Shedding light: A phylotranscriptomic perspective illuminates the origin of photosymbiosis in marine bivalves

doi:10.21203/rs.2.16100/v4

Background: Photosymbiotic associations between metazoan hosts and photosynthetic dinoflagellates are crucial to the trophic and structural integrity of many marine ecosystems, including coral reefs. Although extensive efforts have been devoted to study the short-term ecological interactions between coral hosts and their symbionts, long-term evolutionary dynamics of photosymbiosis in many marine animals are not well understood. Within Bivalvia, the second largest class of mollusks, obligate photosymbiosis is found in two marine lineages: the giant clams (subfamily Tridacninae) and the heart cockles (subfamily Fraginae), both in the family Cardiidae. Morphologically, giant clams show relatively conservative shell forms whereas photosymbiotic fragines exhibit a diverse suite of anatomical adaptations including flattened shells, leafy mantle extensions, and lens-like microstructural structures. To date, the phylogenetic relationships between these two subfamilies remain poorly resolved, and it is unclear whether photosymbiosis in cardiids originated once or twice.

Results: In this study, we establish a backbone phylogeny for Cardiidae utilizing RNASeq-based transcriptomic data from Tridacninae, Fraginae, and other cardiids. A variety of phylogenomic approaches were used to infer the relationship between the two groups. Our analyses found conflicting gene signals and potential rapid divergence among the lineages. Overall, results support a sister group relationship between Tridacninae and Fraginae, which diverged during the Cretaceous. Although a sister group relationship is recovered, ancestral state reconstruction using maximum likelihood-based methods reveals two independent origins of photosymbiosis, one at the base of Tridacninae and the other within a symbiotic Fraginae clade.

Conclusions: The newly revealed common ancestry between Tridacninae and Fraginae brings a possibility that certain genetic, metabolic, and/or anatomical exadaptation existed in their last common ancestor, which promoted both lineages to independently establish photosymbiosis, possibly in response to the modern expansion of reef habitats.

Evolutionary Biology

Marine and Freshwater Biology

Photosymbiosis

Tridacinae

Fraginae

Symbiodiniaceae

Reef habitat

Photosymbiotic associations between marine organisms and photosynthetic algae allow each partner to thrive in nutrient-deficient environments and to constitute highly productive ecosystems [1]. This mutualistic relationship has repeatedly evolved in diverse eukaryotic lineages ranging from single-celled foraminiferans to metazoans, including corals, acoels, sacoglossans and bivalves, etc. [2]. Although the immediate threat of coral reef symbiont loss (i.e., bleaching) has generated extensive efforts in studying the short-term ecological interactions between coral hosts and symbionts, long-term evolutionary dynamics of photosymbiosis in most marine lineages are poorly understood [3-4]. Therefore, it is crucial to start investigating the origination and extinction patterns of photosymbiosis in a diversity of organisms and identify tractable study systems, so we can better understand and predict how such systems will respond to long-term environmental change.

Among photosymbiotic host organisms, bivalve mollusks pose an evolutionary dilemma. Compared to other invertebrates that house symbionts in translucent tissues, bivalves possess opaque, protective shells that represent an inherent obstacle to symbiont exposure to sunlight. Therefore, extensive morphological or behavioral adaptations are needed to establish successful photosymbiosis. Nonetheless, at least 17 modern or extinct bivalve lineages are known/suspected to form photosymbioses, and each possess(ed) distinct morphological traits adaptive to photosymbiosis [3]. Within modern taxa, at least seven lineages have been reported to harbor photosynthetic algae [2]. However, obligate and confirmed mutualistic associations are only found in two clades within the bivalve family Cardiidae: the well-known giant clams in the subfamily Tridacninae and the less-studied heart cockles in the subfamily Fraginae [5-8]. Tridacninae has a Late Eocene to Recent fossil record with appearance of symbiont-bearing members from the Late Oligocene. Symbiont-bearing Fraginae appear slightly later in the fossil record, from the Late Miocene to Recent [3]. Comparative studies between these two lineages can reveal important insights into the evolution of photosymbiosis in complex organisms.

Giant clams (Tridacninae) were the first documented photosymbiotic bivalves [5]. A dual feeding strategy coupling photosymbiosis with filter-feeding is hypothesized to have resulted in the enormous size of Tridacna gigas, one of the heaviest non-colonial invertebrates known [9]. Giant clams acquire a considerable amount of organic carbon through photosymbiosis [10]. All species in the subfamily possess symbiotic algae from the family Symbiodiniaceae (also found in symbiosis with corals), mostly placed in the genera Symbiodinium, Cladocopium, and Durusdinium [8, 11]. The giant clam harbors extracellular symbionts within a tubular network derived from the digestive system that extends into the mantle tissue [12-13]. The tubules only develop in the presence of the symbionts [14], indicating the existence of responsive host-symbiont interactions.

Ecological and morphological adaptations for the symbionts to obtain sufficient sunlight are apparent in Tridacninae. Unlike most species in the family Cardiidae, tridacnines do not bury in sediment. Instead, they are epibenthic (Fig. 1A) with a great expansion of the posterior body, widely gaping shells and a hypertrophied mantle that is exposed to the light (Fig. 1B). The mantle tissues also contain iridophores that scatter photosynthetically productive wavelength to symbionts distributed in the deeper regions [15].

The subfamily Fraginae is a more diverse but less studied group compared to the Tridacninae. It contains more than fifty species, including one symbiotic clade and one non-symbiotic clade, both reciprocally monophyletic [7]. The photosymbiotic species are exclusive to three genera: Fragum, Corculum, and Lunulicardia, comprising at least 23 species [7]. Part or most of their energy source is derived from algal photosynthesis [8]. Symbiont diversity within Fraginae is less explored, but species examined so far harbor algae belonging to Symbiodiniaceae genera Symbiodinium and Cladocopium [8]. Photosymbiotic fragines host algae within not only the mantle, but also the gill and part of the foot. The symbiont-containing structure is remarkably similar to that documented for giant clams – an extracellular tubular network branching into the symbiont-containing tissues [7-8, 16].

Despite the similarities between Fraginae and Tridacninae on symbiont identity and symbiont-containing structure, morphological and behavioral adaptations are otherwise quite variable. Photosymbiotic fragines are relatively small sized (~1-10 cm) and live in a diverse spectrum of habitats, ranging from shallow reef flats with clear waters to deeper lagoons [7]. Some species are entirely epifaunal (Fig. 1A, D), the rest are shallowly buried in the sediment, as in non-symbiotic cardiids (Fig. 1A). Morphologically, some species possess shells that are very similar to those of non-symbiotic fragines and expose symbionts to light via shell gaping (Fig.1A, C). In contrast, others exhibit highly flattened translucent shells with sophisticated microstructures to enhance light acquisition, referred to as “shell windows” (Fig.1A, D; 17). Those morphological variations suggest that fragines may depend on photosymbiosis to different degrees, and have evolved diverse solutions to maintain the associations.

Given that the two modern photosymbiotic bivalve groups both arose within the family Cardiidae but use radically different strategies, it is natural to ask whether they inherited photosymbiosis from a common ancestor or independently evolved the associations. If photosymbiosis were an ancestral character, it would imply a single origin with subsequent losses of symbiosis in non-symbiotic fragines and possibly other cardiids. If the two subfamilies acquired photosymbiosis separately, it would suggest that their symbiont-harboring system and other metabolic adaptations are a result of convergent evolution.

In order to address the acquisition of photosymbiosis in bivalves, a phylogenetic framework is required. Currently, the phylogenetic relationships within Tridacninae and Fraginae are relatively well resolved, and both groups are monophyletic [7, 18-19]. However, the relationship between the two subfamilies remains poorly understood. One of the first cardiid phylogenies, based on a cladistic analysis of shell microstructure and gut morphology, placed Tridacninae and Fraginae as sister groups [20], but later work incorporating more characters resolved the two subfamilies as distantly related lineages within Cardiidae [21]. The first molecular phylogeny constructed to address this issue was based on a single genetic marker (nuclear 18S rRNA), grouping Tridacninae with non-symbiotic cardiids in the subfamilies Trachycardiinae and Laevicardiinae, leaving Fraginae as sister group to the rest [22]. The most recent multi-gene phylogenetic analyses also failed to reliably resolve the positions of Tridacninae and Fraginae [19]. The placement of Tridacninae was not consistent and was recovered as sister group to various subfamilies (e.g., Lymnocardiinae and Cardiinae) in different analyses. The same situation characterizes the placement of Fraginae as its phylogenetic position has shifted with different phylogenetic reconstruction methods. A previous thesis [23] utilizing the same multi-gene dataset, however, did recover a sister relationship between Tridacninae and Fraginae in one Bayesian analysis. Nonetheless, these conflicting results highlight the difficulties in resolving relationships based on three Sanger-based markers, thus hindering our understanding of evolution of photosymbiosis in bivalves.

The recent development of phylogenomic approaches to bivalve phylogenies [24-26] has demonstrated the possibility of resolving difficult nodes according to previous Sanger-based approaches (e.g., [27]). Therefore, we adopted an RNAseq approach and generated transcriptomic data for Tridacninae, Fraginae and other cardiids with the aim to better understand the evolutionary of photosymbiosis in Cardiidae.

Cardiid phylogeny

This is the first phylogenetic study using transcriptomic data that aims to resolve the relationships between the two cardiid subfamilies which contain photosymbiotic taxa. The best corroborated hypothesis supports a sister relationship between Tridacninae and Fraginae, both recovered as monophyletic (Fig. 3). Other than the position of Tridacninae, the placement of all other cardiid subfamilies is consistent with the most recent multi-gene Cardiidae phylogeny comprising 110 species [19].

Our analyses highlighted the pervasive challenge to reconstruct a well-resolved and highly supported cardiid phylogeny. The fundamental reconstruction difficulty stems from the diversification process of Tridacninae, photosymbiotic, and non-symbiotic Fraginae. Our results show that the branches leading to the three crown groups are long and subtended by very short internodes, indicating the divergence among these groups was rapid. These ancient but fast diversification events are inherently difficult to resolve [28]. Some of the major problems include: not enough data to resolve the nodes; molecular data not variable enough at the appropriate level; strong conflicts among genes; and inadequate substitution models [28]. We discuss these concerns in the following paragraphs.

Firstly, current studies on cardiid phylogeny may suffer from some level of data limitation. For example, although Herrera et al. 2015 [19] had excellent taxon sampling through Cardiidae, the gene coverage was low. In the present study, taxon sampling was limited due to the need to obtain high quality transcriptomes, which requires freshly collected specimens. These constraints could be overcome by incorporating DNA-based phylogenomic approaches (e.g., RADseq, exome capture, etc.) using well-preserved museum specimens.

In addition, some of the data may lack the appropriate level of resolution. For example, the ML Analysis on Matrix 2 resulted in poorly resolved topologies with low bootstrap support values. This observation where large, albeit more incomplete matrices provided better resolution than small and more complete matrices is not unique to our dataset (e.g., [24, 29]). It is likely influenced by gene choice in the more complete matrix [29]. If a gene is present in more taxa, it is likely to be relatively conserved; hence lacking enough genetic variation to resolve rapid divergence, as seen in some of our individual gene trees. These very conserved/slow-evolving genes might be overrepresented in a smaller matrix, but contribute minimally to any phylogenetic resolution.

Gene conflicts are also apparent in our dataset based on the supernetwork and submatrices analyses, in part may be explained by incomplete lineage sorting at these rapid-divergence nodes. In addition, genes with different rates of evolution gave rise to divergent topologies. Some faster evolving genes (e.g., Matricis Q-V) produced clearly problematic topologies, such as placing photosymbiotic Fraginae as sister clade to all other cardiids. These rapidly evolving genes might be subject to more genetic saturation and could be under the influence of strong selection, all of which hinder their ability to resolve deep phylogenetic nodes and conflict with other more informative genes.

Lastly, sequence compositional heterogeneity is known to be especially problematic for inferring short internal nodes [30]. However, based on the low level of composition heterogeneity in our dataset and the Dayoff recoding analysis, it is unlikely to produce significant bias in the results.

In sum, we have found that overly conservative genes and fast evolving genes don’t provide informative resolution to the nodes of interest, a phenomenon observed in other taxonomic groups [31]. These genes generate conflicting topologies and could be responsible for the observed low bootstrap values. On the contrary, ML analyses on the large matrix, moderately evolved genes, as well as the Bayesian analysis, all support a sister relationship between Tridacninae and Fraginae.

Implications on photosymbiosis evolution

Our analyses support two independent origins of photosymbiosis in Cardiidae. This result should be robust to phylogenetic uncertainties, because a non-sister relationship between Tridacninae and Fraginae will only reinforce the independent evolution scenario. Our ancestral state reconstruction was model-based; an alternative parsimony-based analyses would deem “two independent origins” or “one origin and one loss (in the non-symbiotic Fraginae clade)” equally likely. However, the latter scenario is not well supported because fossil record indicates that visible shell adaptations in both Tridacninae and Fraginae appeared after Late Oligocene [3, 32]. If the common ancestor of the two subfamilies is photosymbiotic, it would imply that photosymbiosis was established in late Cretaceous but persisted more than 30 Ma without any apparent shell adaptations. It is much more probable that there are two separate origins of photosymbiosis with shell adaptations evolved shortly after.

The repeated evolution of bivalve photosymbiosis suggests its adaptive advantage. The relatively rapid crown group diversification, coupled with morphological responses [3], is consistent with criteria for adaptive radiation - the generation of new species exhibiting pronounced morphological divergence over relatively short timeframes, typically in response to new environmental conditions [33]. In photosymbiotic cardiids, the species number is modest compared to other documented examples. However, this might be due to the lack of systematics research in these groups, as more studies have started to reveal their hidden diversities (e.g., [34]).

As for most cardiid lineages, Tridacninae and Fraginae have inferred ancestral distributions that span the Indo-Pacific; Tridacninae has a wider ancestral range, reaching the western temperate northern Pacific [19]. Given that the origin of photosymbiosis in both subfamilies overlaps with the expansion of modern reefs, it is likely that the formation of shallow marine habitat in the Indo-Pacific is a key environmental driver for bivalve photosymbiotic adaptation. This is further supported by the fact that photosymbiotic fraginaes has a sister non-symbiotic lineages that still inhabits deep sandy bottoms [7], possibly representing the ancestral ecology of these bivalves before they shifted to shallower habitat. Kiessling 2009 [35] proposed a fundamental question regarding the formation of reef biodiversity - Have reef taxa originated within reefs or have they evolved somewhere else then moved into reef habitats? Our results indicate that at least for photosymbiotic bivalves, they have likely originated and diversified within the reef habitats. More biogeographical, palaeontological and phylogenetic data are certainly needed to further corroborate this point.

Both Tridacninae and photosymbiotic Fraginae are thought to exhibit phenotypic adaptations to benefit the symbionts, making some species’ shell and mantle morphologies drastically different from typical cardiids (Fig. 1). What is striking, and little mentioned until now, is the divergent morphological trends in photosymbiotic fragines contrasted with the uniform morphological trends in Tridacninae, essentially two different responses to expose symbionts to optimal irradiance in sister lineages. Previous studies of adaptive radiations have well highlighted examples of divergent morphological responses to newly available niches (e.g., [36]), as seen in fragines. But uniform morphological responses, as seen in tridacnines, are less documented. Both strategies have advantages. While divergent morphologies provide highly specialized adaptations to different fine-scale niches, an uniform/static morphology may enable the lineages to become broadly adapted generalists to mediate environmental fluctuations [37]. The different morphological evolution patterns in photosymbiotic cardiid suggests that in the acquisition of a key innovation, historical morphological contingencies (e.g., opaque heavy shell, unexposed tissues, infaunal habit) and common ancestry do not predict the directionality of morphological evolution, not even for sister lineages that use the same ecological strategy to adapt to similar habitats.

Despite the versatile shell and mantle morphologies in Fraginae and Tridacninae, their symbiont-containing tubular system share striking similarities. Both stem from the digestive system of the hosts and form tertiary tubular networks [13, 16]. The only other similar molluscan structures are found in some marine gastropods who temporarily maintain algae or chloroplasts in their tissues [38]. The development of giant clam tubules are only triggered by the presence of symbionts [14], indicating the acquisition of photosymbiosis is a highly interactive process between hosts and symbionts.

It has long been hypothesized that different photosymbiotic bivalves express homologous genes to build the tubular system, in response to similar symbiont signals [16]. The newly-found sister relationship between Tridacninae and Fraginae lend further support to this theory, suggesting that genes homologous to the tubular-formation ones are ancestral to the two lineages. It is even possible that the genes are ancestral to bivalve and gastropods, as the latter can form similar anatomical structures. Given that the different mollusk lineages evolved photosymbiosis independently, it is likely that the gene/anatomical level similarities are generated from parallel evolution. That is, each lineage independently coopted similar genetic mechanisms for generating symbiont-containing structures. Molecular level parallel evolution has been shown in photosymbiotic systems. For example, both corals and giant clams repurpose the expression of the vacuolar H+-ATPase gene (VHA) to facilitate their carbon concentrating process and promote algal photosynthesis, even though the two lineages are very distantly related [39, 40]. Our analyses further support that VHA is a conserved gene, ancestral to bivalves, corals, and other animals. Photosymbiotic bivalves don’t possess any “special” version of VHA; the amino acid sequence is the same as the ones found in non-symbiotic taxa. It is more likely that the adaptation to photosymbiosis is realized at the regulatory/expression level, where VHA is highly expressed in tissues that harbor symbionts. In contrast with the relatively labile shell and mantle adaptations, it is possible that the evolution of host metabolomic and developmental adaptations are more constrained genetically, resulting in similar mechanisms in diverse animal groups. More in-depth studies on the molecular mechanisms behind photosymbiosis are needed to gain better insights.

To further investigate macroevolution of bivalve photosymbiosis, a better understanding of potential photosymbiotic fossil taxa is essential. However, they are challenging to identify in the fossil record based on shell morphology alone [2]. Although photosymbiotic bivalves possess a suite of morphological traits to enhance light exposure, almost all characters used to support photosymbiotic condition are found in modern non-photosymbiotic bivalves. Examples include permanent shell gaping (Family Limidae, Galeommatidae), enlarged mantle (Galeommatidae), or transparent shells (Placunidae, Pinnidae). The only photosymbiotic-exclusive shell character is the shell “window” microstructure [17], but many photosymbiotic bivalve species do not possess it, and similar features have not been found in fossil taxa. Similarly, other identifiable photosymbiotic ecological traits (shallow water distribution, fast growth, reclining habits) are not unique to photosymbiotic bivalves either [2]. Therefore, alternative methods need to be developed (e.g. better isotopic metrics, metabolite signatures) if we wish to fully understand the evolutionary dynamics of bivalve photosymbiosis.

Lastly, further characterization of host-symbiont diversity and interactions are essential for developing a full picture of animal-algal photosymbiosis. A comprehensive documentation of host-symbiont biodiversity can provide insights into photosymbiotic mechanisms in diverse habitats and with different organismal complexities. For example, Li et al. 2018 [8] demonstrated that fragine species at varying water depths have different dependencies on algal photosynthesis, with deeper host species utilizing less photosynthetically derived carbon and exhibiting less shell modifications. Therefore, it is highly likely that host-symbiont interactions and reliance vary greatly among host-symbiont pairs, depending on symbiont types, as well as degrees of host adaptations. Li et al. 2018 [8] summarized known symbiont diversity in giant clams and showed that several fragine species harbor symbionts from the genus Cladocopium, which also occupies cnidarians, giant clams, and foraminifera. However, this by no means captured the full spectrum of potential symbiont diversity in photosymbiotic bivalves, especially when new host species are discovered regularly (e.g., [34]). In most photosymbiotic bivalve systems, the roles symbionts play in host ontogeny, reproduction, response to environmental fluctuation, etc. are unexplored, all of which require our continued effort to identify and characterizing existing photosymbiotic diversity.

Transcriptome assembly and data quality

The number of used reads, contigs, and other values to assess the quality of the assembled transcriptomes can be found in Table 1. Orthology assessment of this 33-taxon dataset with the OMA stand-alone algorithm recovered 244,752 orthogroups. The two super-matrices (Fig. 2 and Additional file 3: Fig. S1) respectively yielded 1108 (Matrix 1: occupancy of > 50%; 280,086 aa) and 313 (Matrix 2: occupancy of > 75%; 78,272 aa) orthologs. Levels of compositional heterogeneity were low in most orthogroups (Additional file 4: Fig. S2). The relative composition frequency variability (RCFV) per taxon and per amino acid ranged from 0.0002 to 0.001, representing overall compositional homogeneity throughout all amino acids and taxa included in Matrix 1.

Phylogenetic analyses and gene evolution

All 26 analyses (Table 2) recovered strong support for the monophyly of Tridacninae, Trachycardiinae, Laevicardiinae, Cardiinae, Lymnocardiinae, photosymbiotic Fraginae, and non-symbiotic Fraginae. Monophyly of Fraginae (including both symbiotic and non-symbiotic clades) was supported in 63% of the analyses. The clade composed of Trachycardiinae, Laevicardiinae, and Cardiinae was supported in all analyses. The placement of Lymmnocardiinae was less certain, although 60% of the analyses recovered it as the sister group to all other Cardiidae.

The most supported overall topology by different analyses (Fig. 3A) is consistent with the result obtained from the maximum likelihood (ML) analysis on Matrix 1 (Fig. 3C), which revealed a sister group relationship between Tridacninae and Fraginae, although with moderate bootstrap support (65/100). After Dayhoff recoding of Matrix 1, the same topology was obtained, as for the PhyloBayes analysis of Matrix 2, although the two independent chains did not fully converge. The conflict was mostly driven by the position of Lymnocardiinae as the sister group to all other cardiids. The posterior probability for the Tridacninae and Fraginae sister group relationship is 0.99 and does not show conflict between the two chains. Analyses of submatrices C, E, and G-I also recovered this common topology. The second most common topology is largely consistent with the first one, except that Lymnocardinae is placed as the sister group of Tridacninae (Fig. 3B). Other matrices supporting this topology include the slowest evolving submatrix A, and three relatively fast evolving ones (J, M, O).

Besides the most supported topologies, an additional 11 topologies were supported by only one or two matrices each. In particular, ML analysis on Matrix 2 placed Fraginae as sister group to a clade composed of (((Trachycardiinae, Laevicardiinae), Cardiinae), Lymnocardiinae), and recovered Tridacninae as the sister group to all other Cardiidae. However, the bootstrap support values for the backbone topology are low (<60).

The supernetwork analyses on the two main matrices showed relatively long branches leading to Tridacninae, photosymbiotic Fraginae, and non-photosymbiotic Fraginae respectively (Additional file 5: Fig. S3), a result consistent with the strong support for these nodes obtained from the phylogenetic analyses. However, substantial reticulation was observed at the node leading to these three clades, indicating strong gene conflict in resolving their relationships. This is likely the reason for the low bootstrap supports obtained in the ML analyses.

BLAST results for the fastest 10% and slowest 10% genes are shown in Additional file 1 and 2: Table S1 and S2. The slow-evolving genes are composed of a higher proportion of nuclear ribosomal genes compared to the fast evolving ones (13% vs. 0%). The fast-evolving genes have a higher number of mitochondrial genes compared to the conserved genes (23% vs. 1%). This pattern is in line with what is currently known about the molecular evolution rates of these gene groups.

The coral V-type H+-ATPase (VHA, photosymbiosis-related gene) BLAST searches recovered VHA sequences from eight Tridacninae and Fraginae species. Tridacninae and Fraginae VHA amino acid sequences are mostly identical, except for Fragum fragum, which has three (out of 137) amino acid substitutions. Besides F. fragum, there are no sequence differences between the photosymbiotic and non-symbiotic taxa (Additional file 6: Fig. S4). The coral VHA differs from bivalve sequences by 6-11 amino acid substitutions. Both coral and bivalve sequences share ~85% similarity with the Symbiodiniaceae VHA. All animal VHA genes formed a monophyletic group with 100% bootstrap support.

Fossil calibration and ancestral state reconstruction

The cardiid dating estimates based on the most common topology are shown in Figure 4. Tridacninae and Fraginae diverged in the Late Cretaceous. Soon after, the symbiotic and non-symbiotic Fraginae lineages also separated. Those long branches eventually lead to relatively recent and rapid diversification of the photosymbiotic crown groups. The diversification of photosymbiotic fragines is dated at around 15 Ma (SD = 3.1). The diversification of crown Tridacninae is ~27 Ma (SD = 4.4), and the radiation of the genus Tridacna dates back to ~6 Mya (SD = 6.3). Age estimates of the other non-focal subfamilies, including Trachycardiinae, Laevicardiinae, Cardiinae, and Lymnocardiinae, are largely within the range of Herrera et al. 2015 [19].

Ancestral state reconstructions using the Binary State Speciation and Extinction (BiSSE) and mk2 models yield congruent results (Fig. 4). Both identified two independent origins of photosymbioses in Cardiidae, one within Tridacninae and the other within the photosymbiotic Fraginae. Comparison with reef abundance through time shows that the onset of Tridacninae photosymbiosis corresponds to the beginning of a rapid global reef expansion, while radiations of the genus Tridacna and photosymbiotic fragines both occurred within peak reef abundance.