Coral reef ecosystem consists of a flexible consortium of eukaryotic and prokaryotic organisms as a holobiont and are rich in compounds for interacting each other as cross talks, protection of their territory and as triggers for these symbiotic association dynamics (Kooperman et al., 2007; Shnit-Orland and Kushmaro, 2009; Kvennefors et al., 2012). Coral hosts large, diverse and specific microbial populations, which have both important beneficial and harmful roles for the host (Ritchie and Smith, 2004; Rosenberg et al., 2007). More specifically, corals provide three habitats for bacteria (the surface mucus layer, coral tissue, and calcium carbonate skeleton) each of which harbors a distinct bacterial population (Koren and Rosenberg, 2006). Among the bacterial populations, vibrios have been recognized as important members of coral holobionts (Rohwer and Kelly, 2004; Vezzulli et al., 2010, 2012; Baker-Austin et al., 2013; Moreira et al., 2014; Rubio-Portillo et al., 2014; Munn, 2015). Some vibrios may establish mutualistic partnership with corals by releasing nutrients and secondary metabolites (Ritchie, 2006; Chimetto et al., 2008, 2009), while others play a major role in the disruption of coral health (Munn, 2015).

Coral and coral reefs are in steep decline worldwide due to the combined effects of various stressors such as global warming, pollution, overfishing and infectious diseases (Hughes et al., 2003; Weil et al., 2006; Dinsdale et al., 2008; Vezzulli et al., 2012; Baker-Austin et al., 2013). Rising seawater temperature related to global climate change is a big threat to coral health and is ultimately linked to increasing coral disease such as bleaching events (Tout et al., 2015). Impressive progress has been reported regarding characterization of the coral microbiota over the last decade with its altered composition often correlated to the appearance of signs and diseases and/or bleaching that ultimately suggest a link between microbes, global coral health and stability of reef ecosystems (Krediet et al., 2013). Among them, coral vibriosis is a well-known disease, of which occurrences are greatly influenced by increasing seawater temperature (Vezzulli et al., 2012; Baker-Austin et al., 2013; Munn, 2015). In particular, at warm temperatures, V. shiloi expresses a cell-surface adhesion protein that is required for bacterial attachment to the coral surface and simultaneously expresses Toxin-P that ultimately inhibits photosynthesis of the coral-endosymbiotic algae and super-oxide dismutase required for survival of this pathogen inside the coral (Rosenberg et al., 2007; Munn, 2015).

The coral reef ecosystem in Ishigaki Island in southwestern Japan is surrounded by well-developed fringing corals with a variety of mangroves and sandy or rocky shores (Sano, 2000; Shimomura and Naruse, 2015). Recently, these reefs have been severely stressed due to typhoons, coral bleaching and soil pollution. Red soil derived turbid seawater in Okinawa, Ishigaki, and Iriomote coral reef seawater adversely affects the photosynthesis of coral symbiont algae (Zooxanthelae) and also interfered with the settlement of coral larvae on the ocean bed which ultimately influenced coral damage (Kakuma and Kamimura, 2011). Due to this, eutrophication including inflow of red soil is an important threat to Ishigaki corals. More specifically, accumulation of red soil and high nutrient inputs due to land development since 1972, have severely affected the Ishigaki coral reef (Hasegawa, 2011). Rivers from the northern part of Ishigaki coral reef, originating from agricultural watershed may have detrimental effects on corals because exposure to silt and nutrient rich sediments may badly stress corals (Weber et al., 2006). In addition, run-off from soils after typhoons can easily damage coral as well (Harii et al., 2014). Presently, several reports are available regarding coral-microbiota from different parts of the world (Rohwer et al., 2002; Wild et al., 2004; Rosenberg et al., 2007; Chimetto et al., 2009; Alves et al., 2010; Krediet et al., 2013; Chimetto Tonon et al., 2015) but little is known about the Ishigaki coral reef microbial communities.

Therefore, the aims of this study are to characterize the Vibrio diversity spatially and temporally, to elucidate which environmental determinants [temperature or dissolved organic carbon (DOC)] correlate the Vibrio diversity dynamics and to characterize antimicrobial resistance profiles of potential coral pathogens.

Ishigaki coral reef ecosystems are known to be the largest and the most diverse reef ecosystems in Japan, but are in now extremely vulnerable. In this era of global warming, the surrounding seawater of coral reefs are one of the most important ecological niches as well as heat carriers. It is worth investigating the evolution of spatiotemporal Vibrio dynamics in oceanic fields not only for the conservation of fragile coral reefs but also for public health and aquaculture issues (Rubio-Portillo et al., 2014; Tout et al., 2015). We performed a 3 years survey to elucidate Vibrio diversity using well-curated pyrH gene sequence set to achieve fine scale discrimination (Thompson J.R. et al., 2005; Thompson et al., 2005a, 2007; Tall et al., 2013; Chimetto Tonon et al., 2015). The pyrH gene set allows us not only to affiliate Vibrio isolates to known described Vibrionaceae species or currently unknown new species candidates but also to elucidate the structure of regional populations of specific species and how they have diverged. Our study demonstrated that the Vibrio community of the coral surrounding seawater consisted of at least 22 known described species and 12 undescribed species candidates (26 isolates) which is likely to be a more diversified Vibrio community compared to those reported from tropical and temperate coral reef environments (Chimetto et al., 2008; Raina et al., 2009; Alves et al., 2010; Chimetto Tonon et al., 2015). Unfortunately, we did not assess the Ishigaki coral reef microbiota due to lack of coral samples, the seawater around Ishigaki coral reefs is likely to share major Vibrionaceae communities of reported microbiomes V. communis/V. owensii, V. mediterranei (V. shiloi), V. harveyi, V. alginolyticus, V. campbellii, V. maritimus/V. variabilis, V. tubiashii, V. coralliilyticus, V. pelagius consisting of coral holobionts (Alves et al., 2010; Chimetto Tonon et al., 2015). High abundance of Harveyi clade (V. hyugaensis, V. communis, V. owensii, V. harveyi, V. campbellii) and lower abundance of Splendidus clade species (V. pelagius) are the specific clues to help define subtropical Ishigaki coral reef microbiota (Hunt et al., 2008; Tall et al., 2013). Coralliilyticus clade species (V. coralliilyticus, V. neptunis), Mediterranei clade species (V. mediterranei, V. maritimus, V. variabillis) and P. rosenbergii are secondary significant members (>5% constitution) of the Ishigaki coral reef seawaters.

The availability of at least 12 novel species candidates (CV172, CV50, C4I174, CV96, CV58, CV39, CV97, C4II282, C121, C4II259, C4II189, and C4V358) in the Ishigaki coral reef ecosystem suggests important evidence of Vibrionaceae evolution. Relevant subsequent studies (Thompson et al., 2005b; Rosenberg et al., 2007; Reis et al., 2009; Moreira et al., 2014; Chimetto Tonon et al., 2015) reported that coral and surrounding seawater are prime sources in isolating novel vibrios and is an important habitat for studying the microevolution of vibrios. Comparing previously recorded vibrios from coral and reef ecosystems (Chimetto et al., 2009; Alves et al., 2010; Chimetto Tonon et al., 2015) we found mostly diversified populations including numerous vibrios unique to our studied reef locations. The retrieved unique Vibrio populations were V. hyugaensis, V. ishigakensis, V. astriarenae, P. rosenbergii, P. damsella subsp. piscicida, P. aphoticum, V. nigripulchritudo, V. alfacsensis, and V. azureus suggesting that Ishigaki coral reef locations are hotspots for versatile and dynamic Vibrio populations and may serve as diversified ecological niches.

This spatiotemporal survey also revealed that the community and population level dynamics in the surrounding seawater of coral holobionts in Ishigaki reef ecosystems can determine generalists and specialists (Figures 2 and 4). V. owensii and V. hyugaensis populations were considered to be the dominant generalist populations in the coral seawater ecosystems. The other prevalent generalists (Supplementary Table S1; Figure 2) in Ishigaki reef seawater were V. variabillis, V. harveyi, V. campbellii, P. rosenbergii, V. coralliilyticus, V. astriarenae, and V. ishigakensis.

Numerous previous reports suggested that vibrios from Harveyi clade appear to have ecologically diversified passively by invading new niches (Chimetto et al., 2008; Preheim et al., 2011; Chimetto Tonon et al., 2015). The observed diversities of V. owensii and V. hyugaensis populations were likely to be higher than those of V. harveyi and V. campbellii (Figure 2). V. owensii was isolated from cultured crustaceans in Australia as a member of Harveyi clade (Cano-Gomez et al., 2010). This species was also isolated from Acropora white syndrome lesions in American Samoa and is considered to be a putative coral pathogen (Ushijima et al., 2012; Wilson et al., 2012) and was also isolated from ‘Montipora capitata’ diseased-coral (Tissue loss disease: Montipora White Syndrome) in Hawaiian coral reefs. V. hyugaensis was isolated from a seawater sample collected in Miyazaki prefecture in Japan (Urbanczyk et al., 2015). It is a luminous Vibrio and several light-producing bacteria were reported from seawater samples taken from different sites in Miyazaki prefecture between 2010 and 2012 (Urbanczyk et al., 2014).

Specialist Vibrio populations in Ishigaki reef seawater showed site-specific distributions (Supplementary Table S1). V. azureus and V. mediterranei were isolated specifically from Osaki whereas, V. nigripulchritudo, V. alfacsensis, and V. orientalis were noted as region-specific in Miyara, Taketomi and Sekisei, respectively. Bacteria as coral holobionts may be host-specific or may display spatial variability which might show protective activities against pathogens or other detrimental agents by occupying entry niches and space (as competitors) or by production of antibiotic compounds (Rohwer et al., 2002; Mullen et al., 2004; Kelman et al., 2006; Ritchie, 2006). Moreover, the mathematical model AdaptML generated the environmental grouping (Supplementary Table S2) of the Ishigaki reef seawater Vibrio populations and detected both ecologically restricted and ecologically distinctive (Hunt et al., 2008) populations. Two proposed novel vibrios isolated from this location V. astriarenae, Agarivorans clade (Al-saari et al., 2015) and V. ishigakensis, Halioticoli clade (Gao et al., 2016) were found in more ecologically diverse populations (Figure 2). The former is agarolytic whereas the latter is non-motile and alginolytic. Benthic flora might determine the specific niches for these species.

One of the more intriguing findings disclosed by this study was the availability of versatile and dominant putative coral pathogenic vibrios, V. owensii and V. coralliilyticus (Supplementary Table S1; Figure 2) occupied 21.6 and 7.2%, respectively in our total analyzed vibrios. This may be due to higher global oceanic temperatures. Temperature influenced pathogenic potential of coral pathogenic vibrios may lead to an increase in incidences of diseases in marine environments. Swimming motility, swarming motility and protease activity have been shown to be related to the virulence of V. owensii (Ushijima et al., 2012). According to Kimes et al. (2012) for V. coralliilyticus, an increase in seawater temperature to more than 27°C plays a direct role in triggering virulence genes and other factors related to host degradation, secretion, antimicrobial resistance, and motility. The mechanism of virulence includes motility and chemotaxis that involve post-colonization with subsequent production of coral tissue damaging ‘zink-metalloproteases’ (Ben-Haim et al., 2003). Recently, Tout et al. (2015) demonstrated that raised-seawater temperature increase natural Vibrio abundances, particularly the notorious coral pathogen V. coralliilyticus, in coral ecosystems. However, our findings on V. coralliilyticus abundance were not significantly correlated to seawater temperature rises. Beyond the previously mentioned potential coral pathogens, we also identified V. harveyi (4.5%) and V. alginolyticus (0.7%) from the seawater. V. alginolyticus was found only in the single habitat H-2 suggesting that this bacterium may have a new ecological niche-partitioning in Ishigaki reef seawater. Other results associated with coral pathogenic vibrios (Luna et al., 2010; Zhenyu et al., 2013; Munn, 2015) show that most of them are opportunistic in nature and show their virulence under certain environmental conditions by overwhelming the host-defense, overgrowth and tissue destruction.

Environmental variables, particularly temperature are considered to be the prominent triggering factor in Vibrio ecology, population dynamics, physiological stress response and evolution (Sato et al., 2009). Elevated temperatures and high loads of nutrients particularly and DOC have direct impacts on coral holobiont microbial communities and ultimately make them diverse, abundant and even niche or host specific (Kline et al., 2006; Paz et al., 2007). Other findings similarly demonstrate that elevated nutrients (i.e., phosphate, nitrate, ammonia) and DOC in coastal waters have been a cause of reef decline (Bruno et al., 2003; Kline et al., 2006). In our assessment, only specific Vibrionaceae populations correlated with environmental parameters (e.g., temperature, DOC). V. campbellii abundance was significantly correlated to rises in seawater temperatures, V. owensii and C6 group of V. hyugaensis showed significant negative correlations with increasing seawater temperature. The optimum growth temperature of V. hyugaensis was reported to be 26°C and it could not grow at 37°C, which might reflect its low tolerance to high temperatures. These findings corroborated previous reports that thermal anomalies and nutrient-rich environment including DOC are well-known ecological triggers, involved in proliferation and increased abundance of holobiont microbial communities in coastal environments globally (Ducklow et al., 1986; Wild et al., 2004; Barott and Rohwer, 2012) along with reef ecosystems in southern Japan (Goto et al., 2010). Roder et al. (2014) also noted that coral associated bacterial communities including potential pathogens emerge and dominate during environmental stress and the shift of bacterial community may be a direct effect of temperature on growth of specific members of the microbial community (Garren et al., 2014).

Our curated data set of Vibrionaceae pyrH gene sequences allows us to perform the first characterization of Vibrio diversity and assessment of spatiotemporal diversity dynamics of Vibrio populations both at community and population levels along with the relationship to environmental determinants from Ishigaki coral reef seawater. The determined Vibrio community of seawater directly connecting to Ishigaki coral holobionts included at least 22 known described species and 12 as yet undescribed species, which are more diversified compared to reported tropical and temperate reef vibrios. Several vibrios such as, V. campbellii, V. owensii and the C6 group of V. hyugaensis abundances seem to correlate with seawater temperature. V. owensii and C6 group of V. hyugaensis show significant negative correlations with increasing seawater temperature but V. campbellii is positively significant in this regard. This study also demonstrates the presence of most of the globally recognized opportunistic potential coral-pathogenic vibrios (V. owensii, V. harveyi, V. alginolyticus). This pioneering study on Ishigaki coral reef seawater vibrios both at community and population levels will be the important gateway for further deep research on vibrios in coral and reef systems.

Conceived and designed the experiments: AA, FT, and TomooS. Performed the experiments: AA, GF, NA, PM, YY, and SM. Analyzed the data: AA, GF, NA, PM, YY, and TomooS. Contributed reagents/materials/analysis tools: AA, GF, NA, PM, YY, SM, FT, TokoS, and TomooS. Wrote the paper: AA and TomooS. Critical review, ideas and suggestion given during manuscript preparation: GF, NA, PM, YY, SM, FT, and TokoS.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.