands. An environmental DNA metabarcoding (eDNA-M) method has recently been introduced for comprehensive surveys of zooxanthellate scleractinian corals, supplementing conventional visual census by SCUBA diving or snorkeling. In particular, the Scl-eDNA-M system, which targets mitochondrial 12S rDNA for eDNA amplification, has proven to be an efficient tool for taxonomically identifying nearly all scleractinian genera reported in Japan. Moreover, our recent study showed that Heliopora can also be detected using the Scl-eDNA-M system. This study is the first to examine whether the Scl-eDNA-M is also applicable for the eDNA-based identification of Nanipora . Analyses of primer composition for PCR amplification and metabarcoding of eDNAs sampled from the Kerama Islands confirmed that Nanipora can be detected by this method. Importantly, the results of this study demonstrated that the overall distribution of Nanipora differs from that of Heliopora . Whereas Nanipora was distributed along the coasts of the inland sea of the Keramas, Heliopora occurred mainly along the exposed outer coasts, with only one site hosting both species. Although such differential distributions need to be confirmed by further visual census survey, this study highlights the efficiency of eDNA-M system for broad surveys of zooxanthellate octocorals. Helioporidae Anthozoa eDNA metabarcoding Kerama Islands Independent distribution profiles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Many corals grow on shallow reefs in tropical and subtropical oceans. Although coral reefs occupy only about 0.2% of the ocean’s surface, they are estimated to provide habitat for nearly 30% of all marine organisms (Reaka-Kudla 2001 ; Fisher et al. 2015 ). Thus, coral reefs represent one of the most complex and biodiverse ecosystems on Earth. The primary architects of reefs are zooxanthellate anthozoans, which build resilient reef structures through the secretion of aragonite calcium carbonate skeletons. The major contributors are scleractinians (class Hexacorallia, subphylum Anthozoa), and there are other contributing hermatypic anthozoan groups. For example, the hydrozoan genus Millepora Linnaeus, 1758 is a key reef-building cnidarian, characterized by its internal calcareous skeleton (Lewis 2006 ; Takama et al. 2018 ; Schuchert 2023 ). Similarly, octocorals in the genus Heliopora de Blainville, 1830, also known as blue corals, form extensive colonies across diverse reef habitats (Harii and Kayanne 2003 ; Abe et al. 2008 ; Reimer et al. 2024 ). In addition, the recently described genus Nanipora Miyazaki & Reimer, 2015 has been documented from the shallow reefs of the Kerama Islands, Okinawa, Japan (Miyazaki and Reimer 2015 ; Miyazaki et al. 2016 ; Castelló et al. 2023 ). Nanipora is currently composed of only one species, Nanipora kamurai Miyazaki and Reimer, 2015 , and Nanipora and Heliopora are the two sole extant genera in the family Helioporidae (McFadden et al. 2022 ; Poliseno et al. 2017 ; Yoshioka et al. 2025 ). Both produce an aragonite calcium carbonate skeleton, similar to scleractinians (Miyazaki and Reimer 2015 ). Hexacoral scleractinians are primary and major architects of coral reefs. Therefore, most studies of coral reef ecosystem have focused solely on scleractinians. The investigation of scleractinians themselves is challenging, since the order Scleractinia is composed of more than 30 families, at least 249 genera and at least 1726 extant species (WoRMS, September 2025). However, given the recent decline of coral reefs caused by climate changes and other issues, research needs to focus not solely on scleractinians but also on other understudied hermatypic cnidarians such as Helioporidae and Milleporidae. Ecological and taxonomic studies of reef-building corals have traditionally been conducted through SCUBA diving or snorkeling by coral researchers, leading to a substantial accumulation of knowledge about corals. To supplement the visual census method, the eDNA metabarcoding (eDNA-M) technique has been introduced for detecting and taxonomically identifying zooxanthellate scleractinians (Nichols and Marko 2019 ; Alexander et al. 2020 ; Shinzato et al. 2021 ; Nishitsuji et al. 2023 ; Ip Y et al. 2023; Noda et al. 2025 ). This approach involves several fundamental processes, including collecting surface seawater over reefs, followed by DNA filtration and PCR amplification of eDNA with coral-specific primers. The resulting amplicons are sequenced, and sequence variations are used for identifying coral species or genera by referencing pre-existing sequences in official or custom databases. Combined with the visual census, the eDNA-M technique offers a more broad and straightforward approach to taxonomically identifying scleractinians at the genus or species level (Gösser et al. 2023 ; Nishitsuji et al. 2023 ). Partial sequences of mitochondrial cytochrome c oxidase subunit I (COI), 16S rDNA, internal transcribed spacer 2 (ITS2), 16S rDNA or 12rDNA have been used for coral eDNA detection (Nichols and Marko 2019 ; Alexander et al. 2020 ; Dugal et al. 2021; Shinzato et al. 2021 ), and further technical improvements are essential in getting highly reliable results. Our previous studies have developed a scleractinian-specific eDNA-M method targeting 12S rDNA for eDNA sequence-based comparison (Shinzato et al. 2021 ; Nishitsuji et al. 2023 ). Hisata et al. ( 2025 ) established a system called the Scl-eDNA-M, capable of detecting and identifying 82 of the 85 scleractinian genera recorded along the coasts of Japan. It is expected that the incorporation of Heliopora, Nanipora and/or Millepora into eDNA-M surveys alongside scleractinians would significantly enhance our understanding of the dynamic changes of hermatypic corals within coral ecosystems. Heliopora spp. are extensively distributed throughout the Indo-West Pacific, including Australia, Taiwan, and Japan (Zann and Bolton 1985 ; Babcock 1990 ; Liu et al. 2004 ; Nakabayashi et al. 2017 ; Taninaka et al. 2021 ). Our recent investigation into the applicability of the Scl-eDNA-M system to corals other than scleractinians has shown that the system can identify Heliopora (Satoh et al. under review). Thus, this study was conducted with two aims. Our first aim was to examine whether the Scl-eDNA-M system is applicable to Nanipora – in other words, is eDNA-based detection of Nanipora by the Scl-eDNA-M system possible? If so, researchers should be able to elucidate the distribution of often cryptic Nanipora more widely and easily. Such results would demonstrate the ability of the Scl-eDNA-M system to survey different types of other coral taxa alongside scleractinians. The second aim of this study was to compare habitats between two octocoral genera, Nanipora and Heliopora , along the coast of the Kerama Islands. Heliopora is widely distributed, and it currently includes two valid species, H. coerulea Pallas, 1766 and H. hiberniana Richards, Yasuda, Kikuchi, Foster, Mitsuyuki, Stat, Suyama & Wilson, 2018 (Richards et al. 2018 ; Taninaka et al. 2021 ). H. coerulea is prevalent throughout the West Pacific, while H. hiberniana is mainly distributed in the Indian Ocean (Taninaka et al. 2021 ). In the Kerama Islands, visual census surveys in 2005 recorded a comparatively wide distribution of H. coerulea (Nagata et al. 2006 ). On the other hand, Nanipora records remain rare and thus its entire distribution in the Kerama Islands is not yet known (Miyazaki and Reimer 2015 ; Miyazaki et al. 2016 ; Castelló et al. 2023 ). Recently, the distribution of zooxanthellate scleractinian corals around the Kerama Islands was studied by eDNA-M methods (Noda et al. 2025 ). Taking advantage of sequence data obtained by Noda et al. ( 2025 ), here we carried out the Scl-eDNA-M detections of Nanipora and Heliopora , hoping to determine eDNA-based distributions of both Helioporidae genera, so that we could compare their localized distributions along the coasts of the Kerama Island. From these results, we might obtain clues whether the two genera share habitats, or whether they are distributed rather independently of each other. Materials and methods Assembly of Naniopora kamurai mitochondrial genome Raw sequencing data of the Nanipora kamurai genome (SRA accession ID: SRR27337705; BioProject ID: PRJNA1031203) were retrieved. The complete mitochondrial genome was assembled using GetOrganelle v1.7.7.0 (Jin et al. 2020 ). Mitochondrial genes were annotated with MITOS2 (Bernt et al. 2013 ) and subsequently curated manually. The annotated mitochondrial genome was visualized with OrganellarGenomeDRAW (Greiner et al. 2019 ). The assembled mitochondrial genome of N. kamurai has been deposited in DDBJ/EMBL/GenBank under accession number LC000000 (under deposition). Data utilized in this study The Kerama Islands consist of approximately 20 islands, including Tokashiki, Zamami, Aka, Kuba, Yakabi, Geruma, Fukaji, and Amuro islands, and others (Supplementary Fig. S1 ). The primary data used in this study were derived from our previous eDNA-M survey of the coral reefs of the Kerama Islands, Okinawa, Japan (Noda et al. 2025 ). Boat surveys for seawater collection were conducted on September 14–15, 2023, from 19 coral reef sites (Fig. S1 ). Sampling locations included seven sites at Aka Island (A1–A3, A6, A12, A13, A19), one at Kuba Island (A4), one at Yakabi Island (A5), three at Zamami Island (A7–A9), three at Gahi Island (A14–A16), two at Amuro Island (A17, A18), and two at Tokashiki Island (A10, A11) (Fig. S1 ). At each site, 2 × 1.2 L of surface seawater were collected and filtered through 0.45 µm Sterivex filters (Merck) within 15 min of collection. Each filter was treated with 2 mL of RNAlater (Qiagen) to prevent DNA degradation and stored in a 4°C ice box before being transferred to a − 20°C freezer in the laboratory. After extraction, eDNA was PCR-amplified using the primer set Scle_12S_Fw (5’-CCAGCMGACGCGGTRANACTTA-3’) and Scle_12S_Rv (5’-AAWTTGACGACGGCCATGC-3’) (Shinzato et al. 2021 ). Amplicons were sequenced on the MiSeq platform (Illumina), and the resulting data (SAMD00762493-SAMD00762530 under BioProject ID SSUB029137) were processed to construct the zero-radius operational taxonomic units (ZOTU) as described in Hisata et al. ( 2025 ). Additional sequence data were obtained from the NCBI nucleotide database: Heliopora coerulea (OL616236; Kayal et al. 2013 ; Ip J et al. 2023), Nanipora cf. kamurai (OL616249), the octocoral Parasphaerasclera valdiviae (NC_062023), and the octocoral Ideogorgia capensis (NC_062014). These sequences were used for molecular phylogenetic analysis based on the Maximum Likelihood method, following Hisata et al. ( 2025 ). Metabarcoding detection of eDNA from Nanipora and Heliopora The Scl-eDNA-M system incorporates two steps in the metabarcoding to ensure that recovered eDNA sequences correspond to corals (Hexacorallia, Anthozoa) (Hisata et al. 2025 ). First, sequences are compared against the MIDORI2_LONGEST_NUC_GB259_srRNA_BLAST database ( https://www.reference-midori.info/ ), which includes only high-quality eukaryotic sequences. This step removes contamination from bacteria, fungi, and other non-marine invertebrates. Second, sequences that pass the MIDORI filter are analyzed against a pipeline containing 12S rDNA sequences from 83 scleractinian genera. In this study, however, the focus was on detecting Heliopora and Nanipora (family Helioporidae, class Octocorallia), not scleractinians. Therefore, mitochondrial genome sequence information of Heliopora and Nanipora was incorporated into the MIDORI pipeline. Presence/absence of the two genera was then assessed using the ZOTU methodology (Shinzato et al. 2021 ; Noda et al. 2025 ; Hisata et al. 2025 ). Results Mitochondrial genome of Nanipora kamurai The complete mitochondrial genome of Nanipora kamurai was 18,888 bp in length and circular (Fig. 1 ). Its size was similar to that of Nanipora cf. kamurai (18,892 bp) reported by Muthye et al. ( 2022 ). The genome contained 14 protein-coding genes ( nad1–6 , nad4L , cox1–3 , atp6 , atp8 , cob , and mt-mutS ), two rRNA genes ( rrnL and rrnS ), and one tRNA gene ( trnM ). Several intergenic spacers were also present (Fig. 1 ). The gene order pattern corresponded to type “A,” the most common arrangement in octocorals (Brockman and McFadden 2012 ; Poliseno et al. 2017 ; Yoshioka et al. 2025 ). Mitochondrial 12S rDNA genes and possible amplification by the Scl-eDNA-M system The Scl-eDNA-M system relies on PCR amplification of a 5′ region of the mitochondrial 12S rDNA gene. The forward primer is 22 nucleotides long (5′-CCAGCMGACGCGGTRANACTTA-3′) and the reverse primer is 19 nucleotides long (5′-AAWTTGACGACGGCCATGC-3′) (Fig. 2 ). Our previous study showed that the blue coral genus Heliopora (represented by H. coerulea ) shares sequence similarity in both primer-binding regions with representative scleractinians (Satoh et al. under review). Figure 2 shows alignment and comparison of the Scl_12S_Fw and Scl_12S_Rv primer sequences and the corresponding amplicon regions of Acropora tenuis as a representative scleractinian (van Oppen et al. 2000 ), Heliopora coerulea (Kayal et al. 2013 ), and Nanipora kamurai (from data as described above). The forward primer sequence matched perfectly between Heliopora and Nanipora , although one base difference was observed in Acropora (Fig. 2 ). Similarly, the reverse primer sequence matched perfectly between Heliopora and Nanipora but differed at three positions from Acropora . Based on our previous findings (Satoh et al. under review), we judged it highly likely that the Scl-eDNA-M system could amplify 12S rDNA not only from Heliopora but also from Nanipora . The amplicon sequences of Heliopora and Nanipora showed significant differences, suggesting that the two genera can be easily distinguished by this marker (Fig. 2 ). Detection of eDNA corresponding to Nanipora and Heliopora Since Heliopora eDNA was successfully amplified with the Scl-eDNA-M system, we next examined whether Nanipora eDNA had also been inadvertently amplified and deposited in the dataset of Noda et al. ( 2025 ). Noda et al. ( 2025 ) surveyed 19 coral reef sites around the Kerama Islands in 2023 (Fig. 3 ). Using this dataset, we examined the eDNA-based detection of Nanipora and Heliopora . Both genera were successfully detected by this eDNA-M approach (Table 1 ). Ten of the 19 sites yielded positive results, while nine sites showed no ZOTUs for either genus. Heliopora was detected at four sites: A4, A5, A7, and A18, with ZOTU counts of 35, 12, 49, and 79, respectively (Table 1 ). Nanipora was detected at seven sites: A2, A3, A11, A12, A13, A16, and A18, with ZOTU counts of 34, 124, 427, 39, 74, 1, and 53, respectively (Table 1 ). Although site A16 yielded only one ZOTU for Nanipora , we considered this a positive result. Overall, all positive sites showed substantial ZOTU counts for at least one genus, confirming eDNA-based detection of both Heliopora and Nanipora . Interestingly, most sites were positive for either Heliopora or Nanipora , but not both (Table 1 ; Fig. 3 ). Only site A18 at Amuro Island was positive for both genera, with 79 ZOTUs for Heliopora and 53 for Nanipora (Table 1 ; Fig. 3 ). To confirm the taxonomic assignment of these sequences, four ZOTUs for Heliopora and 11 ZOTUs for Nanipora were randomly selected and analyzed by Maximum Likelihood phylogenetic reconstruction (Fig. 4 ). The resulting tree showed that Heliopora ZOTUs clustered with the reference sequence of H. coerulea (OL616236), while Nanipora ZOTUs clustered with N. cf. kamurai (OL6162491) and N. kamurai (LC00000). Thus, both species were independently detected and identified by this study. Distribution profiles of Nanipora a nd Heliop ora in the Kerama Islands As noted above, the Kerama Islands exhibit unique geographic and environmental features (Taniguchi et al. 1999 ), including an inland sea that produces the exceptional water clarity known as “Kerama blue.” In contrast, strong currents occur on the west side of Kuba and Yakabi islands, as well as on the east side of Tokashiki Island. We examined the distribution of Nanipora and Heliopora in relation to these localities (Fig. 3 ). Sites positive for Nanipora kamurai were A2, A3, A12, and A13 around Aka Island; A16 off Gahi Island; A18 off Amuro Island; and A11 off Tokashiki Island. All of these were “inland sea” sites (Fig. 3 ). In contrast, Heliopora coerulea was detected at three sites: A4 (Kuba Island), A5 (Yakabi Island), and A7 (northwest Zamami Island), all located on the exposed, outer reef sites (Fig. 3 ). The only exception was A18 (Amuro Island), an inland site but along the strait between Tokashiki Island and others, where both genera co-occurred (Fig. 3 ). No data were available for the eastern coast of Tokashiki Island in this study (Fig. 3 ). Overall, the distribution pattern suggests two tendencies: (a) habitats of Nanipora and Heliopora are generally non-overlapping, and (b) Nanipora is associated with more “inland” reefs, whereas Heliopora occurs on the exposed reefs of the Kerama Islands. Discussion Detection of Nanipora by eDNA metabarcoding An aim of this study was to examine whether the Scl-eDNA-M system can detect and identify the octocoral genus Nanipora. Since the system uses mitochondrial 12S rDNA sequence for identification, Nanipora mitochondrial genome sequence is inevitably necessary to pursue this analysis. This study obtained the complete mitochondrial genome of N. kamurai (Fig. 1 ), which had the most common and general gene composition and arrangement pattern reported for octocorals (Brockman and McFadden 2012 ; Poliseno et al. 2017 ; Muthye et al. 2022 ; Yoshioka et al. 2025 ), indicating the assembly was carried out properly. We then identified 12S rDNA sequences of Nanipora and compared its primer and amplicon sequences with that of Acropora tenuis and Heliopora coerulea (Fig. 2 ). It became evident that N. kamurai shares identical primer target sequences (the first 22 and the last 19 nucleotides) to those of H. coerulea (Fig. 2 ). Since our previous study revealed that the Scl-eDNA-M system, including PCR amplification of eDNA and metabarcoding, is efficiently applicable in the detection of H. coerulea , we expected that the system would also be highly applicable to N. kamurai too. A survey of eDNA sequence data collected from coral-reef coast of Kerama Islands resulted in obtaining ZOTUs that corresponded to those of Nanipora (Table 1 ), and to those of Heliopora as well (Table 1 ). The accuracy of Nanipora ZOTU sequences was confirmed by molecular phylogenic analysis using the entire sequence of amplicons obtained (Fig. 4 ). Therefore, this study succeeded in application of the Scl-eDNA-M system in eDNA-based detection of the two genera ( Nanipora and Heliopora ) of the family Helioporidae of the suborder Octocorallia. Our results highlight an advantage of the Scl-eDNA-M system utility, namely the eDNA-based detection of octocoral genera Heliopora and Nanipora along with zooxanthellate scleractinians. This can reduce the time and cost of eDNA-M of zooxanthellate cnidarian corals from two different classes, Hexacorallia and Octocorallia. On the other hand, it should be kept in mind that the efficiency in detection of two types of octocorals by PCR amplification using scleractinian-specific primers may be affected or reduced compared to octocoral-specific primers. Improvements in the design of degenerate primers may help solve such potential problems. The feasibility of this approach is largely due to the conservation of the primer-binding sequences among organisms in amplification step via PCR. Although the reverse primer of the Scl-eDNA-M system has a three-nucleotide difference from Heliopora and Nanipora , this apparently did not significantly affect eDNA detection efficiency for the two octocorals. Nevertheless, we reiterate that this difference may still impact amplification efficiency, potentially affecting the levels of detection compared to scleractinians. Differential distribution of Heliopora and Nanipora along the coast of the Kerama Islands The Kerama Islands consist of approximately 20 islands (Fig. 3 ; Fig. S1 ). These islands are clustered together to form an “inland sea”-like structure that opens to the south. Strong currents flow around the outer edges of the archipelago, particularly on the western and eastern sides due to the Kuroshio, and also possibly, from the southern opening, along the strait between Tokashiki Island and others. In contrast, the inland sea is characterized by relatively calm waters. This combination of environmental and geographic features produces the exceptional water clarity known as “Kerama Blue” and supports a high diversity of corals in the region (Taniguchi et al. 1999 ; Noda et al. 2025 ). Nanipora kamurai was formally described by Miyazaki and Reimer ( 2015 ) from specimens collected from Ama Beach on Zamami Island at a depth of 1 m in July 2012 (Fig. 3 , solid triangle at Zamami Island). A subsequent survey showed that Nanipora colonies were comparatively rare in the Kerama Islands, with additional colonies discovered from the south coast of Shinashiru Island (Miyazaki et al. 2016 , Castelló et al. 2023 ) (Fig. 5 a, b). Nanipora forms small encrusting colonies that are mostly observed inside holes and crevices within the coral reef substrate (Castelló et al. 2025 ). This cryptic habitat can likely result in an underestimation of the distribution of Nanipora detected by eDNA in surface water. Nevertheless, this eDNA study showed eDNA-based detection at seven sites in the Kerama Islands, a broader distribution of Nanipora than previously recorded (Table 1 ; Fig. 3 ). For instance, the ZOTU counts for Nanipora reached 427 at site 11, on the southwest side of Tokashiki Island, and 124 at site 3 near Aka Island, suggesting a possible population of Nanipora there. The distribution of Nanipora has recently been studied more broadly across the western Pacific (Castelló et al. 2023 , 2025 ), in which it was discussed that Nanipora is an opportunistic and habitat-generalist octocoral with great plasticity to adapt to a variety of different environments. This variability may support the hypothesis that Nanipora comprises multiple species. Additionally, a potential ecological role of Nanipora was revealed, as it often grows in close association with various algae groups such as turf algae, macroalgae, crustose coralline algae, and Cyanobacteria (Castelló et al. 2025 ). Further research should investigate Nanipora distribution and ecology in Kerama Islands, with the new suggestions obtained by this study. In contrast to Nanipora kamurai , Heliopora coerulea colonies can reach many meters in size (Planck et al. 1988 , Abe et al. 2008 , Reimer et al. 2024 ), and thus easily distinguishable by visual census survey. Indeed, a broad survey carried out in 2005 revealed extensively wide distribution of Heliopora , as well as several scleractinian species, in the Kerama Island (Fig. 5 c, d; Fig. 6 ) (Nagata et al. 2006 ). Interestingly, Heliopora distribution profile was not recorded to be uniform and/or overall, in the Kerama Islands (Fig. 6 ) (Nagata et al. 2006 ). Instead, Heliopora was distributed off Kuba and Yakabi islands, and the eastern coast of Tokashiki Island, but not in inland-sea areas around Zamami and Aka islands as well as western coast of Tokashiki Island (Fig. 6 ). These sites of Heliopora distribution matched well to those demonstrated by this eDNA study (Fig. 3 ), although our study had no data about eastern coast of Tokashiki Island. Among additional three sites of Heliopora distribution shown by 2005 visual census survey, two were off Fukaji Island and a site off Amuro Island (site A18; compare Fig. 3 with Fig. 6 ). Site A18 with Nanipora near Amuro Island is highly likely the same point as shown by Nagata et al. ( 2006 ). An important finding of this eDNA study is difference in the habitat localization between the two zooxanthellate octocorals, Nanipora and Heliopora. Nanipora inhabits the “inland sea” reefs of the Kerama Islands and did not appear along the exposed outer coast of the islands. In contrast, Heliopora was rarely detected in inland reefs but was detected at the exposed coasts of Kerama Islands. Site A18 was an exceptional point due to the occurrence of both genera. It is likely that this site along a strait between Tokashiki island and others has geographic features of both inland sites and open ocean sites (Fig. 6 ). Understanding the ecological and evolutionary mechanisms driving such differential distributions remains intriguing questions that should be addressed in future studies. At the time of our survey, the coral reefs of the Kerama Islands had experienced at least two severe disturbances: bleaching caused by elevated seawater temperatures around 1998, and feeding damage from massive outbreaks of crown-of-thorns starfish (COTS) around 2008. The damage caused by COTS was particularly devastating, with coral coverage on Kerama reefs dropping from 30–80% to an average of 10% (Taniguchi et al. 1999 ; Taniguchi 2010 ). Since then, however, average coral coverage recovered to about 40% by the early 2020s (Terada et al. 2019 ; https://www.biodic.go.jp/moni1000/findings/reports/pdf/2021_coral_reef.pdf ), although many reefs in Okinawa have subsequently suffered damage during the 2024 global bleaching event. In 2014, the Ministry of the Environment, Government of Japan (MEG), designated the “Kerama Islands National Park” as the country’s thirty-first national park in order to preserve these coral reefs. With respect to Heliopora distribution, following the 2005 survey by Nagata et al. ( 2006 ), extensive COTS-related damage occurred, but the reefs gradually recovered over time, leading to the present status shown by this eDNA survey. Heliopora coerulea is an example of a coral species that survives bleaching events (Guzman et al. 2019 ) and COTS out-breaks (Foo et al. 2024 ) better than most scleractinian corals. The observation that the Heliopora distribution profile remained largely unchanged before and after this severe COTS outbreak suggests that coral recovery may tend to restore to pre-disturbance distribution patterns, rather than forming entirely new patterns. This provides a suggestive clue for understanding the processes involved in coral reef formation and maintenance. In other words, most sites of the inland-sea coast of Kerama Islands are occupied by Acropora, Montipora, Cyphastrea, Dipsastraea, Favites, Galaxea, Goniastrea, Herpolitha, Pavona, Pocillopora, Porites and other scleractinians (Taniguchi et al. 1999 ; Nagata et al. 2006 ; Noda et al. 2025 ). It is likely that there was limited space for Heliopora to invade into the inland sea-like areas. In contrast, Nanipora usually forms small encrusting colonies that are mostly observed inside holes and crevices within the coral reef substrate. Such niches were abundant in inland sea-like areas and in gaps among large scleractinian coral colonies. Such ecological or geographical features might allow Nanipora originate and/or inhabit there. In conclusion, this study revealed that the Scl-eDNA-M system is able to detect and identify the zooxanthellate octocoral genus Nanipora together with the other helioporid genus Heliopora. This advance in coral-detection technology suggested that these two Helioporidae genera do not share the same habitats but instead selectively occupy different coral reef environments within the Kerama Islands, highlighting the efficiency of eDNA-M system for broad surveys of zooxanthellate octocorals. The ecological and evolutionary mechanisms driving such differential distributions of two zooxanthellate octocorals remains intriguing questions that should be addressed in future studies. Declarations Acknowledgments We extend our heartfelt thanks to Mayuki Suwa and Haruhi Narisoko for their general assistance with eDNA studies at Marine Genomic Unit of Okinawa Institute of Science and Technology Graduate University (OIST). Dr. Stacy Pirro (Iridian Genomes) is thanked for mitochondrial genome sequence generation. Author contributions NS conceived and designed study. YY, TN and KH carried out sequencing analysis. KH performed bioinformatic analyses. TN and EY incorporated data and discussion points of Heliopora and GMC and JDR Nanipora into the study. NS and JDR prepared the manuscript and all authors commented on it. Funding This study was supported by the JST COI-NEXT project (grant no. JPMPF2205) to OIST and the Okinawa Prefecture Innovation/Ecosystem Joint Research Promotion Program to OIST and OES Conflict of interest declaration We declare that one of the authors of this manuscript (JDR) is on the Editorial Bord of this journal. Data accessibility Electric supplementary material is available online. References Abe M, Ohno M, Kurozumi T, Goto T, Suzuki R, Hasegawa H, Hanawa S, Nakai T, (2008) Report of the Survey of Heliopora coerulea Communities in Oura Bay, Okinawa. (in Japanese) Alexander JB, Bunce M, White N, Wilkinson SP, Adam AAS, Berry T, et al. (2020) Development of a multi-assay approach for monitoring coral diversity using eDNA metabarcoding. 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Frontiers Marine Science 8: 714662. doi: 10.3389/fmars.2021.714662 Terada R, Abe M, Abe T, Aoki M, Dazai A, Endo H, Kamiya M, Kawai H, Kurashima A, Motomura T, Murase N, Sakanishi Y, Shimabukuro H, Tanaka J, Yoshida J, Aoki M (2019) Japan’s nationwide long-term monitoring survey of seaweed communities known as the “Monitoring Sites 1000”: Ten-year overview and future perspectives. Phycological Researc 69: 12–30. https://doi.org/10.1111/pre. 12395 van Oppen MJH, Catmull J, McDonald BJ, Hislop NR, Hagerman PJ, Miller DJ (2000) The Mitochondrial Genome of Acropora tenuis (Cnidaria; Scleractinia) Contains a Large Group I Intron and a Candidate Control Region. J Molecular Evolution 55: 1. DOI: 10.1007/s00239-001-0075-0 WoRMS (2025). Scleractinia. Accessed at: https://www.marinespecies.org/aphia.php?p=taxdetails &id=1363 on 2025-09-11 Yoshioka Y, Kanai M, Gishitomi S, Arakaki N, Koido T, Shinzato C, Inoue J, Nagata T, Satoh N (2025) Extensive mitochondrial genomic analyses reveal dynamic gene order rearrangements in the class Octocorallia (Cnidaria: Anthozoa). Gene Reports 38: 102111. https://doi.org/10.1016/j.genrep.2024.102111 Zann LP, Bolton L (1985) The distribution, abundance and ecology of the blue coral Heliopora coerulea (Pallas) in the Pacific. Coral Reefs 4: 125–134. https://doi.org/10.1007/BF00300871 Additional Declarations Competing interest reported. We declare that one of the authors of this manuscript (JDR) is on the Editorial Bord of this journal. 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16:25:42","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":97155,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure1N.png","url":"https://assets-eu.researchsquare.com/files/rs-7776767/v1/0b9f76e6f7f1eed3c8587a67.png"},{"id":95568249,"identity":"cc3e9e26-612c-4c9c-9a23-13afc58720ec","added_by":"auto","created_at":"2025-11-10 16:25:42","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":119673,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure3N.png","url":"https://assets-eu.researchsquare.com/files/rs-7776767/v1/5aadc2f17faadadc8593a879.png"},{"id":95655668,"identity":"577bb5ee-31c6-4cab-a6d3-fd4c22b4066a","added_by":"auto","created_at":"2025-11-11 16:16:41","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":60298,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure4N.png","url":"https://assets-eu.researchsquare.com/files/rs-7776767/v1/46a04ef25f81a8f4a012a580.png"},{"id":95655911,"identity":"17388932-f3b2-4726-a002-bb9bd8d1723a","added_by":"auto","created_at":"2025-11-11 16:17:13","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":744385,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7776767/v1/ec8cedc3cda79e5264be20a6.png"},{"id":95568262,"identity":"285b0de9-b936-4cf6-9ac3-eff91c0cf9e3","added_by":"auto","created_at":"2025-11-10 16:25:42","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":153680,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7776767/v1/bde337f0c1fd7b17b08f6230.png"},{"id":95568259,"identity":"58fce619-8691-4f53-a40a-11666013e826","added_by":"auto","created_at":"2025-11-10 16:25:42","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":233734,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFirure2N.png","url":"https://assets-eu.researchsquare.com/files/rs-7776767/v1/44f6b280dca5262a34bc22b6.png"},{"id":95568251,"identity":"232424f9-4afc-45c5-a1aa-298d1817c853","added_by":"auto","created_at":"2025-11-10 16:25:42","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":14350,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7776767/v1/b5062a85b74f61e39ac476fd.png"},{"id":95655482,"identity":"74c376ab-91d4-4b01-bbab-00a4a70ef73e","added_by":"auto","created_at":"2025-11-11 16:16:19","extension":"xml","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":125318,"visible":true,"origin":"","legend":"","description":"","filename":"c7bb61a233f2431e9f6d0562edd97ff11structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7776767/v1/4e8e073f147064381a527bb0.xml"},{"id":95568260,"identity":"6b90bbaa-8c33-498d-9533-e81ab09c699a","added_by":"auto","created_at":"2025-11-10 16:25:42","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":137025,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7776767/v1/b661e79f0a24fece591065b8.html"},{"id":95568229,"identity":"105ca190-ecb9-46f5-95f8-e7ef664326a3","added_by":"auto","created_at":"2025-11-10 16:25:42","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":315538,"visible":true,"origin":"","legend":"\u003cp\u003eComplete mitochondrial genome of \u003cem\u003eNanipora kamurai\u003c/em\u003e. Inner grey circle indicates GC content. NADH dehydrogenase genes are shown in yellow, ubiquinol cytochrome \u003cem\u003ec\u003c/em\u003e reductase in light green, cytochrome \u003cem\u003ec\u003c/em\u003e oxidase in pink, ATP synthase in green, tRNAs in blue, and rRNAs in red. The circular genome, 18,888 bp in length, was visualized using OGDRAW.\u003c/p\u003e","description":"","filename":"Figure1N.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7776767/v1/4facbd739a53fe461c9acc43.jpg"},{"id":95568238,"identity":"df0a4812-5a23-41c8-8f74-fc2b3b54aea0","added_by":"auto","created_at":"2025-11-10 16:25:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2789618,"visible":true,"origin":"","legend":"\u003cp\u003eAlignment of mitochondrial 12S rDNA sequences used for PCR amplification in the Scl-eDNA-M analysis. Sequences are shown from top to bottom as follows: Scle-12S-Fw (forward primer, 22 nucleotides), Scle-12S-Rv (reverse primer, 22 nucleotides), and the corresponding sequences from \u003cem\u003eAcropora tenuis\u003c/em\u003e(NC_003522), \u003cem\u003eHeliopora coerulea\u003c/em\u003e (OL616236), and \u003cem\u003eNanipora kamurai\u003c/em\u003e(LC00000). The alignment demonstrates that the forward and reverse primer sequences are conserved across these three genera, supporting the applicability of the Scl-eDNA-M system for detecting the two octocorals. Asterisks indicate nucleotide difference between Acropora and \u003cem\u003eHeliopora/ Nanipora\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Firure2N.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7776767/v1/ba414a34820c643d9f58b1b3.jpg"},{"id":95568234,"identity":"5a764562-cb62-45bb-80be-28e3ef8c1fd7","added_by":"auto","created_at":"2025-11-10 16:25:42","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":669411,"visible":true,"origin":"","legend":"\u003cp\u003eeDNA-based distribution of \u003cem\u003eNanipora kamurai\u003c/em\u003e (red circles) and \u003cem\u003eHeliopora coerulea\u003c/em\u003e (blue circles) at 19 monitoring sites along the coasts of the Kerama Islands. Sites where neither genus was detected are marked with an “X.” The \u003cem\u003eNanipora\u003c/em\u003e record sites at Zamami Island and Aka Archipelagos, are indicated by a black triangle.\u003c/p\u003e","description":"","filename":"Figure3N.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7776767/v1/2024ea57ad249fa4d7d99932.jpg"},{"id":95568231,"identity":"ea49bc39-f9d3-48a1-8f18-a03c43b96679","added_by":"auto","created_at":"2025-11-10 16:25:42","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":218444,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular phylogenetic tree of 15 ZOTUs obtained from this eDNA-M analysis for detecting \u003cem\u003eHeliopora coerulea\u003c/em\u003e (brown) and \u003cem\u003eNanipora kamurai\u003c/em\u003e (blue). The tree was constructed using the 12S rDNA marker and illustrates that the 15 ZOTUs are separated into two distinct groups: one comprising four ZOTUs and the other 11 ZOTUs. Reference sequences include \u003cem\u003eH. coerulea\u003c/em\u003e (OL616236), \u003cem\u003eNanipora cf. kamurai\u003c/em\u003e (OL616249), and \u003cem\u003eN. kamurai\u003c/em\u003e (LC000000). Outgroup sequences used for comparison were \u003cem\u003eParasphaerasclera valdiviae\u003c/em\u003e (NC062023) and \u003cem\u003eIdeogorgia capensis\u003c/em\u003e (NC062914). Raw data for the 15 ZOTUs are available under Project ID PRJDB17825. Each label (e.g., “A18-1_Zotu_434”) indicates the sampling site, biological replicate, and ZOTU number. Notably, A18-1 and A18-2 (yellow highlight) correspond to \u003cem\u003eH. coerulea\u003c/em\u003e and \u003cem\u003eN. kamurai\u003c/em\u003e collected from the same site (site 18), respectively. There was one nucleotide sequence difference between \u003cem\u003eN. cf. kamurai (OL6162491) and N. kamurai (\u003c/em\u003eLC00000\u003cem\u003e) ZOTU, resulting in divergence of the former from the others.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure4N.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7776767/v1/15689e704ff07cf3c500b72c.jpg"},{"id":95568239,"identity":"2b4ba537-47b9-405c-89c0-e0de60052143","added_by":"auto","created_at":"2025-11-10 16:25:42","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2937163,"visible":true,"origin":"","legend":"\u003cp\u003ePhotographs of (a, b) \u003cem\u003eNanipora kamurai \u003c/em\u003eobserved off Shinashiru Island, south of Aka Island and (c, d) \u003cem\u003eHeliopora coerulea\u003c/em\u003e (yellow arrows) at the east reef slope of Amuro Island. These photographs were captured by (a) Dr. G.M. Castelló in 2025 and (b) E. Yamakawa in 2005, respectively.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7776767/v1/c72742cbad97d9301d844bc1.jpg"},{"id":95568233,"identity":"021e245d-0ade-49fd-8f64-830de544e0c9","added_by":"auto","created_at":"2025-11-10 16:25:42","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":434860,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of \u003cem\u003eHeliopora coerulea\u003c/em\u003e at Kerama Islands, surveyed by visual census in 2005 (modified from Nagata et al. 2006). ●, recorded: ✕, not recorded.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7776767/v1/45d89eb8ab86f5f693fa18ed.jpg"},{"id":105223261,"identity":"7bf9b1da-ced4-466f-a16d-e2b147f3b1de","added_by":"auto","created_at":"2026-03-23 16:01:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8230320,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7776767/v1/95882aed-60b6-4509-af31-df1c907ededb.pdf"},{"id":95654566,"identity":"4f3e5ba2-95bd-4f8b-9947-ce700a13379e","added_by":"auto","created_at":"2025-11-11 16:12:28","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1082208,"visible":true,"origin":"","legend":"","description":"","filename":"SupplemantaryFigs.S1S3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7776767/v1/d5736821d397397abec4e2b7.pdf"}],"financialInterests":"Competing interest reported. We declare that one of the authors of this manuscript (JDR) is on the Editorial Bord of this journal.","formattedTitle":"\u003cp\u003eInferring the distribution of octocorals Nanipora and Heliopora along the coast of the Kerama Islands, Okinawa, Japan, by eDNA metabarcoding \u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMany corals grow on shallow reefs in tropical and subtropical oceans. Although coral reefs occupy only about 0.2% of the ocean\u0026rsquo;s surface, they are estimated to provide habitat for nearly 30% of all marine organisms (Reaka-Kudla \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Fisher et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Thus, coral reefs represent one of the most complex and biodiverse ecosystems on Earth. The primary architects of reefs are zooxanthellate anthozoans, which build resilient reef structures through the secretion of aragonite calcium carbonate skeletons. The major contributors are scleractinians (class Hexacorallia, subphylum Anthozoa), and there are other contributing hermatypic anthozoan groups. For example, the hydrozoan genus \u003cem\u003eMillepora\u003c/em\u003e Linnaeus, 1758 is a key reef-building cnidarian, characterized by its internal calcareous skeleton (Lewis \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Takama et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Schuchert \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Similarly, octocorals in the genus \u003cem\u003eHeliopora\u003c/em\u003e de Blainville, 1830, also known as blue corals, form extensive colonies across diverse reef habitats (Harii and Kayanne \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Abe et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Reimer et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In addition, the recently described genus \u003cem\u003eNanipora\u003c/em\u003e Miyazaki \u0026amp; Reimer, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e has been documented from the shallow reefs of the Kerama Islands, Okinawa, Japan (Miyazaki and Reimer \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Miyazaki et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Castell\u0026oacute; et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003eNanipora\u003c/em\u003e is currently composed of only one species, \u003cem\u003eNanipora kamurai\u003c/em\u003e Miyazaki and Reimer, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, and \u003cem\u003eNanipora\u003c/em\u003e and \u003cem\u003eHeliopora\u003c/em\u003e are the two sole extant genera in the family Helioporidae (McFadden et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Poliseno et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yoshioka et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Both produce an aragonite calcium carbonate skeleton, similar to scleractinians (Miyazaki and Reimer \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHexacoral scleractinians are primary and major architects of coral reefs. Therefore, most studies of coral reef ecosystem have focused solely on scleractinians. The investigation of scleractinians themselves is challenging, since the order Scleractinia is composed of more than 30 families, at least 249 genera and at least 1726 extant species (WoRMS, September 2025). However, given the recent decline of coral reefs caused by climate changes and other issues, research needs to focus not solely on scleractinians but also on other understudied hermatypic cnidarians such as Helioporidae and Milleporidae.\u003c/p\u003e\u003cp\u003eEcological and taxonomic studies of reef-building corals have traditionally been conducted through SCUBA diving or snorkeling by coral researchers, leading to a substantial accumulation of knowledge about corals. To supplement the visual census method, the eDNA metabarcoding (eDNA-M) technique has been introduced for detecting and taxonomically identifying zooxanthellate scleractinians (Nichols and Marko \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Alexander et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Shinzato et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Nishitsuji et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ip Y et al. 2023; Noda et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This approach involves several fundamental processes, including collecting surface seawater over reefs, followed by DNA filtration and PCR amplification of eDNA with coral-specific primers. The resulting amplicons are sequenced, and sequence variations are used for identifying coral species or genera by referencing pre-existing sequences in official or custom databases. Combined with the visual census, the eDNA-M technique offers a more broad and straightforward approach to taxonomically identifying scleractinians at the genus or species level (G\u0026ouml;sser et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Nishitsuji et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Partial sequences of mitochondrial cytochrome c oxidase subunit I (COI), 16S rDNA, internal transcribed spacer 2 (ITS2), 16S rDNA or 12rDNA have been used for coral eDNA detection (Nichols and Marko \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Alexander et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Dugal et al. 2021; Shinzato et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and further technical improvements are essential in getting highly reliable results. Our previous studies have developed a scleractinian-specific eDNA-M method targeting 12S rDNA for eDNA sequence-based comparison (Shinzato et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Nishitsuji et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Hisata et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) established a system called the Scl-eDNA-M, capable of detecting and identifying 82 of the 85 scleractinian genera recorded along the coasts of Japan.\u003c/p\u003e\u003cp\u003eIt is expected that the incorporation of \u003cem\u003eHeliopora, Nanipora\u003c/em\u003e and/or \u003cem\u003eMillepora\u003c/em\u003e into eDNA-M surveys alongside scleractinians would significantly enhance our understanding of the dynamic changes of hermatypic corals within coral ecosystems. \u003cem\u003eHeliopora\u003c/em\u003e spp. are extensively distributed throughout the Indo-West Pacific, including Australia, Taiwan, and Japan (Zann and Bolton \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Babcock \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Nakabayashi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Taninaka et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Our recent investigation into the applicability of the Scl-eDNA-M system to corals other than scleractinians has shown that the system can identify \u003cem\u003eHeliopora\u003c/em\u003e (Satoh et al. under review). Thus, this study was conducted with two aims. Our first aim was to examine whether the Scl-eDNA-M system is applicable to \u003cem\u003eNanipora\u003c/em\u003e \u0026ndash; in other words, is eDNA-based detection of \u003cem\u003eNanipora\u003c/em\u003e by the Scl-eDNA-M system possible? If so, researchers should be able to elucidate the distribution of often cryptic \u003cem\u003eNanipora\u003c/em\u003e more widely and easily. Such results would demonstrate the ability of the Scl-eDNA-M system to survey different types of other coral taxa alongside scleractinians.\u003c/p\u003e\u003cp\u003eThe second aim of this study was to compare habitats between two octocoral genera, \u003cem\u003eNanipora\u003c/em\u003e and \u003cem\u003eHeliopora\u003c/em\u003e, along the coast of the Kerama Islands. \u003cem\u003eHeliopora\u003c/em\u003e is widely distributed, and it currently includes two valid species, \u003cem\u003eH. coerulea\u003c/em\u003e Pallas, 1766 and \u003cem\u003eH. hiberniana\u003c/em\u003e Richards, Yasuda, Kikuchi, Foster, Mitsuyuki, Stat, Suyama \u0026amp; Wilson, 2018 (Richards et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Taninaka et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eH. coerulea\u003c/em\u003e is prevalent throughout the West Pacific, while \u003cem\u003eH. hiberniana\u003c/em\u003e is mainly distributed in the Indian Ocean (Taninaka et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the Kerama Islands, visual census surveys in 2005 recorded a comparatively wide distribution of \u003cem\u003eH. coerulea\u003c/em\u003e (Nagata et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). On the other hand, \u003cem\u003eNanipora\u003c/em\u003e records remain rare and thus its entire distribution in the Kerama Islands is not yet known (Miyazaki and Reimer \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Miyazaki et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Castell\u0026oacute; et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRecently, the distribution of zooxanthellate scleractinian corals around the Kerama Islands was studied by eDNA-M methods (Noda et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Taking advantage of sequence data obtained by Noda et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), here we carried out the Scl-eDNA-M detections of \u003cem\u003eNanipora\u003c/em\u003e and \u003cem\u003eHeliopora\u003c/em\u003e, hoping to determine eDNA-based distributions of both Helioporidae genera, so that we could compare their localized distributions along the coasts of the Kerama Island. From these results, we might obtain clues whether the two genera share habitats, or whether they are distributed rather independently of each other.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eAssembly of\u003c/b\u003e \u003cb\u003eNaniopora kamurai\u003c/b\u003e \u003cb\u003emitochondrial genome\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRaw sequencing data of the \u003cem\u003eNanipora kamurai\u003c/em\u003e genome (SRA accession ID: SRR27337705; BioProject ID: PRJNA1031203) were retrieved. The complete mitochondrial genome was assembled using GetOrganelle v1.7.7.0 (Jin et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Mitochondrial genes were annotated with MITOS2 (Bernt et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and subsequently curated manually. The annotated mitochondrial genome was visualized with OrganellarGenomeDRAW (Greiner et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The assembled mitochondrial genome of \u003cem\u003eN. kamurai\u003c/em\u003e has been deposited in DDBJ/EMBL/GenBank under accession number LC000000 (under deposition).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eData utilized in this study\u003c/h2\u003e\u003cp\u003eThe Kerama Islands consist of approximately 20 islands, including Tokashiki, Zamami, Aka, Kuba, Yakabi, Geruma, Fukaji, and Amuro islands, and others (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The primary data used in this study were derived from our previous eDNA-M survey of the coral reefs of the Kerama Islands, Okinawa, Japan (Noda et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Boat surveys for seawater collection were conducted on September 14\u0026ndash;15, 2023, from 19 coral reef sites (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Sampling locations included seven sites at Aka Island (A1\u0026ndash;A3, A6, A12, A13, A19), one at Kuba Island (A4), one at Yakabi Island (A5), three at Zamami Island (A7\u0026ndash;A9), three at Gahi Island (A14\u0026ndash;A16), two at Amuro Island (A17, A18), and two at Tokashiki Island (A10, A11) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). At each site, 2 \u0026times; 1.2 L of surface seawater were collected and filtered through 0.45 \u0026micro;m Sterivex filters (Merck) within 15 min of collection. Each filter was treated with 2 mL of RNAlater (Qiagen) to prevent DNA degradation and stored in a 4\u0026deg;C ice box before being transferred to a \u0026minus;\u0026thinsp;20\u0026deg;C freezer in the laboratory. After extraction, eDNA was PCR-amplified using the primer set Scle_12S_Fw (5\u0026rsquo;-CCAGCMGACGCGGTRANACTTA-3\u0026rsquo;) and Scle_12S_Rv (5\u0026rsquo;-AAWTTGACGACGGCCATGC-3\u0026rsquo;) (Shinzato et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Amplicons were sequenced on the MiSeq platform (Illumina), and the resulting data (SAMD00762493-SAMD00762530 under BioProject ID SSUB029137) were processed to construct the zero-radius operational taxonomic units (ZOTU) as described in Hisata et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAdditional sequence data were obtained from the NCBI nucleotide database: \u003cem\u003eHeliopora coerulea\u003c/em\u003e (OL616236; Kayal et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ip J et al. 2023), \u003cem\u003eNanipora\u003c/em\u003e cf. \u003cem\u003ekamurai\u003c/em\u003e (OL616249), the octocoral \u003cem\u003eParasphaerasclera valdiviae\u003c/em\u003e (NC_062023), and the octocoral \u003cem\u003eIdeogorgia capensis\u003c/em\u003e (NC_062014). These sequences were used for molecular phylogenetic analysis based on the Maximum Likelihood method, following Hisata et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMetabarcoding detection of eDNA from\u003c/b\u003e \u003cb\u003eNanipora\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eHeliopora\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe Scl-eDNA-M system incorporates two steps in the metabarcoding to ensure that recovered eDNA sequences correspond to corals (Hexacorallia, Anthozoa) (Hisata et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). First, sequences are compared against the MIDORI2_LONGEST_NUC_GB259_srRNA_BLAST database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.reference-midori.info/\u003c/span\u003e\u003cspan address=\"https://www.reference-midori.info/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which includes only high-quality eukaryotic sequences. This step removes contamination from bacteria, fungi, and other non-marine invertebrates. Second, sequences that pass the MIDORI filter are analyzed against a pipeline containing 12S rDNA sequences from 83 scleractinian genera. In this study, however, the focus was on detecting \u003cem\u003eHeliopora\u003c/em\u003e and \u003cem\u003eNanipora\u003c/em\u003e (family Helioporidae, class Octocorallia), not scleractinians. Therefore, mitochondrial genome sequence information of \u003cem\u003eHeliopora\u003c/em\u003e and \u003cem\u003eNanipora\u003c/em\u003e was incorporated into the MIDORI pipeline. Presence/absence of the two genera was then assessed using the ZOTU methodology (Shinzato et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Noda et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Hisata et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eMitochondrial genome of\u003c/b\u003e \u003cb\u003eNanipora kamurai\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe complete mitochondrial genome of \u003cem\u003eNanipora kamurai\u003c/em\u003e was 18,888 bp in length and circular (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Its size was similar to that of \u003cem\u003eNanipora cf. kamurai\u003c/em\u003e (18,892 bp) reported by Muthye et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The genome contained 14 protein-coding genes (\u003cem\u003enad1\u0026ndash;6\u003c/em\u003e, \u003cem\u003enad4L\u003c/em\u003e, \u003cem\u003ecox1\u0026ndash;3\u003c/em\u003e, \u003cem\u003eatp6\u003c/em\u003e, \u003cem\u003eatp8\u003c/em\u003e, \u003cem\u003ecob\u003c/em\u003e, and \u003cem\u003emt-mutS\u003c/em\u003e), two rRNA genes (\u003cem\u003errnL\u003c/em\u003e and \u003cem\u003errnS\u003c/em\u003e), and one tRNA gene (\u003cem\u003etrnM\u003c/em\u003e). Several intergenic spacers were also present (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The gene order pattern corresponded to type \u0026ldquo;A,\u0026rdquo; the most common arrangement in octocorals (Brockman and McFadden \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Poliseno et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yoshioka et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eMitochondrial 12S rDNA genes and possible amplification by the Scl-eDNA-M system\u003c/h3\u003e\n\u003cp\u003eThe Scl-eDNA-M system relies on PCR amplification of a 5\u0026prime; region of the mitochondrial 12S rDNA gene. The forward primer is 22 nucleotides long (5\u0026prime;-CCAGCMGACGCGGTRANACTTA-3\u0026prime;) and the reverse primer is 19 nucleotides long (5\u0026prime;-AAWTTGACGACGGCCATGC-3\u0026prime;) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Our previous study showed that the blue coral genus \u003cem\u003eHeliopora\u003c/em\u003e (represented by \u003cem\u003eH. coerulea\u003c/em\u003e) shares sequence similarity in both primer-binding regions with representative scleractinians (Satoh et al. under review).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows alignment and comparison of the Scl_12S_Fw and Scl_12S_Rv primer sequences and the corresponding amplicon regions of \u003cem\u003eAcropora tenuis\u003c/em\u003e as a representative scleractinian (van Oppen et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), \u003cem\u003eHeliopora coerulea\u003c/em\u003e (Kayal et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and \u003cem\u003eNanipora kamurai\u003c/em\u003e (from data as described above). The forward primer sequence matched perfectly between \u003cem\u003eHeliopora\u003c/em\u003e and \u003cem\u003eNanipora\u003c/em\u003e, although one base difference was observed in \u003cem\u003eAcropora\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similarly, the reverse primer sequence matched perfectly between \u003cem\u003eHeliopora\u003c/em\u003e and \u003cem\u003eNanipora\u003c/em\u003e but differed at three positions from \u003cem\u003eAcropora\u003c/em\u003e. Based on our previous findings (Satoh et al. under review), we judged it highly likely that the Scl-eDNA-M system could amplify 12S rDNA not only from \u003cem\u003eHeliopora\u003c/em\u003e but also from \u003cem\u003eNanipora\u003c/em\u003e. The amplicon sequences of \u003cem\u003eHeliopora\u003c/em\u003e and \u003cem\u003eNanipora\u003c/em\u003e showed significant differences, suggesting that the two genera can be easily distinguished by this marker (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetection of eDNA corresponding to\u003c/b\u003e \u003cb\u003eNanipora\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eHeliopora\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSince \u003cem\u003eHeliopora\u003c/em\u003e eDNA was successfully amplified with the Scl-eDNA-M system, we next examined whether \u003cem\u003eNanipora\u003c/em\u003e eDNA had also been inadvertently amplified and deposited in the dataset of Noda et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNoda et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) surveyed 19 coral reef sites around the Kerama Islands in 2023 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Using this dataset, we examined the eDNA-based detection of \u003cem\u003eNanipora\u003c/em\u003e and \u003cem\u003eHeliopora\u003c/em\u003e. Both genera were successfully detected by this eDNA-M approach (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Ten of the 19 sites yielded positive results, while nine sites showed no ZOTUs for either genus. \u003cem\u003eHeliopora\u003c/em\u003e was detected at four sites: A4, A5, A7, and A18, with ZOTU counts of 35, 12, 49, and 79, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eNanipora\u003c/em\u003e was detected at seven sites: A2, A3, A11, A12, A13, A16, and A18, with ZOTU counts of 34, 124, 427, 39, 74, 1, and 53, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although site A16 yielded only one ZOTU for \u003cem\u003eNanipora\u003c/em\u003e, we considered this a positive result. Overall, all positive sites showed substantial ZOTU counts for at least one genus, confirming eDNA-based detection of both \u003cem\u003eHeliopora\u003c/em\u003e and \u003cem\u003eNanipora\u003c/em\u003e. Interestingly, most sites were positive for either \u003cem\u003eHeliopora\u003c/em\u003e or \u003cem\u003eNanipora\u003c/em\u003e, but not both (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Only site A18 at Amuro Island was positive for both genera, with 79 ZOTUs for \u003cem\u003eHeliopora\u003c/em\u003e and 53 for \u003cem\u003eNanipora\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cimg 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\" width=\"609\" height=\"166\"\u003e\u003c/p\u003e\u003cp\u003eTo confirm the taxonomic assignment of these sequences, four ZOTUs for \u003cem\u003eHeliopora\u003c/em\u003e and 11 ZOTUs for \u003cem\u003eNanipora\u003c/em\u003e were randomly selected and analyzed by Maximum Likelihood phylogenetic reconstruction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The resulting tree showed that \u003cem\u003eHeliopora\u003c/em\u003e ZOTUs clustered with the reference sequence of \u003cem\u003eH. coerulea\u003c/em\u003e (OL616236), while \u003cem\u003eNanipora\u003c/em\u003e ZOTUs clustered with \u003cem\u003eN.\u003c/em\u003e cf. \u003cem\u003ekamurai\u003c/em\u003e (OL6162491) and \u003cem\u003eN. kamurai\u003c/em\u003e (LC00000). Thus, both species were independently detected and identified by this study.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eDistribution profiles of\u003c/b\u003e \u003cb\u003eNanipora\u003c/b\u003e a\u003cb\u003end\u003c/b\u003e \u003cb\u003eHeliop\u003c/b\u003e\u003cb\u003eora in the Kerama Islands\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs noted above, the Kerama Islands exhibit unique geographic and environmental features (Taniguchi et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), including an inland sea that produces the exceptional water clarity known as \u0026ldquo;Kerama blue.\u0026rdquo; In contrast, strong currents occur on the west side of Kuba and Yakabi islands, as well as on the east side of Tokashiki Island. We examined the distribution of \u003cem\u003eNanipora\u003c/em\u003e and \u003cem\u003eHeliopora\u003c/em\u003e in relation to these localities (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Sites positive for \u003cem\u003eNanipora kamurai\u003c/em\u003e were A2, A3, A12, and A13 around Aka Island; A16 off Gahi Island; A18 off Amuro Island; and A11 off Tokashiki Island. All of these were \u0026ldquo;inland sea\u0026rdquo; sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In contrast, \u003cem\u003eHeliopora coerulea\u003c/em\u003e was detected at three sites: A4 (Kuba Island), A5 (Yakabi Island), and A7 (northwest Zamami Island), all located on the exposed, outer reef sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The only exception was A18 (Amuro Island), an inland site but along the strait between Tokashiki Island and others, where both genera co-occurred (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). No data were available for the eastern coast of Tokashiki Island in this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOverall, the distribution pattern suggests two tendencies: (a) habitats of \u003cem\u003eNanipora\u003c/em\u003e and \u003cem\u003eHeliopora\u003c/em\u003e are generally non-overlapping, and (b) \u003cem\u003eNanipora\u003c/em\u003e is associated with more \u0026ldquo;inland\u0026rdquo; reefs, whereas \u003cem\u003eHeliopora\u003c/em\u003e occurs on the exposed reefs of the Kerama Islands.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cb\u003eDetection of\u003c/b\u003e \u003cb\u003eNanipora\u003c/b\u003e \u003cb\u003eby eDNA metabarcoding\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAn aim of this study was to examine whether the Scl-eDNA-M system can detect and identify the octocoral genus \u003cem\u003eNanipora.\u003c/em\u003e Since the system uses mitochondrial 12S rDNA sequence for identification, \u003cem\u003eNanipora\u003c/em\u003e mitochondrial genome sequence is inevitably necessary to pursue this analysis. This study obtained the complete mitochondrial genome of \u003cem\u003eN. kamurai\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which had the most common and general gene composition and arrangement pattern reported for octocorals (Brockman and McFadden \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Poliseno et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Muthye et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yoshioka et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), indicating the assembly was carried out properly.\u003c/p\u003e\u003cp\u003eWe then identified 12S rDNA sequences of \u003cem\u003eNanipora\u003c/em\u003e and compared its primer and amplicon sequences with that of \u003cem\u003eAcropora tenuis\u003c/em\u003e and \u003cem\u003eHeliopora coerulea\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). It became evident that \u003cem\u003eN. kamurai\u003c/em\u003e shares identical primer target sequences (the first 22 and the last 19 nucleotides) to those of \u003cem\u003eH. coerulea\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Since our previous study revealed that the Scl-eDNA-M system, including PCR amplification of eDNA and metabarcoding, is efficiently applicable in the detection of \u003cem\u003eH. coerulea\u003c/em\u003e, we expected that the system would also be highly applicable to \u003cem\u003eN. kamurai\u003c/em\u003e too. A survey of eDNA sequence data collected from coral-reef coast of Kerama Islands resulted in obtaining ZOTUs that corresponded to those of \u003cem\u003eNanipora\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and to those of \u003cem\u003eHeliopora\u003c/em\u003e as well (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The accuracy of \u003cem\u003eNanipora\u003c/em\u003e ZOTU sequences was confirmed by molecular phylogenic analysis using the entire sequence of amplicons obtained (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Therefore, this study succeeded in application of the Scl-eDNA-M system in eDNA-based detection of the two genera (\u003cem\u003eNanipora\u003c/em\u003e and \u003cem\u003eHeliopora\u003c/em\u003e) of the family Helioporidae of the suborder Octocorallia.\u003c/p\u003e\u003cp\u003eOur results highlight an advantage of the Scl-eDNA-M system utility, namely the eDNA-based detection of octocoral genera \u003cem\u003eHeliopora\u003c/em\u003e and \u003cem\u003eNanipora\u003c/em\u003e along with zooxanthellate scleractinians. This can reduce the time and cost of eDNA-M of zooxanthellate cnidarian corals from two different classes, Hexacorallia and Octocorallia. On the other hand, it should be kept in mind that the efficiency in detection of two types of octocorals by PCR amplification using scleractinian-specific primers may be affected or reduced compared to octocoral-specific primers. Improvements in the design of degenerate primers may help solve such potential problems. The feasibility of this approach is largely due to the conservation of the primer-binding sequences among organisms in amplification step via PCR. Although the reverse primer of the Scl-eDNA-M system has a three-nucleotide difference from \u003cem\u003eHeliopora\u003c/em\u003e and \u003cem\u003eNanipora\u003c/em\u003e, this apparently did not significantly affect eDNA detection efficiency for the two octocorals. Nevertheless, we reiterate that this difference may still impact amplification efficiency, potentially affecting the levels of detection compared to scleractinians.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDifferential distribution of\u003c/b\u003e \u003cb\u003eHeliopora\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eNanipora\u003c/b\u003e \u003cb\u003ealong the coast of the Kerama Islands\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe Kerama Islands consist of approximately 20 islands (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These islands are clustered together to form an \u0026ldquo;inland sea\u0026rdquo;-like structure that opens to the south. Strong currents flow around the outer edges of the archipelago, particularly on the western and eastern sides due to the Kuroshio, and also possibly, from the southern opening, along the strait between Tokashiki Island and others. In contrast, the inland sea is characterized by relatively calm waters. This combination of environmental and geographic features produces the exceptional water clarity known as \u0026ldquo;Kerama Blue\u0026rdquo; and supports a high diversity of corals in the region (Taniguchi et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Noda et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eNanipora kamurai\u003c/em\u003e was formally described by Miyazaki and Reimer (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) from specimens collected from Ama Beach on Zamami Island at a depth of 1 m in July 2012 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, solid triangle at Zamami Island). A subsequent survey showed that \u003cem\u003eNanipora\u003c/em\u003e colonies were comparatively rare in the Kerama Islands, with additional colonies discovered from the south coast of Shinashiru Island (Miyazaki et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Castell\u0026oacute; et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). \u003cem\u003eNanipora\u003c/em\u003e forms small encrusting colonies that are mostly observed inside holes and crevices within the coral reef substrate (Castell\u0026oacute; et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This cryptic habitat can likely result in an underestimation of the distribution of \u003cem\u003eNanipora\u003c/em\u003e detected by eDNA in surface water. Nevertheless, this eDNA study showed eDNA-based detection at seven sites in the Kerama Islands, a broader distribution of \u003cem\u003eNanipora\u003c/em\u003e than previously recorded (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). For instance, the ZOTU counts for \u003cem\u003eNanipora\u003c/em\u003e reached 427 at site 11, on the southwest side of Tokashiki Island, and 124 at site 3 near Aka Island, suggesting a possible population of \u003cem\u003eNanipora\u003c/em\u003e there. The distribution of \u003cem\u003eNanipora\u003c/em\u003e has recently been studied more broadly across the western Pacific (Castell\u0026oacute; et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), in which it was discussed that \u003cem\u003eNanipora\u003c/em\u003e is an opportunistic and habitat-generalist octocoral with great plasticity to adapt to a variety of different environments. This variability may support the hypothesis that \u003cem\u003eNanipora\u003c/em\u003e comprises multiple species. Additionally, a potential ecological role of \u003cem\u003eNanipora\u003c/em\u003e was revealed, as it often grows in close association with various algae groups such as turf algae, macroalgae, crustose coralline algae, and Cyanobacteria (Castell\u0026oacute; et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Further research should investigate \u003cem\u003eNanipora\u003c/em\u003e distribution and ecology in Kerama Islands, with the new suggestions obtained by this study.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn contrast to \u003cem\u003eNanipora kamurai\u003c/em\u003e, \u003cem\u003eHeliopora coerulea\u003c/em\u003e colonies can reach many meters in size (Planck et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1988\u003c/span\u003e, Abe et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Reimer et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and thus easily distinguishable by visual census survey. Indeed, a broad survey carried out in 2005 revealed extensively wide distribution of \u003cem\u003eHeliopora\u003c/em\u003e, as well as several scleractinian species, in the Kerama Island (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) (Nagata et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Interestingly, \u003cem\u003eHeliopora\u003c/em\u003e distribution profile was not recorded to be uniform and/or overall, in the Kerama Islands (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) (Nagata et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Instead, \u003cem\u003eHeliopora\u003c/em\u003e was distributed off Kuba and Yakabi islands, and the eastern coast of Tokashiki Island, but not in inland-sea areas around Zamami and Aka islands as well as western coast of Tokashiki Island (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These sites of \u003cem\u003eHeliopora\u003c/em\u003e distribution matched well to those demonstrated by this eDNA study (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), although our study had no data about eastern coast of Tokashiki Island. Among additional three sites of \u003cem\u003eHeliopora\u003c/em\u003e distribution shown by 2005 visual census survey, two were off Fukaji Island and a site off Amuro Island (site A18; compare Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e with Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Site A18 with \u003cem\u003eNanipora\u003c/em\u003e near Amuro Island is highly likely the same point as shown by Nagata et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAn important finding of this eDNA study is difference in the habitat localization between the two zooxanthellate octocorals, \u003cem\u003eNanipora\u003c/em\u003e and \u003cem\u003eHeliopora. Nanipora\u003c/em\u003e inhabits the \u0026ldquo;inland sea\u0026rdquo; reefs of the Kerama Islands and did not appear along the exposed outer coast of the islands. In contrast, \u003cem\u003eHeliopora\u003c/em\u003e was rarely detected in inland reefs but was detected at the exposed coasts of Kerama Islands. Site A18 was an exceptional point due to the occurrence of both genera. It is likely that this site along a strait between Tokashiki island and others has geographic features of both inland sites and open ocean sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Understanding the ecological and evolutionary mechanisms driving such differential distributions remains intriguing questions that should be addressed in future studies.\u003c/p\u003e\u003cp\u003eAt the time of our survey, the coral reefs of the Kerama Islands had experienced at least two severe disturbances: bleaching caused by elevated seawater temperatures around 1998, and feeding damage from massive outbreaks of crown-of-thorns starfish (COTS) around 2008. The damage caused by COTS was particularly devastating, with coral coverage on Kerama reefs dropping from 30\u0026ndash;80% to an average of 10% (Taniguchi et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Taniguchi \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Since then, however, average coral coverage recovered to about 40% by the early 2020s (Terada et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.biodic.go.jp/moni1000/findings/reports/pdf/2021_coral_reef.pdf\u003c/span\u003e\u003cspan address=\"https://www.biodic.go.jp/moni1000/findings/reports/pdf/2021_coral_reef.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), although many reefs in Okinawa have subsequently suffered damage during the 2024 global bleaching event. In 2014, the Ministry of the Environment, Government of Japan (MEG), designated the \u0026ldquo;Kerama Islands National Park\u0026rdquo; as the country\u0026rsquo;s thirty-first national park in order to preserve these coral reefs. With respect to \u003cem\u003eHeliopora\u003c/em\u003e distribution, following the 2005 survey by Nagata et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), extensive COTS-related damage occurred, but the reefs gradually recovered over time, leading to the present status shown by this eDNA survey. \u003cem\u003eHeliopora coerulea\u003c/em\u003e is an example of a coral species that survives bleaching events (Guzman et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and COTS out-breaks (Foo et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) better than most scleractinian corals. The observation that the \u003cem\u003eHeliopora\u003c/em\u003e distribution profile remained largely unchanged before and after this severe COTS outbreak suggests that coral recovery may tend to restore to pre-disturbance distribution patterns, rather than forming entirely new patterns. This provides a suggestive clue for understanding the processes involved in coral reef formation and maintenance.\u003c/p\u003e\u003cp\u003eIn other words, most sites of the inland-sea coast of Kerama Islands are occupied by \u003cem\u003eAcropora, Montipora, Cyphastrea, Dipsastraea, Favites, Galaxea, Goniastrea, Herpolitha, Pavona, Pocillopora, Porites\u003c/em\u003e and other scleractinians (Taniguchi et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Nagata et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Noda et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). It is likely that there was limited space for \u003cem\u003eHeliopora\u003c/em\u003e to invade into the inland sea-like areas. In contrast, \u003cem\u003eNanipora\u003c/em\u003e usually forms small encrusting colonies that are mostly observed inside holes and crevices within the coral reef substrate. Such niches were abundant in inland sea-like areas and in gaps among large scleractinian coral colonies. Such ecological or geographical features might allow \u003cem\u003eNanipora\u003c/em\u003e originate and/or inhabit there.\u003c/p\u003e\u003cp\u003eIn conclusion, this study revealed that the Scl-eDNA-M system is able to detect and identify the zooxanthellate octocoral genus \u003cem\u003eNanipora\u003c/em\u003e together with the other helioporid genus \u003cem\u003eHeliopora.\u003c/em\u003e This advance in coral-detection technology suggested that these two Helioporidae genera do not share the same habitats but instead selectively occupy different coral reef environments within the Kerama Islands, highlighting the efficiency of eDNA-M system for broad surveys of zooxanthellate octocorals. The ecological and evolutionary mechanisms driving such differential distributions of two zooxanthellate octocorals remains intriguing questions that should be addressed in future studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003eWe extend our heartfelt thanks to Mayuki Suwa and Haruhi Narisoko for their general assistance with eDNA studies at Marine Genomic Unit of Okinawa Institute of Science and Technology Graduate University (OIST). Dr. Stacy Pirro (Iridian Genomes) is thanked for mitochondrial genome sequence generation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eNS conceived and designed study. YY, TN and KH carried out sequencing analysis. KH performed bioinformatic analyses.\u003c/p\u003e\n\u003cp\u003eTN and EY incorporated data and discussion points of \u003cem\u003eHeliopora\u003c/em\u003e and GMC and JDR \u003cem\u003eNanipora\u003c/em\u003e into the study. NS and JDR prepared the manuscript and all authors commented on it.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis study was supported by the JST COI-NEXT project (grant no. JPMPF2205) to OIST and the Okinawa Prefecture Innovation/Ecosystem Joint Research Promotion Program to OIST and OES\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest declaration\u0026nbsp;\u003c/strong\u003eWe declare that one of the authors of this manuscript (JDR) is on the Editorial Bord of this journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData accessibility\u0026nbsp;\u003c/strong\u003eElectric supplementary material is available online.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbe M, Ohno M, Kurozumi T, Goto T, Suzuki R, Hasegawa H, Hanawa S, Nakai T, (2008) Report of the Survey of \u003cem\u003eHeliopora coerulea\u003c/em\u003e Communities in Oura Bay, Okinawa. (in Japanese)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlexander JB, Bunce M, White N, Wilkinson SP, Adam AAS, Berry T, et al. 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Accessed at: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.marinespecies.org/aphia.php?p=taxdetails\u003c/span\u003e\u003cspan address=\"https://www.marinespecies.org/aphia.php?p=taxdetails\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u0026amp;id=1363 on 2025-09-11\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYoshioka Y, Kanai M, Gishitomi S, Arakaki N, Koido T, Shinzato C, Inoue J, Nagata T, Satoh N (2025) Extensive mitochondrial genomic analyses reveal dynamic gene order rearrangements in the class Octocorallia (Cnidaria: Anthozoa). 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Coral Reefs 4: 125\u0026ndash;134. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF00300871\u003c/span\u003e\u003cspan address=\"10.1007/BF00300871\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"coral-reefs","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"core","sideBox":"Learn more about [Coral Reefs](http://link.springer.com/journal/338)","snPcode":"338","submissionUrl":"https://submission.nature.com/new-submission/338/3","title":"Coral Reefs","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Helioporidae, Anthozoa, eDNA metabarcoding, Kerama Islands, Independent distribution profiles","lastPublishedDoi":"10.21203/rs.3.rs-7776767/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7776767/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTwo zooxanthellate octocorals, \u003cem\u003eHeliopora coerulea\u003c/em\u003e and \u003cem\u003eNanipora kamurai\u003c/em\u003e (family Helioporidae, order Scleralcyonacea, class Octocorallia), produce aragonite calcium carbonate skeletons similar to those of scleractinian hexacorals and contribute to the formation of complex coral reef ecosystems. \u003cem\u003eH. coerulea\u003c/em\u003e is known to occur along the coasts of the Kerama Islands, Okinawa, Japan and \u003cem\u003eN. kamurai\u003c/em\u003e was originally described from the Kerama Islands. An environmental DNA metabarcoding (eDNA-M) method has recently been introduced for comprehensive surveys of zooxanthellate scleractinian corals, supplementing conventional visual census by SCUBA diving or snorkeling. In particular, the Scl-eDNA-M system, which targets mitochondrial 12S rDNA for eDNA amplification, has proven to be an efficient tool for taxonomically identifying nearly all scleractinian genera reported in Japan. Moreover, our recent study showed that \u003cem\u003eHeliopora\u003c/em\u003e can also be detected using the Scl-eDNA-M system. This study is the first to examine whether the Scl-eDNA-M is also applicable for the eDNA-based identification of \u003cem\u003eNanipora\u003c/em\u003e. Analyses of primer composition for PCR amplification and metabarcoding of eDNAs sampled from the Kerama Islands confirmed that \u003cem\u003eNanipora\u003c/em\u003e can be detected by this method. Importantly, the results of this study demonstrated that the overall distribution of \u003cem\u003eNanipora\u003c/em\u003e differs from that of \u003cem\u003eHeliopora\u003c/em\u003e. Whereas \u003cem\u003eNanipora\u003c/em\u003e was distributed along the coasts of the inland sea of the Keramas, \u003cem\u003eHeliopora\u003c/em\u003e occurred mainly along the exposed outer coasts, with only one site hosting both species. Although such differential distributions need to be confirmed by further visual census survey, this study highlights the efficiency of eDNA-M system for broad surveys of zooxanthellate octocorals.\u003c/p\u003e","manuscriptTitle":"Inferring the distribution of octocorals Nanipora and Heliopora along the coast of the Kerama Islands, Okinawa, Japan, by eDNA metabarcoding","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-10 16:25:37","doi":"10.21203/rs.3.rs-7776767/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-27T15:59:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-29T16:04:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"243181935926971959968348434129900441131","date":"2026-01-22T07:48:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"332824905487309319995392903568298154037","date":"2026-01-21T15:58:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3622761819620001674494845823195365979","date":"2025-11-03T12:15:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"291520866741567649793671939744341627834","date":"2025-10-31T14:09:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-29T13:30:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-14T15:02:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-06T06:47:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Coral Reefs","date":"2025-10-04T01:32:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"coral-reefs","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"core","sideBox":"Learn more about [Coral Reefs](http://link.springer.com/journal/338)","snPcode":"338","submissionUrl":"https://submission.nature.com/new-submission/338/3","title":"Coral Reefs","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8fabd934-8622-4747-9da8-60dee10d9516","owner":[],"postedDate":"November 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T16:00:21+00:00","versionOfRecord":{"articleIdentity":"rs-7776767","link":"https://doi.org/10.1007/s00338-026-02857-4","journal":{"identity":"coral-reefs","isVorOnly":false,"title":"Coral Reefs"},"publishedOn":"2026-03-20 15:57:32","publishedOnDateReadable":"March 20th, 2026"},"versionCreatedAt":"2025-11-10 16:25:37","video":"","vorDoi":"10.1007/s00338-026-02857-4","vorDoiUrl":"https://doi.org/10.1007/s00338-026-02857-4","workflowStages":[]},"version":"v1","identity":"rs-7776767","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7776767","identity":"rs-7776767","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}