Fish stable isotope community structure of a Bahamian coral reef

Study site

Four accessible and conservation-protected reef sites (Fig. 1) on the Exuma side of Cape Eleuthera (the Bahamas) with relatively high structural complexity and a diverse fish community were selected for visual surveys and fish sampling. These sites were close to each other and to shore and represented both bommies and patch reefs. In spite of the conservation status of the study sites, the reefs have been subject to chronic overfishing and otherwise impacted by cyclones and coral bleaching episodes.

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Fish survey

Underwater visual census (UVC) was conducted by two divers using eight single-sweep 30 m × 5 m transects at each site to record fish species, individual total length (L, to nearest cm) and numbers of individuals. Surveyors’ length estimation precision was repeatedly measured by conducting underwater fish-shaped object length estimation training (Bell et al. 1985) to minimise error (± 5%). Transects ran parallel to each other to avoid intersection. Transects were carried out in the morning (9:30–11:30) or afternoon (14:00–16:00), while swimming at a steady speed for 30 min. Highly mobile transient individuals (e.g. sharks and jacks) were excluded since they had large home range and were not necessarily reef associated, and also large schools of fishes were excluded since they were sighted only sporadically (Ferreira et al. 2001).

Sampling for stable isotope analysis

After landing, approximately 2 g of dorsal white muscle tissue near the dorsal fin were dissected, rinsed with water and stored in individual whirlpack bags in a − 20 °C freezer, and algal samples were only rinsed and stored in a freezer. All samples were dried in individual tin trays in an oven at 40 °C for ~ 12 h until fully dried and then in individual sealed Eppendorf tubes in zip-lock bags.

Stable isotope analysis preparation

All dried samples were imported to Newcastle University, freeze dried and then manually ground with mortar and pestle. Fish samples were weighed to 1.0 ± 0.1 mg in tin capsules with a Mettler MT5 microbalance. The prepared samples were analysed by Iso-Analytical Ltd (Crewe, UK) by Elemental Analysis-Isotope Ratio Mass Spectrometry (EA-IRMS). The ¹⁵N/¹⁴N ratio (δ¹⁵N) was expressed relative to N₂ in air for nitrogen, while that of ¹³C/¹²C (δ¹³C) was relative to Pee Dee Belemnite (PDB) of CO₂. Reference material used for this analysis was IA-R042 (δ¹³C = − 21.6 ± 0.1‰, δ¹⁵N = 7.6 ± 0.1‰), with quality control check samples IA-R042, IA-R038 (δ¹³C = − 25.0 ± 0.1‰, δ¹⁵N = − 0.4 ± 0.1‰), a mixture of IA-R006 (δ¹³C = − 11.7 ± 0.0‰) and IA-R046 (δ¹⁵N = 21.9 ± 0.2‰). IAR042 and IA-R038 were calibrated against and traceable to IAEA-CH-6 (δ¹³C = − 10.4‰) and IAEA-N-1 (δ¹⁵N = 0.4‰), IA-R006 to IAEA-CH-6 and IA-R046 to IAEA-N-1. External standards (fish white muscle tissue, δ¹³C = − 18.9 ± 0.0‰, δ¹⁵N = 12.9 ± 0.1‰) were also used for future reference. The precision of analysis for δ¹³C, δ¹⁵N, %C and %N was ± 0.1‰, ± 0.2‰, ± 4% and ± 1%, respectively. For individual samples, no lipid extraction was needed because their C/N ratios were less than 3.7 (Fry et al. 2003; Sweeting et al. 2006).

Data analysis

All data were tested for normality and homogeneity of variance prior to analysis and analysed in R 3.24 (R Core Team 2016) using the package siar (Parnell and Jackson 2013) between δ¹³C and δ¹⁵N data, linear regression (Wilkinson and Rogers 1973; Bates et al. 1992) between δ¹⁵N and log₂ body mass and mvtnorm to simulate values from a bivariate normal distribution. Results were visualised using ggplot2 (Wickham and Chang 2016). The assumptions of the ordinary least squares linear regression analyses were assessed with QQ plots, histograms of standardised residuals and plots of standardised residuals versus fitted values. Pearson’s correlation coefficient was used to test correlations between variables. Significance was set at p = 0.05 in all cases. All errors are reported as ± 1SE unless otherwise stated.

Species and trophic guild stable isotope analyses

The bulk δ¹³C and δ¹⁵N data were used to interpret δ¹⁵N–δ¹³C relationships in each species and trophic guild (Froese and Pauly 2017). Isotopic niches of five trophic guilds (four benthic: benthivore, herbivore, omnivore and piscivore and one pelagic: planktivore) were investigated using SIBER in the siar package. This was achieved by investigating standard ellipse parameters: eccentricity (E) and the angle in degrees (θ, 0°–180°) between semi-major axis and the x axis, the sample size corrected standard ellipse areas (SEA_C) and Bayesian standard ellipse areas (SEA_B) (Jackson et al. 2011). θ and E values potentially distinguish among isotopic niches where different trophic guilds have similar sized SEA_C but different relationships between δ¹³C and δ¹⁵N (Reid et al. 2016). θ values close to 0° or 90° suggest dispersion in only one axis: θ values close to 0° represent relative dispersion along the x axis (δ¹³C), indicating multiple production sources, while θ values close to 90° highlight relative dispersion along the y axis (δ¹⁵N), indicating feeding across multiple trophic positions from a uniform basal source. E explains the variance on the x and y axes: low E refers to similar variance on both axes with a more circular shape, while high E indicates that the isotopic niche is stretched along either x or y axis. The overlap of the standard ellipses between guilds was calculated using “sea.overlap” using SIBER. In order to compare isotopic niche areas among trophic guilds, a Bayesian approach was used that calculated 20,000 posterior estimates of SEA_B based on the data set. The mode and 95% credible intervals (CI) were reported. A significant difference among SEA_B was interpreted graphically whereby if the 95% CI did not overlap, then the SEA_B were deemed to be significantly different (Parnell and Jackson 2013).

δ¹⁵N–body size relationship

Cross-species relationships between stable isotope data and M were analysed using linear regression between mean bulk δ¹³C and δ¹⁵N of each species and their maximum body mass (M_max) recorded by Humann and DeLoach (1989). Comparing fishes at a fixed proportion of maximum size (here ≥ 55% of L_max) reduced the risk of comparing different life stages (Charnov 1993; Jennings et al. 2001; Galván et al. 2010).

For each species, the δ¹⁵N–log₂M relationship was generated using linear regression, the slope and intercept values being used to calculate δ¹⁵N values of individuals of the same species with other body mass values. To derive the community relationship between δ¹⁵N and log₂B (B was used instead of M to differentiate the community-level analysis from the species-level analysis), the mean weighted δ¹⁵N value of each log₂B class was derived by calculating (1) the mass ratio (r) of each individual (i) in each log₂B class, using \(r_{i} = M_{i} /\sum\nolimits_{i = 1}^{n} {M_{i} }\), where n is the total number of individuals in the log₂B class; and (2) mean weighted δ¹⁵N for each log₂B class j of the whole community as \(\updelta^{15} {\text{N}}_{j} = \sum\nolimits_{i = 1}^{n} {\updelta^{15} } {\text{N}}_{i} \times r_{i}\) (Al-Habsi et al. 2008). The δ¹⁵N of each individual (δ¹⁵N_i) was either calculated using the species-level linear δ¹⁵N–log₂M relationship if it is significant or equivalent to the mean δ¹⁵N of the species if not.

Because of the species richness of these reefs and limitation of fishing techniques, not all species could be sampled. To obtain δ¹⁵N values of such species, several methods were used (full details in “Appendix B” supplementary materials): (1) using samples from similar species within the same genus if possible (order of priority: same genus, family, site, trophic position, diet and feeding habit from fishbase.org) by comparing 3 criteria: (a) length to weight conversion factors, (b) dietary similarities and (c) their feeding behaviours in this area; and (2) using existing data in the literature from nearby locations or elsewhere in the Caribbean with baseline adjustment (D. cavernosa to D. cavernosa if possible, otherwise D. cavernosa to turf algae) to reduce temporal and/or spatial baseline variations (Cabana and Rasmussen 1996).

Predator–prey mass ratio

The uncertainty of PPMR was estimated by (1) simulating 10,000 times of independent variables s (the mean and standard deviation from the linear regression statistics) and Δδ¹⁵N values (mean and standard deviation of 3.4 ± 1.0‰; Post 2002) from a bivariate normal distribution (ρ = 0) and (2) calculating PPMR estimates using Eq. 3. The median, 25th and 75th quantiles were reported. The same method was applied to existing studies for comparison.

Isotopic niches at species and trophic guild levels

In total 9055 individuals (L from 1 to 120 cm, M from 0.01 to 2742 g) were recorded in 32 UVCs over 4800 m² of reef. Of 41 fish species collected and analysed for δ¹⁵N, 11 had sample sizes under three, and three (Coryphopterus glaucofraenum, Balistes vetula, Holocentrus adscensionis) were sampled to represent certain uncollected species. The size range cover ratio (r_L) ranged from 0 to 100% (mean = 31.0 ± 5.0%). Mean species δ¹³C ranged from − 17.4 ± 0.2‰ (Halichoeres pictus) to − 9.5 ± 0.0‰ (Lutjanus griseus), while mean δ¹⁵N ranged from 3.9 ± 0.8‰ (S. aurofrenatum) to 9.9 ± 1.5‰ (Sphyraena barracuda). δ¹⁵N values were significantly but weakly correlated with δ¹³C (p < 0.05, \(r_{\text{adjusted}}^{2} = 0.29\)) at the species level (Fig. 2). Some species had large SE values in δ¹³C (≥ 1‰) (e.g. S. barracuda) or δ¹⁵N (≥ 0.5‰) (e.g. Aulostomus maculatus), or both δ¹³C and δ¹⁵N (e.g. Elacatinus genie). Mean δ¹³C and δ¹⁵N values of strict pelagic planktivores (e.g. Chromis cyanea, TP = 3.7, δ¹³C = − 17.0 ± 0.1‰, δ¹⁵N = 5.2 ± 0.1‰) and strict benthivores of similar TP (e.g. B. vetula, TP = 3.8, δ¹³C = − 12.4 ± 0.3‰, δ¹⁵N = 7.7 ± 0.1‰) were significantly different.

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δ¹⁵N–body mass relationships at species and community levels and PPMR

There were significant but weak relationships across species between log₂M_max (maximum body mass) and both δ¹⁵N (\(r_{\text{adjusted}}^{2} = 0.12\), p < 0.05; Fig. 3a) and δ¹³C data (\(r_{\text{adjusted}}^{2} = 0.17\), p < 0.05; Fig. 3b). The δ¹⁵N values of several species did not scale positively with log₂M_max (e.g. E. genie, Sparisoma viride, Scarus iserti and S. aurofrenatum). The SE values of δ¹³C were generally higher than those of δ¹⁵N regardless of M_max. δ¹⁵N tended to vary with log₂M for 29 species (Fig. 4) with n ≥ 3; of these 24 relationships were positive (significantly so, e.g. C. cyanea), while five were negative (significantly so, e.g. Pomacanthus arcuatus, “Appendix C” supplementary materials). There was considerable variability around the regression line for nine species (\(r_{\text{adjusted}}^{2} < 0.5\), e.g. S. barracuda), whereas this was not the case for others (e.g. Clepticus parrae, Calamus pennatula, Halichoeres garnoti). At the community level, the combined isotope data demonstrated a strong positive linear relationship between mean δ¹⁵N and log₂B, the regression equation being δ¹⁵N = 0.34 ± 0.04log₂B + 4.03 ± 0.24 (\(r_{\text{adjusted}}^{2} = 0.64\), p < 0.05, Fig. 5). The slope value of TP–log₂B was b = 0.10 (\(r_{\text{adjusted}}^{2} = 0.80\), p < 0.05), and the PPMR estimates were 1047:1 (Table 5).

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Table 5

Mean PPMR values of different communities from the literature using the additive framework

Community	PPMR estimates		Reference
	Median	25th and 75th quantiles
Central North Sea	104:1	42 to 75:1	Jennings et al. (2002a, b)
Northern North Sea	1037:1	239 to 4503	Jennings et al. (2001)
Cape Eleuthera	1047:1	250 to 4833:1	Present study
Puget Sound	4320:1	779 to 2.71 × 104:1	Reum et al. (2015)
North Sea	8349:1	1235 to 6.73 × 104:1	Jennings and Warr (2003)
Western Arabian	8935:1	1287 to 6.60 × 104:1	Al-Habsi et al. (2008)
Iberian Peninsula	2.02 × 107:1	2.27 × 105 to 1.61 × 1010:1	Bode et al. (2006)

Species and trophic guild trophodynamics

Stable isotope data at both species and trophic guild levels indicated that at the Cape Eleuthera site there were large differences in trophic ecology within and among species, and species utilising a range of production source types were common.

High within-species variability in δ¹³C and δ¹⁵N values for some species suggested the existence of individual specialisation (Matthews and Mazumder 2004) in the food web where different individuals of the same species were consistently sampling different production sources. For example, similarly sized individuals of the apex predator S. barracuda had similar δ¹⁵N values but differed greatly in δ¹³C values. The δ¹³C value of approximately − 16‰ was close to that within the planktivore trophic guild, while the δ¹³C value of − 10‰ was more consistent with the piscivore trophic guild. Trophic position omnivory indicated by differences in δ¹⁵N also occurs, for example, in the parasitivore E. genie which feeds on parasites from fish at different trophic positions.

The SIBER analysis indicated at least two types of production sources, namely benthic (e.g. algae) and pelagic (e.g. plankton), and mixed-feeding patterns for some species that are typically regarded as relying solely on single types of source materials (e.g. herbivorous fish; Plass-Johnson et al. 2013; Dromard et al. 2015). In this study, isotopic niche areas of the planktivores were significantly smaller than other guilds even though plankton can have highly variable isotopic signatures (McClelland and Montoya 2002; Kürten et al. 2013), which indicated a level of dietary strictness or consistency. High θ, low E and low SEA values of the planktivore guild nevertheless suggested TP omnivory, with these fish feeding at different TPs albeit from the same type of pelagic source (e.g. phytoplankton and zooplankton). The omnivore guild had high E, low θ and SEA values, the δ¹³C data indicating that the two species are supported by plankton and benthic algae with similar δ¹⁵N baselines. Although based on only two species, the omnivores may be connecting these two pathways to some extent (McMeans et al. 2016). The benthivore, piscivore and herbivore trophic guilds, which share mostly benthic production sources, had similar isotopic niche areas which were much greater than those of the planktivores and omnivores, with their isotopic niches spread along the x axis as indicated by E and θ values, suggesting source omnivory within the benthic producer category. Overlapping isotopic niches among the trophic guilds (e.g. piscivore and benthivore) suggested that they might share dietary resources to some extent; for example, some lutjanids are both piscivorous and feed on zoobenthos (Allen 1985; Kulbicki et al. 2005; Layman and Allgeier 2012). The vertical distribution in the isotopic niches for the four benthic trophic guilds reflected the herbivorous fish feeding at low trophic positions, while the omnivores, benthivores and piscivores utilised a wider range of energy sources from different TPs. There were species with stable isotope values outside the isotopic niches of their assumed trophic guilds, which suggested feeding on different food sources than previously known or those derived from snapshot diet studies. For example, the four benthic feeders (H. pictus, A. coeruleus, C. personatus and T. bifasciatum) were likely relying on plankton sources, and the piscivore A. maculatus might be predating on smaller herbivores. Some herbivores came partly within the isotopic niches of other trophic guilds, indicating feeding on food sources in addition to algae such as invertebrates or planktivore faeces (Robertson 1982; Wulff 1997; Dunlap and Pawlik 1998; Chen 2002; Plass-Johnson et al. 2013). This can only be confirmed with detailed dietary analysis including baseline variation (i.e. during the 3–6 months isotopic turn over period).

δ¹⁵N–body mass relationship

The majority of species had a positive trend between δ¹⁵N and log₂M indicating that they tend to feed at higher TPs as size increases. This could be a result of increasing gape size, predatory skill and fitness level allowing individuals to feed on higher TP prey as they grow (Peters 1986; Munday 2001; Newman et al. 2012). Those with negative or highly variable stable isotope-size relationships potentially have dietary shifts from isotopically high value production sources to low or multi-pathway (e.g. exploitation of short food chain) feeding patterns in their sampled size ranges, or otherwise assimilating significantly different isotopic baselines across the population (Jennings et al. 2002a; Layman et al. 2005). Unlike Robinson and Baum (2015) where δ¹⁵N–log₂M relationships of all individuals in two separate trophic pathways (herbivore and carnivore) were investigated, species-level and whole-community-level analyses were conducted in this study. The variation among species is attributable to differences in the trophic pathways supporting them, but to understand better how trophic pathways affect such relationships, more data are clearly needed. At community level, a positive linear relationship between δ¹⁵N and log₂B across the combined sites was found, indicating that TP tended to increase with body mass regardless of taxonomy and larger coral reef fish in this Cape Eleuthera community on average fed at higher TPs.

The weak cross-species relationship between isotopic signatures and log₂M_max suggested that maximum body mass could scarcely constrain species’ trophic capabilities in this food web in which there were small-bodied benthivores and planktivores and large-bodied herbivores. The body-size structuring is similar to that of North Sea and Western Arabian Sea community data (Jennings et al. 2001; Al-Habsi et al. 2008). Here, the small size class biomass data were dominated by herbivores rather than higher TP omnivores such as Labridae (Graham et al. 2017), while the large size classes were dominated by piscivores and omnivores rather than large-bodied herbivores such as Scarinae (Hughes et al. 2007; Zhu 2019). Although the surveyed Cape Eleuthera sites are now legally protected, they were previously fished and are structurally degraded. The linearity of the δ¹⁵N–log₂B relationship at Cape Eleuthera may not be generic; it could be influenced by the loss of habitat structural complexity and aspects of past overfishing (e.g. removal of large herbivores).

The present study also had limitations. All individuals were treated as if they had the same isotopic baseline, yet significant isotopic differences between benthic and pelagic sources are expected (McConnaughey and McRoy 1979; Polunin and Pinnegar 2002), whereas other baselines would have been taken into consideration when estimating the trophic positions of consumers with significantly mixed diets. Also, for some species, sample sizes failed to adequately fulfil requirements for confidently describing stable isotope changes as a function of body mass (Galván et al. 2010), yet linear regression was still applied to these species to explore their δ¹⁵N–log₂M relationships. Low sample sizes and/or size ranges (r_L) meant that for some species, stable isotope data could not be derived across whole UVC size ranges; for these stable isotope data were assumed to be size invariant. For missing species, the methods used to infer stable isotope values had limitations including species within the same genus or family having ontogenetic and/or dietary differences; some not meeting all three criteria and using published data from the same species could be subject to feeding strategies varying ontogenetically (Plass-Johnson et al. 2013) or spatially (Jennings et al. 1997; Matthews and Mazumder 2004). Unlike studies using combined baselines (Mill et al. 2007), here the benthic alga D. cavernosa was the sole baseline and this might not adequately represent the whole benthic assemblage, which includes turf algae, cyanobacteria and other potential production sources.

Predator–prey mass ratio

The mean PPMR at Cape Eleuthera indicates a relatively long food chain in this coral reef system compared with other aquatic systems (Table 5), suggesting potentially greater ecosystem size and stability (Jennings and Warr 2003). However, surveys at adjacent non-protected sites failed to show the presence of large predators there; thus, the PPMR data we report seem to be specific to the protected sites.