The ISME Journal (2015) 9, 2620–2629 © 2015 International Society for Microbial Ecology All rights reserved 1751-7362/15 OPEN www.nature.com/ismej ORIGINAL ARTICLE Productivity links morphology, symbiont specificity and bleaching in the evolution of Caribbean octocoral symbioses

David M Baker1,2,3, Christopher J Freeman4,5, Nancy Knowlton2, Robert W Thacker4, Kiho Kim6 and Marilyn L Fogel3,7 1School of Biological Sciences, Department of Earth Science, and Swire Institute of Marine Science, The University of Hong Kong, Hong Kong, PRC; 2Smithsonian Marine Science Network, National Museum of Natural History, Washington, DC, USA; 3Geophysical Laboratory, Carnegie Institution of Washington, Washington, DC, USA; 4Department of Biology, University of Alabama at Birmingham, Birmingham, AL, USA; 5Smithsonian Marine Station, Fort Pierce, FL, USA and 6Department of Environmental Science, American University, Washington, DC, USA

Many cnidarians host endosymbiotic dinoflagellates from the Symbiodinium. It is generally assumed that the symbiosis is mutualistic, where the host benefits from symbiont photosynthesis while providing protection and photosynthetic substrates. Diverse assemblages of symbiotic gorgonian octocorals can be found in hard bottom communities throughout the Caribbean. While current research has focused on the phylo- and population genetics of gorgonian symbiont types and their photo- physiology, relatively less work has focused on biogeochemical benefits conferred to the host and how these benefits vary across host species. Here we examine this symbiosis among 11 gorgonian species collected in Bocas del Toro, Panama. By coupling light and dark bottle incubations (P/R) with 13C-bicarbonate tracers, we quantified the link between holobiont oxygen metabolism with carbon assimilation and translocation from symbiont to host. Our data show that P/R varied among species, and was correlated with colony morphology and polyp size. Sea fans and sea plumes were net autotrophs (P/R41.5), while nine species of sea rods were net heterotrophs with most below compensation 13 13 (P/Ro1.0). C assimilation corroborated the P/R results, and maximum δ Chost values were strongly correlated with polyp size, indicating higher productivity by colonies with high polyp SA:V. A survey of gorgonian-Symbiodinium associations revealed that productive species maintain specialized, obligate symbioses and are more resistant to coral bleaching, whereas generalist and facultative associations are commonamongsearodsthathavehigher bleaching sensitivities. Overall, productivity and polyp size had strong phylogenetic signals with carbon fixation and polyp size showing evidence of trait covariance. The ISME Journal (2015) 9, 2620–2629; doi:10.1038/ismej.2015.71; published online 19 May 2015

Introduction (Yellowlees et al., 2008; Houlbreque and Ferrier- Pages, 2009; Pernice et al., 2014). We now know that Since the discovery of coral-hosted Symbiodinium, Symbiodinium translocate carbon-based photo- nutrition has been a major focus of coral symbiosis. synthates in excess of host needs and nitrogen in Hand and Muscatine (1958) were among the first to the form of amino acids derived from host wastes demonstrate that Symbiodinium provide photo- and dissolved inorganic nitrogen from the water synthates to their hosts, thus settling debate over column. By coupling the coral polyp’s opportunistic the functional significance of the intracellular algae. heterotrophy with Symbiodinium photosynthesis, Since then, numerous studies have quantified the the coral holobiont can thrive despite the severe contribution of autotrophic and heterotrophic nutrient limitation typical of tropical seas (Yellowlees nutrition to the coral holobiont for a number of et al., 2008; Houlbreque and Ferrier-Pages, 2009). scleractinian species, even to sub-cellular scales However, holobiont performance and host benefits are poorly understood across different host–symbiont Correspondence: DM Baker, School of Biological Sciences, interactions and changing environmental conditions. Kadoorie Biological Sciences Bldg 3 S-11, The University of Hong Our understanding of how coral–Symbiodinium Kong, Pokfulam Rd, Hong Kong, PRC. interactions vary across diverse associations remains E-mail: [email protected] limited. Most studies have focused on several model 7Current address: School of Natural Sciences, University of California, 5200 North Lake Rd, Merced, CA 95343, USA coral species such as Stylophora pistillata, Pocillo- Received 15 August 2014; revised 23 February 2015; accepted pora damicornis and members of the genus Acropora 1 April 2015; published online 19 May 2015 (Muller-Parker et al., 1994; Hoegh-Guldberg and Productivity links morphology, symbiont specificity and bleaching DM Baker et al 2621 Williamson, 1999; Grover et al., 2008). These species limited. Indeed, symbiotic gorgonians differ in their of photophilic corals all possess branching colony ability to capture particles from the water column morphologies and small polyps and are thus too (Lasker, 1981). Although some species obtain suffi- similar in their morphology and nutritional physiol- cient carbon from particulate organic matter ogy to reveal the full breadth of coral–Symbiodinium (Murdock, 1978; Coffroth, 1984), they still require functional diversity (Tremblay et al., 2012; Baker nitrogen supplemented via Symbiodinium photo- et al., 2013). Fewer nutritional studies have com- synthesis (Wainwright, 1967). Unlike Indo-Pacific pared species within and between genera and even soft corals (Fabricius and Klumpp, 1995) previous fewer in non-scleractinian anthozoans (Lasker, 1981; studies of Caribbean gorgonian productivity (as Lasker et al., 1983; Wilkerson and Muscatine, 1984; determined by oxygen metabolism) support a pre- Ribes et al., 1998; Swanson and Hoegh-Guldberg, dominantly autotrophic nutrition in many species. 1998). Such critical gaps limit our understanding of Here we expand on previous gorgonian productiv- the functional, ecological and evolutionary signifi- ity studies by using light and dark bottle incubations cance of these symbioses. to estimate P/R in combination with inorganic stable The evolution of feeding structures occurs when carbon isotope tracer additions to assess autotrophic species compete for limited nutritional resources productivity among 11 species of symbiotic gorgo- (Grant and Grant, 2006). For corals, niche partition- nians. We test the following null hypotheses: (1) all ing and competition likely drive the diversity of gorgonian symbioses are mutualistic where the host colony and polyp morphologies. Divergence towards benefits from symbiont photosynthesis, (2) the mutu- the utilization of novel resources at the extremes of alisms are invariant with respect to the amount of the heterotrophic – autotrophic continuum (that is, carbon translocated to the host and (3) the host- from plankton to dissolved inorganic nutrients) can derived benefits of mutualism are unrelated to reduce competitive interactions and drive specia- morphology, (that is, polyp size) of a species. Further, tion. Porter (1976) argued that variation in coral we examined the functional significance of varied colony morphology, specifically the ratio of surface productivity through aposterioricorrelations between area to volume (SA:V), is central to understanding productivity measures and eco-physiology, including the nutritional strategies of scleractinian corals. symbiont specificity and thermal bleaching resistance. Indeed, Porter predicted that autotrophy should be maximized in corals with high SA:V, with the specific idea that branching corals would exhibit Materials and methods higher photosynthesis/respiration (P/R) relative to massive (non-branching) species. Moreover, such a Field collections relationship would promote the evolution of Five whole colonies of 11 gorgonian species were 'layered' colony morphologies, which Porter identi- collected using SCUBA from reefs nearby the fied as a convergent character in terrestrial plants. At Smithsonian Tropical Research Institute’s (STRI) a smaller scale, variation in polyp size is often field station in Bocas del Toro, Panama in September posited as an indicator of heterotrophic capacity as 2010. A total of three sites (depths of 3–8 m) were larger polyps possess larger tentacles and gastro- visited to obtain a diverse collection. The sea plume, vascular cavities for prey capture and digestion. Antillogorgia acerosa (AACE), and sea rods Ptero- Conversely, small polyps have higher SA:V, which gorgia anceps (PANC) and Eunicea succinea (ESUC) maximizes the light received by Symbiodinium, were collected from Boca del Drago (9°25'21.52"N; inhabiting the gastroderm at densities up to 106 cells 82°19'35.78"W). The sea rods Plexaura homomalla cm− 2, and increases the surface area for nutrient (PHOM), Plexaurella fusifera (PFUS), Plexaurella uptake. Taken together, increasing or decreasing SA: nutans (PNUT), Eunicea calyculata (ECAL), Eunicea V can occur at the colony and polyp scale. Here we flexuosa (EFLE), Eunicea mammosa (EMAM), sea test Porter’s hypothesis by quantifying productivity plume Antillogorgia acerosa and sea fan Gorgonia (P/R and 13C assimilation) and utilizing morphologi- ventalina (GVEN) were sampled from Crawl Cay cal, phylogenetic and ecological data on a diverse Channel (9°15'36.88"N; 82°6'32.78"W). The sea rod collection of Caribbean octocorals (gorgonians). Briareum asbestinum (BASB) was collected near In the Caribbean Sea, gorgonians hosting Symbio- South Carenaro Island (9°20'36.19"N; 82° dinium are especially diverse with at least 51 14'17.15"W). A subsample from each collected speci- described species (Sánchez and Wirshing, 2005). men was treated with dilute bleach to isolate Moreover, there is great variation in gorgonian sclerites, which were used to establish species morphology, with many reefs dominated by species identification following Bayer (1961). Calyx depth, with fan, plume or rod morphologies. Perhaps due to the distance from the surface edge of the aperture the morphological similarity to aposymbiotic deep (where the polyp emerges from the colony) to the water species, and the paucity of symbiotic species base where the polyp is anchored within the colony, in the Indo-Pacific, there is a perception that was used as a proxy for polyp size (Thibaudeau, symbiotic gorgonians gain most of their nutrition 1983). The calyx is the anatomical space occupied by via passive suspension feeding (Leal et al., 2014). To a single polyp, from which the polyp can extend into date, the evidence to support this contention is the environment.

The ISME Journal Productivity links morphology, symbiont specificity and bleaching DM Baker et al 2622 P/R and 13C Incubation Experiment isolated from diverse invertebrate hosts including Detailed experimental procedures are provided as gorgonians (Franklin et al., 2012), and (2) Prada et al. Supplementary Online Material. In short, five colo- (2010), the most comprehensive report on gorgonian nies of each gorgonian species were fragmented into thermal bleaching (loss of Symbiodinium) from the three clonal replicates, with one replicate preserved 2005 mass coral bleaching event in Puerto Rico. as an initial control. The remaining replicates were These data were used to test the hypotheses that incubated in light and dark bottles for an assessment symbiont specificity and bleaching resistance are of net primary production and respiration, respec- correlated with our productivity measurements (P/R, δ13 δ13 tively, measured as change in dissolved oxygen Chost and Csymbiont). concentration following Freeman et al. (2013). Sea- water in each bottle was amended with 98% 13 NaH CO3, such that the resulting carbon stable isotope ratios (δ13C) could be used to trace photo- Results synthetic assimilation by Symbiodinium and Gorgonian productivity subsequent transfer to host cells (Baker et al., 2013; P/R values varied significantly among the 11 Freeman et al., 2013). species of Caribbean gorgonians measured (ANOVA; F = 5.26; Po0.0001; Figure 1). All sea rods had average P/R values o1.5, which is Stable isotope analysis below a conservative threshold for autotrophy For details of sample preparation please refer to the (Wilkinson, 1983). Five out of nine sea rod species Supplementary Online Methods. In brief, each dried had mean values below compensation (P/R = 1.0). gorgonian fragment was physically homogenized, In contrast, a sea fan (G. ventalina)andsea and the host and symbiont fractions were separated plume (A. acerosa) were both net autotrophs using centrifugation of a suspended homogenate. with P/R ~ 1.8 with some replicate fragments The supernatant (host) and pellet (symbiont) were exceeding 2.5. P/R was strongly correlated with dispensed into silver capsules, acidified and dried 2 o autotrophy (NPPlight; R =0.84, P 0.0001, n =11), before stable isotope analysis. 2 not respiration (NPPdark; R =0.16, P = 0.22, n =11; All samples were analyzed using a Thermo Delta V Supplementary Table S1). However, the highest isotope ratio mass spectrometer coupled to a Carlo- dark bottle respiration was observed in A. acerosa Erba NC2500 elemental analyzer via a Conflo III − − 1 − 1 1− (mean NPP = 2.03 mg O2 l g h ) and generally open-split interface at the Geophysical Laboratory. declined with increasing colony volumes with Analytical precision was determined by repeated P. nutans and P. fusifera having the lowest observed ‘ ’ analysis of an internal acetanilide standard ( acet 6 ; respiration (Supplementary Table S1). 70% C). Standard precision during runs of initials and dark incubated enriched samples was +/ − 0.2‰,and +/ − 0.4‰ for the light incubated enriched samples. δ13C - natural abundance and tracer experiment Natural abundance δ13C ranged from − 20.4 to Gorgonian phylogenetic signal and trait correlation − 14.9‰ with a mean of − 17.3‰. There was a strong Sequences of two mitochondrial protein-coding correlation between the mean natural abundance 13 13 genes, ND2 and msh1, were obtained from GenBank δ Chost and δ Csymbiont inclusive of all species to construct a phylogeny of the species sampled in (R2 = 0.67; P = 0.002; Supplementary Figure S2). this study ( McFadden et al., 2006; for details of tree δ13C values increased to a mean of − 6.9‰ and construction see Supplementary Online Methods; 261‰ in the dark and light incubations, respectively Supplementary Table S2). Phylogenetic signal (ANOVA; F = 252, Po0.001; Figure 2). In most describes the degree to which more closely related species, 13C enrichments in the light treatment were organisms share more similar traits. We used the similar between the host and symbiont fractions δ13 'phylosignal' function of the R package Picante to test (Figure 2a), but the light bottle Csymbiont was a δ13 δ13 whether P/R ratios, Chost, Csymbiont, C:Nhost,C: better predictor of holobiont P/R than the corre- δ13 2 Nsymbiont and calyx depth displayed significant sponding Chost (R = 0.84 vs 0.67, respectively; phylogenetic signal given the host phylogeny (that Supplementary Figure S2). Fragments incubated in is, whether more similar values were associated with the dark took up less 13C owing to a lack of more closely related gorgonians more often than photosynthesis (Figure 2a). Yet, significant 13C expected by chance; refer to Supplementary Online assimilation was observed in the host fractions of δ13 Methods for further detail and references). many species (Figure 2b). The Chost of P. anceps, A. acerosa and B. asbestinum incubated in the dark were all o10‰, greater than initial values, but much δ13 A posteriori tests lower than the Chost values from the light bottle Following our experimental work, we obtained data treatment. In fact, all host fractions from the dark from (1) GeoSymbio, a quality controlled database treatment were, on average, +11.8‰ enriched rela- containing phylogenetic data on Symbiodinium tive to their Symbiodinium.

The ISME Journal Productivity links morphology, symbiont specificity and bleaching DM Baker et al 2623

2.2 a,b 2.0 a

1.8 a, b, c 1.6 autotrophy

1.4 c, d, e 1.2 c, d, e

P:R b, c, d d, e compensation 1.0 mm = calyx depth d, e 0.8 c, d, e e

0.6 = colony morphology c, d, e

0.4 0.15 0.3 0.5 0.25 1.5 2.5 1.0 0.75 3.0 1.6 3.5

0.2

0 GVEN AACE EMAM PANC ESUC ECAL EFLEX PHOM PNUT BASB PFUS species Figure 1 Least-squared mean P/R values from 11 species of symbiotic Caribbean gorgonians ordered from highest to lowest values. Dashed line defines compensation (where P/R = 1.0), and the solid line a conservative threshold for autotrophy (P/R41.5; (Wilkinson, 1983)). P/R was calculated from at least four pairs (one light, one dark treatment) of similarly sized clonal fragments from independent colonies (n ≧ 4). Error bars represent s.e.m. and letters indicate significant differences between groups as determined by Tukey’s post hoc test (Po0.05). For species abbreviations refer to the Methods section. Colony morphology (not to scale) and calyx depth (to scale in mm) are shown for reference.

600 host - dark zoox - dark host - light 400 zoox - light

200

0 13

10

0

10

20 GVEN AACE EMAM PANC ESUC ECAL EFLE PNUT PHOM BASB PFUS species Figure 2 (a) Mean δ13C of 11 species of gorgonians and their Symbiodinium after a ~ 7.5 h incubation in dark bottles (filled shapes) or light bottles (open shapes). (b) The same data for host and symbiont fractions incubated in darkness and with enlarged scale to illustrate differences. Gray horizontal lines indicate the upper and lower 95% confidence intervals of the mean natural abundance δ13C values for all species (initial samples). Species abbreviations are listed in the Methods section and are presented in order of decreasing P/R. Error bars represent the 95% confidence interval of the mean.

δ13 δ13 Phylogenetic signals in metabolic traits the enriched Chost and Csymbiont values, P:R and The phylogeny of used in this study is a calyx depth (Supplementary Table S3). The two δ13 subset of the phylogeny reported by McFadden et al. most significant traits, Chost and calyx depth, (2006) based on ND2 and msh1 sequences. We found demonstrated a significant negative correlation strong support for clades representing the genera under both models of trait covariance (BM: r = Plexaurella and Eunicea, as well as a clade contain- − 0.450, P = 0.044; OU: r = − 0.83, Po0.001; ing Antillogorgia and Gorgonia (Figure 3). Four traits Figure 3). Calyx depth was negatively correlated displayed significant phylogenetic signal, including with P/R (R2 = 0.65; P = 0.005; Figure 4).

The ISME Journal Productivity links morphology, symbiont specificity and bleaching DM Baker et al 2624 Sym. types Morph.

B1, B19, B1a

no record

B1

B1

B1, B1m

B1, B1b, B2, unsp.

A4, B1, B1a, C1, unsp.

no record

B1, B9

B19, B33, C1, C3, C3L

Figure 3 Phylogeny of Octocorallia used in this study, derived from sequences reported by McFadden et al. (2006). The scale bar indicates number of substitutions per site, while numbers at nodes indicate percentage Bayesian posterior probabilities. To the right of the δ13 δ13 phylogeny, four columns of circles represent values of photosynthesis to respiration ratios (P/R), Chost, Csymbiont and calyx depth (used as a proxy for polyp size). The size and shading of each circle are proportional to the values reported in Supplementary Table S1. In addition, the known Symbiodinium associations are shown as well as the colony morphology (not to scale). ‘unsp.’ Indicates a unique Symbiodinium genotype with no clade designation.

A posteriori tests – specificity and bleaching Using quality-controlled records obtained from the y = -139 Log(x) + 197; 500 2 GeoSymbio database (Franklin et al., 2012), we AACE R = 0.84; p < 0.0001 found that the gorgonian species studied herein PANC host one to five unique Symbiodinium types 400 throughout the Caribbean Sea (Figure 3; GVEN Supplementary Table S1). G. ventalina and Antil-

logorgia have only been found in association with (‰) 300

clade B1 suggesting a highly specific partnership. host C

In contrast, records from all sea rods suggest more 13 EFLE 200 generalist associations with low specificity for a ESUC

given Symbiodinium type, ranging from two EMAM reported species in Pterogorgia and E. mammosa, ECAL 100 up to five in B. asbestinum including B19, B33, C1, BASB C3 and C3L (Figure 3; Supplementary Table S1). PFUS PNUT Taken together, 85% of the variation in holobiont 0 P/R was explained by the number of known 0.1 1 Symbiodinium genotypesassociatedwitheachhost calyx depth (mm)

species (Figure 5). δ13 Figure 4 Enriched Chost values as a function of calyx depth Using data extracted from Prada et al.(2010)for (used as a proxy for polyp size) for eight species of gorgonians 8 of 11 of our studied species, we found that P/R (data from Thibaudeau, 1983). Solid line represents a significant explained 33% of the variation in bleaching relationship between the variables. For species abbreviations refer prevalence (Figure 6). However, when P. anceps to the Methods section. was excluded from this analysis, the explained variation improved to 96%. Excluding P. anceps Discussion maybeappropriateasthis species often exhibits white polyps that could be mistaken for a Gorgonian productivity varies with morphology bleached condition (pers. obs.). The logistic fit Our data show that the sea fan G. ventalina and illustrates that species with P/R values o1.1 are plume A. acerosa produced 42 times their required highly susceptible to bleaching. Conversely, corals carbon demand with P/R values similar to many bearing small polyps with high P/R appear to resist scleractinian corals. As such, we conclude that these bleaching. species are net autotrophs, acquiring the majority of

The ISME Journal Productivity links morphology, symbiont specificity and bleaching DM Baker et al 2625 2.0 morphologies (encrusting, fan, dish) that are con- 2 AACE* R = 0.85; p < 0.001 vergent traits in corals (Wilkinson, 1983). 1.8 GVEN In one of the first studies of gorgonian metabolism, Cary (1918) measured respiration in 12 Caribbean 1.6 species and found that the highest rates of respira- EMAM tion were observed in species with the highest SA:V 1.4 (that is, Gorgonia, Antillogorgia and Pterogorgia). Thus, we hypothesize that some gorgonian species P:R ** 1.2 PANC are more metabolically active than others, as positive NPP is maintained by high rates of photosynthesis,

1.0 EFLE despite substantial respiration. Therefore, observed PHOM variations in P/R are due to overall metabolic rates PNUT 0.8 rather than variation in either photosynthesis or BASB respiration alone. Like Kanwisher and Wainwright 0.6 (1967), we found a wide range of P/R values for 12345 Caribbean gorgonians, which followed a general # Symbiodinum genotypes hosted pattern of increasing P/R with SA:V. We stress, Figure 5 Observed Symbiodinium genotypes (based on ITS2) however, that comparisons among disparate studies obtained from the GeoSymbio database, representing eight should be interpreted with caution, as the method of gorgonian species analyzed in this study. All n ≧ 3. *Indicates oxygen measurement and environmental conditions P/R data for A. acerosa, using symbiont records for A. elisabethae can vary considerably between studies. Neverthe- and A. bipinnata as there are no records for A. acerosa. **Indicates P/R for P. anceps and symbiont records for all less, it is interesting that Caribbean gorgonians have Pterogorgia as there was only one record for P. anceps. For a similar range of P/R values to some Indo-Pacific species abbreviations refer to the methods section. hard corals, although our data are lower than previous reports (Roffman, 1968). Porter (1976) put forth several hypotheses about PANC 0.8 functional morphology of corals that are consistent with our observations on gorgonians. First, the PNUT parallels drawn between tree morphology (sensu 0.6 incl. PANC R2 = 0.33 Horn (1971)) are particularly striking as the most excl. PANC R2 = 0.95 autotrophic species (G. ventalina and A. acerosa)have ECAL morphologies similar to the broad leaves and needles 0.4 of trees, respectively, which are plant adaptations for enhancing photosynthesis while moderating transpira- tion. In sea plumes, alternating branches reduce self- 0.2 proportion bleached shading and permit light transmission to lower EFLE portions of the colony. Sea fans have a reticulate and

EMAM planar morphology with high surface area. Over time, 0 ESUC sea fans twist their axial skeleton to a position AACE GVEN perpendicular to water flow (Wainwright and Dillon, 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1969). It has been suggested that fan morphologies aid P/R in particle capture (Leversee, 1976). While this may be true for deep-water sea fan species, for G. ventalina Figure 6 Observed prevalence of bleaching of eight gorgonian species during the 2005 mass bleaching in Puerto Rico (data from found in high energy, shallow environments this Prada et al. (2010)) as a function of gorgonian P/R. The dashed line orientation facilitates the oscillation of the colony from represents a logistic function fit to all data, whereas the solid line full illumination to shade in synchrony with the wave represents a logistic function fit to all data excluding P. anceps. period. This also permits the photosynthetic centers to For species abbreviations refer to the Methods section. recover from light saturation and maximize photosyn- thetic efficiency over the course of the day. Colonies uprooted or twisted from this position often show their nutrition from Symbiodinium photosynthesis. heavy melanization (purpling) in response to excessive This contrasts with all sea rod species that had light stress to one face of the fan, whereas adjacent P/Ro1.5 and all Indo-Pacific alcyonaceans that have colonies with normal skeletal development/position- negative P/R values (Fabricius & Klumpp, 1995). ing exhibit normal coloration (pers. obs.). Species with fan or plume morphologies and small Alternatively, the cylindrical or bladed, digitate polyps might maximize light exposure and optimize and branching forms common to the various genera symbiont densities and nutrient exchange via of sea rods may be more suited to capturing plankton increased SA:V. Similar observations have been and particulate matter, especially those with larger made for Indo-Pacific sponges, which are more polyp sizes. Thick, rounded colony branches may productive than their Caribbean counterparts with function to generate eddies on the side of the branch highly autotrophic species exhibiting flattened opposite an oncoming current. Such eddies can

The ISME Journal Productivity links morphology, symbiont specificity and bleaching DM Baker et al 2626 δ13 cause plankton and particulate matter to linger, The relative enrichment of Chost attained versus δ13 affording a polyp more opportunity for capture Csymbiont reflects the variation in photosynthate (Leversee, 1976). There is limited evidence to translocated from source (symbiont) to sink (host). suggest that larger polyps facilitate prey capture. Eunicea mammosa and E. succinea had the lowest δ13 δ13 Lasker et al. (1983) found that two sea rods, Plexaura relative Chost enrichment. Such low Chost could homomalla and P. nina, found on the shallow and result from several factors including (1) higher deep forereef, respectively, differed in their prey symbiont metabolism relative to the host, (2) limited capture abilities. With twice the polyp size, P. nina translocation of photosynthate due to lower host SA:V, was able to capture more particles. It is interesting (3) differential host and symbiont biomass, and that the difference in polyp size and distribution of similarly, (4) a relatively larger structural pool of these congeners occurs over a depth gradient, which carbon in the host fraction, which ‘dilutes’ any suggests that larger polyps are advantageous in translocated 13C. However, if these factors were deeper habitats where light is limiting. important in determining the final 13C assimilation in the host, then similar patterns would have been observed in the Plexaurella, which have the largest δ13C as an indicator of trophic position polyps, lowest SA:V, and thickest host tissues among The natural abundance isotope values of gorgonians the species studied. Given that this is not the case, an are useful indicators of both diet and localized alternative explanation is that Symbiodinium within variation in the base of the marine food web. Our E. mammosa and E. succinea are less mutualistic, data show a wide range in δ13C values (nearly 3.5‰) retaining more of their photosynthates for their own δ13 and a strong correlation between the Chost and metabolism. Such observations of cheating have δ13 Csymbiont, indicating either a common carbon been reported for a variety of microbial symbioses source or tight cycling of carbon between host and (Bronstein, 2001; Porter and Simms, 2014). This symbiont (Supplementary Figure S1). Interestingly, contrasts with species like P. anceps that achieved 13 13 there is little support for utilization of POM in the higher δ Chost than δ Csymbiont, suggesting a larger gorgonian diet. POM from Bocas del Toro had a δ13C carbon subsidy to the host and therefore a more of − 23‰ (Freeman and Thacker, 2011), which is a mutualistic relationship. Interestingly, P. anceps had reflection of a large terrestrial contribution to the highest natural abundance δ13C values reported Almirante Bay (Aronson et al., 2014). The average in this study (Supplementary Figure S1), suggesting δ13C of gorgonians (−17.4‰) greatly exceeded the 1‰ a large contribution of autotrophically derived DIC. trophic enrichment expected between a consumer Although 13C assimilation was not observed in any and its diet. Instead, these values may be enriched dark-incubated Symbiodinium, it was curiously via photosynthetic assimilation of dissolved inorganic evident in all host fractions (Figure 2b). The former δ13 ≈ ‰ δ13 carbon (DIC; CHCO3- 0 ). As such, gorgonian Cis is easily explained by a lack of light to drive a weighted average of DIC and POM contributions photosynthesis. In the dark, host enrichment in the to the auto- and heterotrophic diet, respectively. absence of symbiont photosynthesis must be pro- Ultimately, we limit our interpretation of these data ceeding by anaplerotic pathways, such as fatty-acid as different environmental sources and conditions synthesis and oxidation (DeNiro and Epstein, 1977) among the three collection sites preclude a detailed or by associated microbiota. Microbes other than view of the trophic position of gorgonians based on Symbiodinium could contribute to 13C enrichment natural abundance δ13C. In this regard, the 13Ctracer through similar biosynthetic pathways, as their study was effective in highlighting the relative utiliza- signal is inseparable from the host fraction. Quanti- tion of DIC via photosynthesis among these species. fying the contributions of associated chemoauto- trophic microbes such as cyanobacteria and archaea to holobiont productivity and biogeochemical 13C Tracer reveals the benefits of symbiosis cycling warrants further study. However, the large δ13 4 We have shown that Caribbean gorgonians receive differences in observed Chost enrichments ( 3to important carbon resources from their associated 30-fold) between the light and dark treatment suggest Symbiodinium, which drives significant variation in that associated microbe 13C assimilation was mini- productivity among species. The autotrophic assim- mal relative to Symbiodinium. 13 _ ilation of H CO3 in the light bottles was evident in We found that not all hosts benefit from Symbio- δ13 δ13 Chost and Csymbiont, suggesting a tightly coupled dinium autotrophy. Interestingly, there was no effect 13 δ13 and rapid translocation of C-labeled carbohydrates of light on Plexaurella or Briareum Chost and δ13 δ13 and other biomolecules during the brief incubation Csymbiont (except P. fusifera Csymbiont). Given that 2 δ13 δ13 (Figure 2a). The higher R observed between Csymbiont marginal enrichments of the Chost can be attributed and P/R (Supplementary Figure S2) may indicate to light-independent assimilation (Figure 2b), we superior precision of direct quantification of 13C conclude that Symbiodinium hosted by P. fusifera, assimilation by the holobiont versus the relatively P. nutans and B. asbestinum did not translocate carbon indirect calculation of productivity based on oxygen to their hosts. Indeed, the three species were among metabolism from clonal fragments within separate the lowest with regards to P/R with mean values bottles. o1.0. As such, the interaction between

The ISME Journal Productivity links morphology, symbiont specificity and bleaching DM Baker et al 2627 Symbiodinium and large polyp gorgonian hosts may needed to test these hypotheses among other be more commensal than mutualistic, at least as it Symbiodinium – cnidarian symbioses. pertains to carbon metabolism. However, even though observed 13C assimilation was low in many sea rod species, the role of Symbiodinium may still Gorgonian metabolism and the evolution of symbiosis be critically important for obtaining growth-limiting Across the diversity of gorgonians, there is a strong resources such as nitrogen (Ribes et al., 1998). negative correlation between traits associated with heterotrophy and autotrophy, reflecting strong evo- lutionary and ecological trade-offs between their Symbiont identity and gorgonian productivity divergent life-history strategies. Indeed, we found a Caribbean gorgonians generally associate with mem- link between productivity and the evolutionary bers of Symbiodinium clade B (Baker et al., 2013), trajectories of host species, which may be driven by which is common on Caribbean reefs (Pettay and habitat specialization and niche partitioning within Lajeunesse, 2007). Certain sub-clade types such as B1 highly competitive coral reef environments. The are especially common, leading to the conclusion that strong negative phylogenetic correlation observed this is a generalist symbiont associated with many between polyp size and carbon translocated from hosts (LaJeunesse, 2002). However, Finney et al. symbiont to host suggests that there is an evolu- (2010) revealed that this clade contains substantial tionary trade-off between heterotrophic and auto- genetic variation with evidence of highly specialized trophic modes of nutrition. Species like G. ventalina symbionts among various host species. Such specia- and A. acerosa possess very small polyps compared lization could occur during divergence among host with some sea rods like Eunicea and Plexaurella, species, such has been reported for depth partitioned whose polyps are up to an order of magnitude larger lineages of E. flexuosa and co-evolution of their (Thibaudeau 1983). As a consequence, the density Symbiodinium (Prada et al., 2014). Our comparison and surface area of each polyp, relative to the overall of known Symbiodinium types found evidence of colony size is much higher for the sea fans and symbiont specificity increasing with holobiont pro- plumes than the sea rods we studied. Thus, selection ductivity (Figure 5). Generalist hosts had lower may favor the reduction of feeding structure SA:V if productivities in comparison with specialists, which it increases the amount of light exposure to produc- exhibited a higher degree of autotrophy. The mechan- tive symbionts in the host polyp tissues (Kanwisher ism underlying the enhanced metabolism afforded by and Wainwright, 1967; Wainwright and Dillon, specialized symbioses, including the functional sig- 1969). The converse appears to be true for most sea nificance of octocoral-associated Symbiodinium clade rods, where larger and fewer polyps and the thick, B warrants further investigation (Baker et al., 2013; rod shaped colony morphology would serve to limit Pernice et al., 2014). productivity per unit of biomass but ensure that feeding structures are ready for heterotrophy during changing environmental conditions. Bleaching and gorgonian productivity Over deep time, gorgonian diversification in the During the 2005 mass coral bleaching in Puerto Rico Caribbean was less inhibited by competitive interac- only 8 out of 24 gorgonian species showed visible tions with scleractinians than in the Pacific. With signs of bleaching among four monitored reefs. more niche space available, any species that Remarkably, with the exception of Muricea,no acquired mutualistic Symbiodinium could boost its species suffered subsequent mortality (Prada et al., ability to compete for space. In doing so, selection 2010). However, the corals that readily bleached may have favored more obligate associations with were species with larger polyps and, as we have Symbiodinium with more efficient metabolisms reported here, lower rates of productivity (Figure 6). leading to greater productivity and growth (and thus, Considering the observed specificity among highly competitive abilities) in oligotrophic environments. productive species, we predict that such resistance Such divergence is obvious within the , to bleaching is reflective of the obligate nature of which display a stark contrast between clades these symbioses. Essentially, bleaching may be formed by Gorgonia and Antillogorgia versus the disadvantageous to an obligate host if nutrition is Plexaurella (Figure 3). These two host species are derived exclusively from symbionts. This explains apparent specialists with Symbiodinium clade B1 as the difficulty in experimentally bleaching G. ventalina reported by substantial sampling efforts throughout by elevated temperatures (Kirk et al., 2005), the Caribbean Sea (Andras et al., 2011). Moreover, prolonged incubation in darkness, or with the this specialization may have accelerated the selec- herbicide DCMU (Baker, unpublished data). tion for reduced polyp size and colony morphologies Conversely, in more facultative interactions hosts to enhance light capture (Figure 3). A shared are more likely to expel symbionts when the costs trajectory toward symbiont specificity and autotro- (such as nutrient retention, increased respiration, or phy is further supported by the observation that oxidative damage) outweigh the nutritional benefits, Gorgonia and Antillogorgia are capable of hybridiza- especially if the host can alternatively acquire tion (McFadden et al., 2010). In contrast, we have resources easily by heterotrophy. Further work is shown that Plexaurella do not benefit substantially

The ISME Journal Productivity links morphology, symbiont specificity and bleaching DM Baker et al 2628 from Symbiodinium and hosts are less specific in constructive feedback. Financial support was provided by their Symbiodinium associations (Figures 2 and 3). the Smithsonian Marine Science Network Fellowship to This may reflect a divergence from the Gorgonia/ DMB, the Keck Foundation to MLF, and the U.S. National Antillogorgia sister group to avoid competition for Science Foundation under grants 0829986 and 1208340 space in high light environments. to RWT. While we have examined the benefits of the gorgonian-Symbiodinium symbiosis, further work must address the potential costs of these interac- tions. For example, with changing environmental References conditions (higher SST, eutrophication, acidifica- tion), do the costs of symbiosis outweigh the Andras JP, Kirk NL, Harvell CD (2011). Range-wide benefits? 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