Spatial distribution and feeding substrate of butterflyfishes (family Chaetodontidae) on an Okinawan coral reef

Study species

Although 45 butterflyfish species were recorded in Okinawan region (Nakabo, 2000), 26 species were found in the study site (Table S1). Among the 26 species, 14 species were examined in the present study, since these species showed higher density. The 14 species include three dietary groups (Table S2): seven species of obligate coral polyp feeders (C. baronessa, C. bennetti, C. lunulatus, C. ornatissimus, C. plebeius, C. punctatofasciatus, and C. trifascialis), six species of facultative coral polyp feeders (C. argentatus, C. auriga, C. citrinellus, C. ephippium, C. kleinii, and C. vagabundus) and one non-coralline invertebrate feeder (Forcipiger flavissimus).

Spatial distribution

Underwater visual observations were conducted at Sekisei Lagoon and Nagura Bay in the Yaeyama Islands, Okinawa, Japan (Figs. 1A and 1B). In order to include almost the total area in the study site, multiple sites with inter-site distance was about 2 km were established. As a result, 68 study sites including 32 sites on exposed reefs and 36 sites on inner reefs were established (Fig. 1C). The observations were conducted between June 2016 and February 2017.

Underwater visual survey was conducted in accordance with Nanami (2018). In brief, at each site, one 20-min observation was conducted along a 5-m wide transect using SCUBA. A portable GPS receiver was used to measure the length of each transect. The average distances covered were 343.1 ± 43.9 m [mean ± standard deviation (SD), minimum length = 233 m maximum length = 439 m]. On each transect, the number of individuals and total length of butterflyfishes were recorded. Then, density of each of the 14 butterflyfish species (number of individuals per 500 m²) was obtained by using above-mentioned data (number of individuals and length of 20-min transect) for each site. Depth profiles were obtained by using a diving computer. The interval of depth data recording was 30-s, providing 40 depth values for each site (two depth points per1 min × 20 min). Then, the 40 depth profiles were averaged for each site. The water depths ranged from 2.6 m to 12.4 m (average ± SD = 7.66 ± 1.95 m).

Spatial variation in substrates

The substrate characteristics at each site were recorded by a digital camera. A digital camera was attached to the data collection board using PVC pipes (Fig. S2 ), making it possible to collect digital images and fish data simultaneously. The distance between the video camera and substrate was about 2 m. Static images were obtained at 10-s intervals, providing 121 static images per 20-min transect. For each image, the substrate occupying the center of the image was identified. Substrates were divided into 17 categories for analysis modified from Nanami (2018): (1) branching Acropora (e.g., A. formosa), (2) bottlebrush Acropora (e.g., A. carduus), (3) tabular Acropora (e.g., A. hyacinthus), (4) corymbose Acropora (e.g., Acropora nasuta), (5) Pocillopora, (6) branching corals other than genera Acropora and Pocillopora. (e.g., branching Montipora and Porites), (7) encrusting corals (e.g., encrusting Pectinidae, Faviidae, Poritidae, Echinophyllia, Hydnophora, Scapophyllia, and Montipora), (8) leafy corals (e.g., Echinopora lamellosa), (9) massive corals (e.g., massive Poritidae, Oculinidae, Faviidae, and Mussidae), (10) mushroom corals, (11) other living corals (e.g., Pavona and Goniopora), (12) soft corals, (13) dead corals, (14) coral rubble, (15) rock (calcium carbonate substratum with lower substrate complexity), (16) sand, and (17) macroalgae (e.g., Padina minor and Sargassum spp.). By using the total number of images for each substrate category, the percent cover for the each of the 17 categories was calculated.

Spatial distribution analysis

To test whether fish density or substrate coverage differed between exposed and inner reefs, a generalized linear mixed model (GLMM) with zero-inflated negative binominal error distribution were performed for each fish species and each substrate. In the analysis, glmmTMB package in R statistical computing language were used (R Core Team, 2017) with site as a random term and reefs as the fixed term. Bonferroni correction was applied to test the significant difference. The relationship between the spatial distribution of the 14 butterflyfish species and environmental characteristics (17 substrates plus depth) was analyzed using redundancy analysis (RDA) using CANOCO software (Ter Braak & Smilauer, 2002). Since time-transect (a 20-min swimming at each site) was applied, one density data and one substrate coverage data were obtained at each site. Prior to the analysis, the fish density data were square-root transformed. Forward selection was applied to identify the environmental characteristics which have significant effects on the spatial distributions of the 14 butterflyfish species.

Feeding behavior

To elucidate the feeding behavior, underwater visual observations were conducted at 21 study sites between June 2016 and February 2020 (Fig. 1D). In order to collect the data of feeding behavior effectively, the 21 sites were selected due to relatively greater density of the 14 species.

Five-minute focal observations of individual fish were made by an observer following at a distance of about 3–5 m. The observer recorded the number of bites on each substrate type and estimated the total length of the individual. At the end of the observation period, a new focal individual was chosen, taking care not to choose the same individual. Most individuals did not change their behavior and continued feeding as the observer approached. However, if individuals changed behavior (e.g., fleeing from the observer) when approached, the observation was aborted. To examine the substrate characteristics, 50 quadrats (50 × 50 cm) were haphazardly placed at least 10 m apart in each site. Substrates within quadrats were photographed using a digital camera. In the laboratory, the photographs of each 50 × 50 cm quadrat were divided into 100 subsections (5 × 5 cm) providing totally 5,000 subsections were obtained at each study site. Then, the above-mentioned 17 categories of substrates in each subsection were identified. For each subsection, only the predominant substrate (>50% in the subsection) was recorded. Finally, the average coverage of each substrate was calculated by dividing the number of subsections containing that substrate by the total number of subsections.

Feeding behavior analysis

For each species, the proportion of bites on a given substrate was calculated for each site and then an average proportion over all sites was calculated. Shannon’s diversity index was applied to assess substrate use diversity after calculating the proportion of all bites for each substrate: ${\displaystyle H^{\prime }=-\sum p_i{\log }_{\mathrm{e}}\left(p_i\right)}$ where H’ is the diversity index, and p_i is the proportion of bites on the i th substrate. To evaluate what types of substrates were used positively or negatively in relation to their availability, Strauss’ linear resource index was used (Strauss, 1979; Smith et al., 2018): ${\displaystyle L_i=r_i-P_i}$ where L_i is Strauss’ linear resource index for the i th substrate, r_i is the proportion of bites on the i th substrate, and P_i is the proportion of the i th substrate in the environment. The indices were calculated for each site separately and then the average L_i value with 95% confidence interval (CI) was obtained for each substrate. The 95% CI was calculated as: ${\displaystyle 95\text{\%}{\mathrm{CI}}_i=t\times \mathrm{standard error of}{L}_i}$ where 95% CI_i is the 95% confidence interval for the i th substrate, and t is the t-statistic with Bonferroni correction (p = 0.05/17). L_i ± 95% CI_i >1 indicates a significant positive use of the i th substrate, and <1 indicates a significant negative use. L_i ± 95% CI_i, including 0 indicates no significant selectivity for the i th substrate. Individuals for which the total number of bites was less than 10 were omitted from the analysis.

To summarize species-specific differences in feeding behavior, a principal component analysis (PCA) was conducted based on the numbers of bites and average L_i values for all substrates.

Spatial distributions

Among the 14 species, C. lunulatus appeared to be the most abundant overall regardless of habitat, and C. baronessa and C. ornatissimus the least abundant (Table S1). Significantly greater densities were found on exposed than on inner reefs for two species (C. citrinellus and F. flavissimus: Fig. 2, Table S3). The remaining twelve species did not differ significantly between exposed and inner reefs. On exposed reefs, there was significantly higher cover of encrusting corals and rock. On inner reefs there was significantly greater coverage of dead corals, coral rubble and sand (Fig. S1, Table S4).

RDA revealed the species-specific spatial distributions among the 14 species (Fig. 3). Four species (C. argentatus, C. vagabundus, C. citrinellus and F. flavissimus) were found at sites with greater coverage of rock, soft corals and Pocillopora. Three species (C. punctatofasciatus, C. kleinii and C. ornatissimus) were found at sites with greater coverage of rock, encrusting corals, massive corals, leafy corals, and greater depth. Three species (C. plebeius, C. baronessa and C. bennetti) were found at sites with greater coverage of encrusting corals, massive corals, and leafy corals. Three species (C. ephippium, C. trifascialis and C. auriga) were found at sites with greater coverage of branching Acropora, bottlebrush Acropora, tabular Acropora, coral rubble and mushroom corals. One species (C. lunulatus) was found at sites with greater coverage of branching Acropora, encrusting corals, and massive corals.

Feeding behavior

Among the seven species of obligate coral polyp feeders, five species had higher H’-values (1.63–1.84) indicating higher feeding plasticity, but selected different coral species. Most of the bites of C. baronessa were directed to tabular Acropora, corymbose Acropora, Pocillopora and massive corals (Fig. 4A). C. lunulatus, C. plebeius, and C. punctatofasciatus directed most of their bites to encrusting corals and massive corals (Figs. 4C, 4E and 4F). C. ornatissimus directed most of its bites toward corymbose Acropora and encrusting corals (Fig. 4D). In contrast, the remaining two species had lower H’-values (1.15–1.41) indicating lower feeding plasticity. C. bennetti directed most of the bites to encrusting corals and massive corals (Fig. 4B), whereas C. trifascialis directed most of the bites to tabular Acropora and corymbose Acropora (Fig. 4G).

Among six species of facultative coral polyp feeders, three species (C. argentatus, C. citrinellus and C. kleinii) had relatively high H’-values (1.52–1.80), indicating high plasticity of substrate use for foraging. However, different coral species were selected among the three species. Most of the bites of C. argentatus were directed to encrusting corals, massive corals and rock (Fig. 5A). C. citrinellus directed most of its bites to corymbose Acropora, encrusting corals, massive corals and rock (Fig. 5C). C. kleinii directed most of its bites to encrusting corals and massive corals (Fig. 5E). In contrast, the remaining three species (C. auriga, C. ephippium and C. vagabundus) exhibited smaller H′-values (1.01–1.23), indicating relatively limited substrate use. C. auriga directed most of its bites towards coral rubble and rock (Fig. 5B). C. ephippium directed most of its bites to dead corals and rock (Fig. 5D). Most of bites of C. argentatus were directed to rock (Fig. F).

Non-coralline invertebrate feeder (F. flavissimus) exhibited smaller H’-value (1.29), indicating relatively limited substrate use. Most of the bites of F. flavissimus were directed to rock, but feeding bites were also notable on tabular Acropora, corymbose Acropora and Pocillopora (Fig. 5G).

Substrate preference

Among obligate coral polyp feeders, C. baronessa exhibited significant positive use of tabular Acropora, corymbose Acropora and massive corals (Fig. 6A). Four species ( C. bennetti, C. lunulatus, C. plebeius and C. punctatofasciatus) showed significant positive use of encrusting corals and massive corals (Figs. 6B, 6C, 6E and 6F). C. ornatissimus showed significant positive use of corymbose Acropora and encrusting corals (Fig. 6D). In contrast, C. trifascialis showed significant positive use of tabular Acropora and corymbose Acropora (Fig. 6G). All seven species exhibited significant negative use of dead corals and rocks. Most species showed significant negative use of soft corals and coral rubble.

For the facultative coral polyp feeders, three species (C. argentatus, C. auriga and C. vagabundus) showed no significant positive use of any substrate (Figs. 7A, 7B and 7F). C. citrinellus and C. kleinii showed significant positive use of encrusting corals and massive corals and significant negative use of rock (Figs. 7C and 7E). C. ephippium showed significant positive use of dead corals (Fig. 7D).

For the non-coralline invertebrate feeder (F. flavissimus), no significant positive use was found for any substrate (Fig. 7G). However, a positive tendency were found for tabular Acropora and corymbose Acropora.

Overall trends in feeding behavior

For the number of bites, PCA identified that six species (C. bennetti, C. lunulatus, C. plebeius, C. punctatofasciatus, C. citrinellus and C. kleinii) exhibited similar behavioral characteristics showing greater number of bites on encrusting corals and massive corals (Figs. 8A and 8B), whereas two species (C. baronessa and C. ornatissimus) directed most of their bites to encrusting corals or massive corals with somewhat frequent bites on branching Acropora, Pocillopora, corymbose Acropora and tabular Acropora. C. trifascialis directed most of its bites to corymbose Acropora and tabular Acropora. Five species (C. argentatus, C. auriga, C. ephippium, C. vagabundus and F. flavissimus) exhibited similar behavioral characteristics showing greater numbers of bites to rock, dead corals, and coral rubble.

For substrate selection, eight species (C. bennetti, C. lunulatus, C. plebeius, C. punctatofasciatus, C. citrinellus, C. baronessa, C. ornatissimus and C. kleinii) exhibited similar trends showing positive use of encrusting corals and massive corals (Figs. 8C and 8D). In contrast, five species (C. argentatus, C. auriga, C. ephippium, C. vagabundus and F. flavissimus) exhibited similar trends showing positive use of rock, dead corals and coral rubble. The trend of substrate selection of C. trifascialis was very different from the other 14 species, showing positive use of corymbose Acropora and tabular Acropora.

Relationship between spatial distribution and feeding substrates

Some previous studies have shown the relationships between the spatial distribution of butterflyfish species and benthic communities at Great Barrier Reef. Three obligate coral polyp feeders (C. baronessa, C. ornatissimus and C. plebeius) were found at sites with greater coverage of acroporid corals (Pratchett & Berumen, 2008; Emslie et al., 2010). In contrast, few previous studies have shown the spatial distribution of two species of obligate coral polyp feeders (C. bennetti and C. punctatofasciatus). Thus, present study is the first study that revealed their spatial distributions. These five species were found at sites with greater coverage of encrusting corals, massive corals and rocks in the present study. The five species also exhibited greater proportion of bites on encrusting corals and massive corals. Thus, it is suggested that relatively close relationships between spatial distribution and feeding substrates were found for the five species. Since both encrusting corals and massive corals are less structurally complex, these corals are unlikely to provide refuges for these five species. In contrast, rocky surfaces inherently possess significant structural complexity (e.g., uneven surfaces and large holes). Thus, it is suggested that rocky areas with greater coverage of encrusting corals and massive corals may be suitable for these species as both habitat and foraging sites.

Emslie et al. (2010) showed that C. lunulatus was found at sites where various types of corals (Acroporidae, Faviidae, Musiidae, Pocilloporidae, Poritidae and Pectiniidae) were sympatrically found. Similar result was obtained in the present study, i.e., C. lunulatus was found at sites with greater coverage of encrusting corals, massive corals and branching Acropora. In the present study, C. lunulatus exhibited a greater proportion of feeding bites on encrusting corals and massive corals, but not on branching Acropora. Thus, C. lunulatus did not show a close relationship between spatial distribution and feeding substrate. Since branching Acropora has greater structural complexity, the corals can be suitable habitat for the species, rather than foraging substrate. Thus, among these three types of corals that affected the spatial distribution of C. lunulatus, the function might be different. Namely, branching Acropora is habitat and two types of corals (encrusting and massive corals) are foraging substrate, suggesting that sympatric distribution of these three types of corals would be suitable for this species.

At Great Barrier Reef, C. trifascialis was found at sites with greater coverage of tabular Acropora (Pratchett & Berumen, 2008; Emslie et al., 2010). In contrast, this species was found at sites with greater coverage of branching Acropora whereas exhibited greater proportion of bites on corymbose Acropora and tabular Acropora in the present study. This suggests the highly specialized nature of feeding, which has been shown to feed on a narrow range of corals (Berumen & Pratchett, 2008; Pratchett, 2005; Pratchett, 2007). A similar result was obtained at Lord Howe Island (Pratchett et al., 2014): a high density of C. trifascialis was encountered at sites with greater coverage of arborescent Acropora, whereas the species exhibited a significant preference for feeding on tabular Acropora. Thus, branching Acropora may serve as habitat whereas corymbose and tabular Acropora are suitable for foraging substrate for C. trifascialis, suggesting that the sympatric distribution of these three types of corals would be a suitable environment for this species.

For four facultative coral polyp feeders (C. citrinellus, C. kleinii, C. vagabundus and C. argentatus), C. citrinellus was found at sites with greater coverage of branching Acropora and isoporid corals whereas C. kleinii and C. vagabundus was found at greater coverage of non-acroporid corals (Pratchett & Berumen, 2008; Emslie et al., 2010). Since few studies clarified spatial distribution of C. argentatus, the present study is the first study that revealed the relationship between spatial distribution and foraging substrates for the species. These four species were found at sites with greater coverage of rock, that was different results from the previous studies at Great Barrier Reef. Since these four species exhibited greater proportion of bites on rock, encrusting corals and massive corals in the present study, rocky areas with greater coverage of encrusting corals and massive corals may be suitable for these species as both habitat and foraging sites. Remaining two species of facultative coral polyp feeders (C. auriga and C. ephippium) were found at sites with greater coverage of non-acroporid corals at Great Barrier Reef (Pratchett & Berumen, 2008; Emslie et al., 2010) whereas these species were found at the sites with greater coverage of branching Acropora, coral rubble and dead corals in the present study. Since C. auriga and C. ephippium respectively exhibited greater proportions of bites on coral rubble and dead corals, substrate characteristics would affect both the spatial distribution and foraging for these species.

The present study is the first study that clarified the spatial distribution of Forcipiger flavissimus, a non-coralline invertebrate feeder. The results suggest that there is a close relationship between spatial distribution and feeding substrates, since this species was found at sites with greater coverage of rock as well as exhibited greater proportion of bites on rock.

Feeding substrates

Encrusting corals and massive corals were positively used by eight species (C. baronessa, C. bennetti, C. lunulatus, C. ornatissimus, C. plebeius, C. punctatofasciatus, C. citrinellus and C. kleinii) as feeding substrates. Corymbose Acropora and tabular Acropora were also positively used by three species (C. baronessa, C. ornatissimus and C. trifascialis). In contrast, no species of butterflyfishes exhibited a significant positive use of branching Acropora or bottlebrush Acropora in the present study. Pratchett et al. (2014) presented similar results, in which no significant positive use for arborescent Acropora was found among five species of butterflyfishes in Australia. An explanation for these findings may be found in the morphological characteristics of corals. The morphological complexity of four types of corals (encrusting corals, massive corals, corymbose Acropora and tabular Acropora) is less than that of branching and bottlebrush corals. It is suggested that butterflyfishes feed more easily on polyps of less complex corals than on corals with greater morphological complexity and this behavior is the reason why butterflyfishes prefer the above-mentioned four types of corals. Similar results are reported in previous studies (Tricas, 1989). Tricas (1989) showed that C. multicinctus preferred to feed on the massive coral, Porites lobata, rather than on the branching coral, P. compressa. Cole, Pratchett & Jones (2008) suggested that feeding efficiency is related to coral morphology. Thus, corals with lower morphological complexity (encrusting, massive, corymbose, and tabular corals) might be suitable foraging substrates on which butterflyfishes can easily feed.

Another explanation for coral preferences might be microstructural and nutritional characteristics of corals. Cole, Pratchett & Jones (2008) have suggested that polyp distribution, nematocyst size and lipid content could affect foraging substrate selection. For nutritional aspects, Pratchett (2013) has suggested that preferred corals by butterflyfishes as foraging substrate did not necessarily have greater nutrition among the various coral species. Quantitative comparison for such physical characteristics and nutrition among different coral species should be examined in the future.

In contrast, four species of facultative coral polyp feeders (C. argentatus, C. auriga, C. ephippium and C. vagabundus) exhibited greater numbers of bites on rock, although rock was not a significantly preferred substrate. Coral rubble and dead corals were also used as feeding substrates for C. auriga and C. ephippium, respectively. The diet for these species includes coral polyps and other benthic organisms, such as filamentous algae, polychaetes, sea anemones, hydroids, and sponges (Harmelin-Vivien & Bouchon-Navaro, 1983; Sano, Shimizu & Nose, 1984). Kramer, Bellwood & Bellwood (2014) demonstrated greater density of polychaetes in coral rubble and dead coral compared with living Acropora colonies. Thus, coral rubble and dead corals might be suitable feeding substrate.

One non-coralline invertebrate feeder (F. flavissimus) exhibited greater number of bites on rock. However, some of bites on tabular Acropora, corymbose Acropora and Pocillopora were also shown in the present study. The primary diet of this species consists of sponges, hydroids and polychaetes (Harmelin-Vivien & Bouchon-Navaro, 1983; Sano, Shimizu & Nose, 1984). As suggested by Cole & Pratchett (2013), this species might consume small invertebrates inhabiting corals. Further, Harmelin-Vivien & Bouchon-Navaro (1983) found small volume of scleractinians in the stomach contents of this species. Thus, although the species is categorized as invertebrate feeder, it might occasionally feed on coral polyps.

Implications of coral bleaching

Coral bleaching has been frequently observed on Okinawan coral reefs and the decrease of living coral coverage and its negative effects on coral reef ecosystem have been concerning (e.g., Yara et al., 2009; Yara et al., 2014). In addition, coral bleaching due to global climate change is likely to increase on Okinawan coral reefs in the future (Okamoto, Nojima & Furushima, 2007; Yara et al., 2009; Hongo & Yamano, 2013). Susceptibility to coral bleaching varies among coral taxa in Okinawa as well as other regions (e.g., Fujioka, 1999; Yamazato, 1999; Marshall & Baird, 2000; Loya et al., 2001; Pratchett et al., 2013). Greater susceptibilities and higher rates of subsequent death have been reported for corals in the genera Acropora and Pocillopora (e.g., Loya et al., 2001; McClanahan et al., 2004). In contrast, greater tolerance and higher survival rates against coral bleaching have been reported for other coral taxa, such as members of the families Faviidae, Merulinidae, Mussidae, Poritidae, and Oculinidae (Fujioka, 1999; Yamazato, 1999; Marshall & Baird, 2000). These coral families mainly consist of encrusting and massive corals (Nishihira & Veron, 1995).

Thus, on Okinawan coral reefs, one species of obligate coral polyp feeder (C. trifascialis) would be negatively affected by coral bleaching for both aspects of habitat and foraging substrates since greater densities were found at areas dominated by branching Acropora and the species shows a significant positive use of tabular Acropora and corymbose Acropora. Pratchett et al. (2008) and Wilson, Graham & Pratchett (2013) reached a similar conclusion for this species, since the species is ecologically specialized to particular coral species. Wilson, Graham & Pratchett (2013) summarized the susceptibility of butterflyfishes to habitat disturbance and showed that C. trifascialis is the most vulnerable to living coral loss by bleaching.

On Okinawan coral reefs, feeding behavior of two obligate coral polyp feeders (C. baronessa and C. ornatissimus) would also be negatively affected by coral bleaching, since these species exhibited significant positive use of tabular Acropora and corymbose Acropora. However, the negative effect may not be severe since these two species inhabit areas with less coverage of acroporid corals and show positive feeding selectivity for massive corals or encrusting corals. In addition, three species (C. lunulatus, C. ephippium and C. auriga) would be negatively affected by habitat loss, since these three species inhabit sites with greater coverage of branching Acropora. If extensive coral loss by global bleaching events were seen, most of butterflyfish species might feed on less preferred substrates and expand foraging ranges to compensate the lost of their ecological requirements

Since the other eight species (C. argentatus, C. bennetti, C. plebeius, C. punctatofasciatus, C. citrinellus, C. kleinii, C. vagabundus and F. flavissimus) inhabit sites with greater coverage of rock, encrusting corals and massive corals, a major negative impact from coral bleaching may not occur on Okinawan coral reefs. However, since six species (C. argentatus, C. plebeius, C. punctatofasciatus, C. citrinellus and C. kleinii) exhibited feeding bites on acroporid corals, there is likely to be some negative effect. Based on the findings from the present study, the key corals for conservation would be branching Acropora as habitat and four types of coral (massive corals, encrusting corals, tabular Acropora and corymbose Acropora) as foraging substrates. However, as suggested by Pratchett et al. (2008), the long-term effects of coral loss causing less structural complexity should be carefully considered. Diverse coral communities should be conserved to maintain high species diversity of butterflyfish assemblages in Okinawan coral reefs.