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Mycosporine-like amino acid content in four species of sea anemones in the genus Anthopleura reflects phylogenetic but not environmental or symbiotic relationships.

We examine the occurrence of UV-absorbing, mycosporine-like amino acids (MAAs) in four sympatric species of sea anemones in the genus Anthopleura, all collected from intertidal habitats on the Pacific Coast of temperate North America. We compare patterns of MAAs in A. elegantissima of several types: specimens having predominately zooxanthellae (dinoflagellates comprising at least two species) or zoochlorellae as symbionts; those containing algal endosymbionts of both kinds, and naturally occurring aposymbiotic specimens that lack the endosymbionts typically found in most specimens. We also compare MAAs in zooxanthellate specimens of A. sola and A. xanthogrammica, and specimens from the asymbiotic species A. artemisia. Our findings indicate that the complements of the four major MAAs in these species of Anthopleura (mycosporine-taurine, shinorine, porphyra-334, and mycosporine-2 glycine) broadly reflect phylogenetic differences among the anemones rather than the taxon of endosymbionts, presence or absence of s ymbionts, or environmental factors. An exception, however, occurs in A. elegantissima, where mycosporine-2 glycine increases in concentration with the density of zooxanthellae. Our evidence also shows that A. elegantissima can accumulate MAAs from its food, which may explain the occasional occurrence of minor MAAs in some individuals.

Introduction

Mycosporine-like amino acids (MAAs) are small (244-374 Da), ultraviolet-absorbing ([lambda].sub.max] = 309-360 nm) molecules that occur in virtually all taxa of marine and freshwater cyanobacteria, algae, and metazoans (reviewed by Bandaranayake, 1998; Dunlap and Shick, 1998; Karentz, 2001; Shick and Dunlap, 2002). Structural diversity among the 20-plus known MAAs and their precursors results from substitutions of various amines, amino acids, and amino alcohols at two conjugated positions of the UV-absorbing chromophore of the MAA base structure (see aforementioned reviews). The often-observed correlation between the exposure of organisms to solar or artificial ultraviolet radiation (UVR) and their concentration of MAAs (see reviews), plus optical considerations (Garcia-Pichel, 1994), suggest a role as UV sunscreens, a contention that has been verified experimentally (Adams and Shick, 1996, 2001; Neale et al., 1998).

As products of the early shikimic acid pathway, MAAs (Shick et al., 1999) and the closely related fungal mycosporines (Favre-Bonvin et al., 1987) are expected to be synthesized by algae, cyanobacteria, fungi, and perhaps bacteria, but not by metazoans. This is because animals lack this biochemical pathway, as inferred from their inability to synthesize shikimate-derived aromatic amino acids (Kishore and Shah, 1988; Bentley, 1990; Haslam, 1993; Herrmann and Weaver, 1999), albeit apparently not from any demonstration that metazoans lack the enzymes of the shikimic acid pathway or the nucleotide sequences coding these enzymes (Shick and Dunlap, 2002). Therefore, the MAAs found in diverse marine microbial-invertebrate phototrophic symbioses were thought at first simply to originate in the algal or cyanobacterial partner (reviewed by Shick and Dunlap, 2002).

Carroll and Shick (1996) first confirmed experimentally the suggestion by Chalker et al. (1988) that non-symbiotic metazoans also containing MAAs obtain them from their diet. But the MAA complement in consumers does not always coincide with that in their diet, nor does the MAA complement in symbioses always match that produced by their microbial endosymbionts in vitro. Thus, MAAs in the diet may be modified by bacteria in the consumer's digestive tract, and MAAs synthesized by a symbiont could be modified after translocation to the host (see: Dunlap and Shick, 1998; Shick and Dunlap, 2002; and references therein).

Accounting for the MAAs found in a given organism or symbiotic consortium is therefore complicated and requires a detailed knowledge of the diet and the MAAs available therein; the MAAs that the symbionts can produce; and the capacity of the animal to absorb, retain, and modify dietary MAAs and those synthesized by its endosymbionts. These factors may vary spatially (Adams et al., 2001), temporally (including ontogenetically [Adams and Shick, 1996, 2001] and during growth [Sebens, 1981]), and genotypically (Banaszak et al., 2000).

Sea anemones in the genus Anthopleura contain taxonomically diverse microalgal endosymbionts that vary with latitude (LaJeunesse and Trench, 2000; Secord and Augustine, 2000), and four species of anemones in this genus overlap in intertidal and shallow subtidal habitats along the Pacific Coast of temperate North America (Fig. 1). From this array, we have selected sympatric populations to test the effects of the foregoing factors (especially the role of exposure to solar radiation, endosymbionts, and diet) on the number, profile, and concentration of MAAs.

We recognized early on that symbiotic and aposymbiotic individuals of A. elegantissima present exceptions to the empirically derived paradigms that concentrations of MAAs are determined by exposure to UVR, and that endosymbiotic dinoflagellates (zooxanthellae, Symbiodinium spp.) are the source of the MAAs. Exposing both symbiotic and aposymbiotic anemones to natural or artificial UVR does not affect the concentrations or complement of MAAs (Scelfo, 1988; Stochaj et al., 1994; Banaszak and Trench, 1995), nor does S. californium isolated from A. elegantissima produce MAAs when cultured under UVR (Banaszak and Trench, 1995). A broader study of the MAAs in other species in this genus would provide a comparative context in which to examine these exceptions and clarify the contribution of the several factors involved in the accumulation of the observed complements of MAAs.

Populations of A. elegantissima and A. xanthogrammica north of Central California host two taxa of phototrophic endosymbionts: zoochlorellae, which are green Chiorellalike chlorophytes (Muscatine, 1971), and the dinofiagellate S. muscatinei (LaJeunesse and Trench, 2000). An individual anemone may contain exclusively one or the other type of alga, or a combination of both (Muscatine, 1971). The balance between these algae may depend on their differential sensitivity to temperature and sunlight (O'Brien, 1980; O'Brien and Wyttenbach, 1980; Saunders and MullerParker, 1997; Secord and Augustine, 2000). Moreover, zoochlorellae translocate, to the host, photosynthetic products that differ qualitatively from zooxanthella photosynthate (Minnick and McCloskey, cited by Verde and McCloskey, 1996), and zooxanthellae contribute more reduced carbon to the host's nutrition than do zoochlorellae (Verde and McCloskey, 1996). Such physiological differences in the algae, and the great evolutionary distance between these algal phyla, might be expected to yield rather different MAA complements in individual sea anemones harboring one or the other endosymbiont--if indeed zoochlorellae (like some other chlorophytes: Karentz, 2001) and S. muscatinei can produce MAAs. In addition, populations of A. elegantissima in Central and Southern California host only zooxanthellae (Secord and Augustine, 2000), in this case both S. muscatinei and S. californium (LaJeunesse and Trench, 2000).

In Central California, A. sola and A. xanthogrammica are sympatric with A. elegantissima, often cohabiting the same sites, where all three species harbor only zooxanthellae (Pearse and Francis, 2000). The species of Symbiodinium that occur in A. sola and A. xanthogrammica have not been determined, so these anemones may not host algae capable of synthesizing MAAs.

The asymbiotic species A. artemisia rarely (or, more likely, never: see discussion in Pearse and Francis, 2000) contains endosymbiotic algae. Therefore, it should contain only MAAs obtained or modified from its diet. The diet of A. artemisia is probably similar to that of A. elegantissima, because these two species co-occur at the same sites in Central California and have a similar body size (which would enable them to capture similar prey). Thus, any differences in the MAAs in these two species might arise from secondary modification of dietary MAAs and not from the presence or absence of symbionts. This is suggested by the finding that symbiotic and aposymbiotic specimens of A. elegantissima contain an identical complement of MAAs (Stochaj et al., 1994; Banaszak and Trench, 1995). Size-dependent dietary differences between zooxanthellate A. elegantissima and A. xanthogrammica (Sebens, 1981), and, presumably, between A. elegantissima and the larger A. sola, may affect the MAAs they ingest in their prey, and result in different MAA complements among the anemones independently of those derived from their endosymbionts. We present the results of a simple experiment to show that A. elegantissima can indeed absorb and retain MAAs from its food.

Finally, a phylogeny based on mitochondrial DNA sequences is available for many species in the genus Anthopleura (Geller and Walton, 2001). This provides the opportunity to examine the provenance and presumed adaptive role of MAAs in a phylogenetic context (Mangum and Hochachka, 1998). Moreover, MAAs might be useful characters to help resolve the phylogenetic trichotomy of A. elegantissima, A. sola, and A. xanthogrammica, the interrelationships of which have not yet been clarified by DNA sequencing.

Materials and Methods

Collection of sea anemones

Specimens of four species of Anthopleura were collected in the summers of 1993 and 1996 in the States of Washington and California. Except as noted, all sea anemones were shipped alive to Orono, Maine, immediately after collection. The anemones were blotted, weighed, minced with a razor blade, and either analyzed immediately for MAAs, or lyophilized. If the material was lyophilized, its dry weight ([W.sub.d]) was first measured, and it was then stored at -79 [degrees]C prior to biochemical analysis.

Green, brown, and mixed specimens of Anthopleura elegantissima (for descriptions, see Saunders and Muller-Parker, 1997) were taken from a single rock at about +0.6 m (MLLW) tidal height at Anaco Beach, Fidalgo Island, Washington (48[degrees]29'N, 122[degrees]42'W). We chose this location (Fig. 1) because the anemones there harbor both zooxanthellae and zoochlorellae (Saunders and Muller-Parker, 1997). Differences in their oral disc pigmentation indicated that most of the anemones belonged to different clones, and we used this criterion to select the specimens for analysis, precluding clonemates.

Sea anemones from California were collected in 1996 from Santa Cruz County, a location that is south of the southern limits of zoochlorellae in A. xanthogrammica (Bodega Bay area) and A. elegantissima (central Oregon) (Fig. 1). Specimens of A. artemisia, A. elegantissima, and A. sola, and large specimens of A. xanthogrammica, were taken from various shore levels at Opal Cliffs near the seaward end of 41St Avenue, Capitola (36[degrees]58'N, 121[degrees]57'W); small individuals of A. xanthogrammica were collected at Scott Creek Beach (37[degrees]02'N, 122[degrees] 13'W). Specimens of A. elegantissima came from distinct clonal aggregations of individuals differing in oral disc pattern, and one individual per clone was analyzed. The small specimens of A. xanthogrammica were collected in May and held in outdoor tanks without feeding until being shipped to Orono with the other sea anemones collected in August. Aposymbiotic individuals of A. elegantissima that had been collected locally and maintained in darkness in the laboratory in flowing seawater at seasonal temperatures for at least a year were also included. They were fed previously frozen adult brine shrimps every week or 2 weeks.

Determination of algal complement

Several tentacles were snipped from each sea anemone before it was analyzed for MAAs. The tentacles were placed in a drop of artificial seawater on a microscope slide and crushed with a cover slip; the slurry was transferred to a hemacytometer, diluted if necessary, and 500-1000 algal cells were counted. Zoochlorellae and zooxanthellae were readily distinguishable by size and color (Secord and Augustine, 2000). Because the proportions of zoochlorellae and zooxanthellae may vary between the tentacles and column (G. Muller-Parker, Western Washington University, pers. comm.), specimens of A. elegantissima from Washington that contained 100% of either algal type in their tentacles were examined further. A section of column wall of such specimens was excised, crushed, and examined for its algal complement to confirm that only one algal taxon was present. As noted above, zoochlorellae do not occur in Anthopleura spp. from Santa Cruz. Therefore, to confirm the singular presence of zooxanthellae in specimens from thi s location, or the lack of algal endosymbionts in A. artemisia and aposymbiotic specimens of A. elegantissima, only the tentacles were examined.

Feeding experiment

The distinctive complement of MAAs in the ocular lenses of fishes (Dunlap et al., 1989) suggested the use of such lenses as sources of unambiguous dietary markers for a feeding study. The lenses were excised from the lumpfish, Cyclopterus lumpus, from Mt. Desert Island, Maine, and their wet weight ([W.sub.w]) was measured. Preliminary analyses by D.S. Mason (University of Maine) indicated the presence of an unknown MAA. Based on its high absorbance at 357 nm and its co-elution with an identically absorbing compound from the red macroalga Palmaria palmata, this unknown MAA was tentatively identified as usujirene (Sekikawa et al., 1980; Adams et al., 2001). Because no quantitative standard was available for the presumed usujirene, its concentrations were approximated by matching its extinction coefficient to that of its trans isomer palythene (Adams et al., 2001). Because the cis isomer will have a lower molar extinction coefficient than the trans isomer, this method will slightly overestimate the concentration of usujirene. Preliminary determinations indicated that both lenses from a single fish have the same complement and concentrations of MAAs, so that the amount of MAA available to an anemone ingesting one lens could be estimated from the MAA content of the contralateral lens from the same fish. One lens from each of two fish was frozen at -- 79[degrees]C for later analysis of MAAs, and the other lens was fed to one of two clonemates of A. elegantissima, which were collected from the south jetty at the entrance to Bodega Bay, California (38[degrees]19'N, 123[degrees]02'W) and held in the laboratory at Orono for 2 weeks at 15 [degrees]C, without feeding, prior to the experiment. One anemone was frozen and lyophilized 4 days after feeding, and the second 8 days after feeding. The MAA concentrations in the fed anemones were compared with those in three additional clonemates (controls) that were not fed lenses.

Analyses of MAAs

Fresh, minced sea anemones or frozen lumpfish lenses were extracted in a refrigerator for 1 h in each of three changes of 100% HPLC-grade methanol; the material was sonicated for 30 s during the first extraction. Lyophilized tissues were similarly extracted, but in 80% aqueous methanol. To remove lipids and some lipophilic pigments, extracts of sea anemones were passed through a C18 Sep Pak Plus (Waters Corp., Milford, MA) before being injected onto the HPLC column.

Mycosporine-like amino acids were analyzed using the HPLC method described by Stochaj et al. (1994) and using quantitative standards prepared by W.C. Dunlap. The present analyses were carried out on a 250 mm X 4.6 mm Phenosphere 5-[micro]m C8 column (Phenomenex, Torrance, CA). The mobile phase for methanolic extracts of sea anemones consisted of 55% aqueous methanol with 0.1% acetic acid, this concentration of methanol providing the best separation of mycosporine-2 glycine from porphyra-334 (Stochaj et al., 1994). MAAs from extracts of lumpfish lenses, and of anemones consuming them, were also separated using a mobile phase of 25% aqueous methanol and 0.1% acetic acid to resolve the less polar MAAs. Detection was at 334 and 310 nm for all specimens, and also at 357 nm for lenses and anemones used in the feeding experiment.

Taxonomic affinities of MAAs

Relationships among the four sympatric species of Anthopleura from Santa Cruz based on their MAA complements were examined by cluster analysis. Only the four MAAs that occurred in every specimen of all species studied were used in this analysis. Each of the four MAAs common to all species was expressed, for each specimen, as a molar fraction of the total MAA pool, and a matrix of distances was calculated using the average Euclidean distance (Sneath and Sokal, 1973). Distances were clustered using the unweighted pair group method algorithm (UPGMA), and the phenogram was constructed with Statistica, release 5.1 (StatSoft, Inc., Tulsa, OK).

Results

MAA and algal complements in A. elegantissima

In the 1993 collection of A. elegantissima from Washington, five MAAs were present in quantifiable amounts: mycosporine-taurine, shinorine, porphyra-334, mycosporine-2 glycine, and palythine, with mycosporine-2 glycine being the most concentrated. When the data for the four predominant MAAs were pooled for all specimens regardless of their algal complement, mycosporine-2 glycine accounted for half of the total concentration of MAAs (Fig. 2A). The highly variable level of palythine was always less than 10% of that of the next-most-concentrated MAA and is not shown here. There was no obvious trend in the concentration of individual MAAs in anemones having mixed algal complements up to about 50% zooxanthellae: 50% zoochlorellae. However, the concentration of mycosporine-2 glycine in anemones harboring zooxanthellae exclusively was substantially greater than in anemones having mixed symbionts (Fig. 2B). There was a significant, positive relation between the concentration of this MAA and the arcsinetransformed pro portion of zooxanthellae in the anemones ([r.sup.2] = 0.816, P = 0.036, n = 5). No other MAA concentration showed any significant relationship to the proportion of zooxanthellae (P > 0.05 in all cases).

In the 1996 collection from the same site, the sea anemones had a more balanced distribution of mycosporinetaurine and mycosporine-2 glycine, and intermediate, similar proportions of shinorine and porphyra-334, regardless of whether they harbored >90% zoochlorellae or >90% zooxanthellae (Fig. 3A and 3B). Of these four MAAs, Student's t tests on arcsine-transformed data indicated that the molar proportions of three of them did not differ significantly between the predominantly zoochlorellate and predominantly zooxanthellate specimens; only shinorine showed a difference, its proportional concentration being marginally greater in the zoochlorellate anemones (t = 2.852, d.f. = 4, P = 0.046).

Complements and concentrations of MAAs in sympatric species of Anthopleura

Microscopic examination of the sea anemones from Santa Cruz confirmed that A. artemisia and aposymbiotic specimens of A. elegantissima lacked algal endosymbionts, and that A. elegantissima, A. sola, and A. xanthogrammica harbored only zooxanthellae. Collectively, these four species (including zooxanthellate and aposymbiotic individuals of A. elegantissima and large and small specimens of A. xanthogrammica) contained seven MAAs in quantifiable amounts, but only four of these (mycosporine-taurine, shinorine, porphyra-334, and mycosporine-2 glycine) occurred in all individuals of all species (Fig. 4). Palythine and palythene occurred in some specimens of all species except for A. artemisia, which never contained these MAAs but uniquely contained mycosporine-glycine:valine. The total concentration of the four common MAAs (Fig. 5) varied interspecifically [ANOVA, [F.sub.5, 12]) = 11.923, P < 0.001] among the anemones from Santa Cruz. Both zoochlorellate and zooxanthellate specimens of A. elegantissima from Washing ton (1996 collection) had the same apparent concentration of these MAAs as their zooxanthellate conspecifics from Santa Cruz. (Data for the Washington specimens were included in Fig. 5 for heuristic reasons, but were excluded from the statistical analysis of the sympatric populations from California. Zooxanthellate and zoochlorellate specimens from Washington did not differ in their total concentration of MAAs [t test, P > 0.05].) Student-Newmann-Keuls multiple comparison tests revealed the following relationships in the anemones from Santa Cruz. The asymbiotic species, A. artemisia, had the lowest absolute concentration of MAAs, overlapping with that in A. sola, which in turn overlapped with that in large specimens of A. xanthogrammica. The highest concentration occurred in A. elegantissima (whether zooxanthellate or aposymbiotic) and small specimens of A. xanthogrammica.

To facilitate interspecific comparisons, the molar proportions of the four common MAAs in the Santa Cruz specimens are shown in Figure 6, where several patterns are evident. A. elegantissima presented the same relative distribution of MAAs as seen in the 1996 collection in Washington: approximately equal proportions of mycosporinetaurine and mycosporine-2 glycine, and intermediate proportions (~0.05-0.15 molar fraction) of shinorine and porphyra-334. A. xanthogrammica was distinctly different, with mycosporine-taurine and shinorine each representing about 0.45 of the total MAAs. A. artemisia differed from both of the foregoing species in that mycosporine-taurine accounted for 0.8 of the total MAAs, and mycosporine-2 glycine was scarcely present. Aposymbiotic individuals of A. elegantissima showed an intermediate situation, having a proportion of mycosporine-2 glycine that was smaller than in zooxanthellate conspecifics but larger than in A. artemisia or A. xanthogrammica. A. sola evinced a proportion of mycos porine-2 glycine intermediate to that in A. elegantissima and the other species in the genus, amid much variation among individuals.

The phenogram (Fig. 7) based on the data in Figure 6 displays discrete clusters consistent with the foregoing qualitative description. Specimens of both A. artemisia and A. xanthogrammica formed distinct clades. A. sola was not clearly separated from A. elegantissima. Aposymbiotic individuals of A. elegantissima presented a separate clade, and the occurrence of one specimen of A. sola in this clade was owing to its exceptionally low concentration of mycosporine-2 glycine (less than half the mean value for the group), yielding an MAA profile distinctly like that of aposymbiotic specimens of A. elegantissima. Large and small specimens of A. xanthogrammica were not separated by their molar fractions of MAAs used to construct the phenogram, but were separable by their absolute concentrations of MAAs (Figs. 4 and 5).

Feeding experiment

Specimens of A. elegantissima readily ingested and retained the ocular lenses of C. lumpus. The fed anemones produced no visible fecal pellets or egesta, perhaps because the proteinaceous lenses (average [W.sub.w] = 23 mg, which was [approximately equal to] 1% of the anemones' average fresh weight) were completely absorbed (a 1% ration of brine shrimps would be at least 90% absorbed: Zamer, 1986). These lenses contained principally palythene and usujirene, with a smaller amount of palythine and a trace of asterina-330 (not quantified). Unfed specimens of A. elegantissima from Bodega Bay contained the usual complement of MAAs seen in this species from the other locations, including palythine, and, exceptionally, usujirene, but no palythene. Clonemates of A. elegantissima given lumpfish lenses to eat did contain palythene 4 and 8 days after feeding (Fig. 8). Perhaps because of the relatively high endogenous concentrations of palythine in control anemones compared with the small amount available in a lens (Fig. 8A, B, top panel), the level of this MAA did not change significantly in fed anemones. The concentration of usujirene rose significantly by 4 days after feeding on a lens rich in this MAA, and declined to control levels by 8 days after a single meal of lumpfish lens. In those cases where ingesting a lumpfish lens led to a significant increase in the concentration of a particular MAA, the total change in that MAA was compared with the amount of that MAA available in the meal (Fig. 8B). For palythene, anemones eating a lens still contained 88% of this compound initially available in the lens (estimated from the MAA content of the second lens from the same fish) at 4 days, and 33% at 8 days after the meal. Perhaps because of the suboptimal chromatographic resolution of usujirene in the anemone extracts, the significant elevation of this MAA at 4 days after feeding was apparently 20% greater than the amount available in the meal.

Discussion

Our data show clearly, and for the first time, that the complement of MAAs in the four North American species of Anthopleura broadly reflects the taxonomic and phylogenetic differences among these species. This conclusion is supported by our other findings: that the profiles and concentrations of the major MAAs are independent of the presence or absence of algal endosymbionts, the taxon of the symbionts that are present, and of several environmental factors. Nevertheless, our evidence does suggest that A. elegantissima can accumulate MAAs from its food, which may explain the temporal and local variations seen in the MAA profile, and the occurrence of minor MAAs in some individuals. These findings and others (Shick and Dunlap, 2002) lead us, finally, to consider the possibility that MAAs are synthesized de novo by the sea anemones.

Phylogeny of Anthopleura spp. as deduced from their complements of MAAs

In the phenogram (Fig. 7) based on the proportional complements of the four major MAAs in Anthopleura spp. from California, specimens of A. artemisia and A. xanthogrammica form distinct clades. However, unlike the phylogenetic tree of Geller and Walton (2001) based on mitochondrial DNA sequences, in which the three species having algal endosymbionts form an unresolved trichotomy, the MAA complements indicate that A. elegantissima and A. sola are more closely related to each other than either is to A. xanthogrammica.

A. elegantissima and A. sola are difficult to distinguish morphologically (Francis, 1979), and Smith and Potts (1987) were unable to distinguish them by allozymic analysis. The reproductive isolation of the two species was confirmed only by a more thorough genetic study using allozymes (McFadden et al., 1997), and by the analysis of Geller and Walton (2001), which was able to separate them by fixed differences in base pairs. Our finding that A. elegantissima and A. sola cluster together, distinct from A. xanthogrammica, supports the view of Pearse and Francis (2000) that they are sibling species and the view of McFadden et al. (1997) that the speciation was probably relatively recent.

Although it is gratifying, the finding that MAAs may contribute to phylogenetic understanding renders problematic any interpretations of the environmental factors affecting the MAA composition in a species of Anthopleura--that is, discriminating between the expression of this character as enforced by natural selection or as an ancestral legacy (Mangum and Hochachka, 1998). Until the biochemical steps involved in synthesizing and modifying individual MAAs in vivo have been elucidated and the factors controlling their expression have been determined, the issue will not be resolved.

Role of algal endosymbionts in determining the complements of MAAs in the anemones

The foregoing says nothing of the possible role of the algal endosymbionts -- another layer of genotypic complexity to be considered -- although the adoption of symbiosis itself is a trait that has been important in the evolution of the genus (Geller and Walton, 2001). Out data for the Washington specimens of A. elegantissima that harbored exclusively zoozanthellae or zoochlorellae (the extremes of the continuum in Fig. 2B) clearly demonstrate that the identity of the alga does not qualitatively affect the MAAs that are present in the consortium. This in turn strongly suggests that, in these anemones, the algal endosymbionts are not the source of the MAAs in the consortium, although the algae might procide a necessary precursor (such as deoxygadusol: Shick and Dunlap, 2002). That the algae do not determine the MAA complement is also indicated by our finding that the same proportional MAA complement occurs in anemones harboring predominantly (>90%) one alga or the other (Fig. 3). It is conceivable that one typ e of alga produces MAAs and the other does not, so that the former, even if a minor proportion of the total algal complement, could produce the MAAs detected in the mixed symbiosis, but this would be inconsistent with our interpretation of the data for anemones harboring exclusively one alga or the other. Unfortunately, neither zoochlorellae nor S. muscatinei (the species of zooxanthella that occurs in Washington speciments of A. elegantissima: LaJeunesse and Trench, 2000) has been cultured successfully, so it is not known whether either can indeed produce MAAs. The placement of S. muscatinei in lineage B of the established phylotypes of Symbiodinium spp. (e.g., Rowan, 1998; LaJeunesse, 2001) suggests that is does not produce MAAs, because none of the other species in this clade do (Banaszak et al., 2000; LaJeunesse, 2001).

Because MAAs that have accumulated in animal tissues have a long residence time (Carroll and Shick, 1996; Adams and Shick, 1996, 2001; Newman et al., 2000), it is also conceivable that the MAAs present in an anemone at a particular time are products not just of its population of symbionts at that time, but also of an earlier suite of symbionts. As has been shown in A. elegantissima (Saunders and Muller-Parker, 1997) and A. xanthogrammica (Bates, 2000), the symbiont population can change temporally, according to environmental factors. However, complete symbiont-switching has not been reported in A. elegantissima, and Weis and Levine (1996) found that naturally aposymbiotic specimens could not be experimentally infected with algae, and that anemones rendered aposymbiotic in the laboratory could be reinfected only with difficulty, and then only with the original algae. The most parsimonious explanation remains that zoochlorellae and S. muscatinei in Washington specimens of A. elegantissima are not the source of MAAs in those sea anemones.

In contrast to the foregoing conclusion, there are indications, at least in A. elegantissima, that the level of mycosporine-2 glycine in an anemone is associated with its density of zooxanthellae. Whereas the concentrations of MAAs did not differ among most Washington anemones having very different proportions of zoochlorellae and zooxanthellae, mycosporine-2 glycine was significantly elevated in individuals harboring exclusively zooxanthellae (Fig. 28). Similarly, in the California anemones, aposymbiotic specimens of A. elegantissitna had a lower proportion of mycosporine-2 glycine than zooxanthellate specimens (Fig. 6), which clearly separated the former from the latter, and, furthermore, placed the apozooxanthellate individuals nearer to the asymbiotic A. artemisia (Fig. 7), which scarcely contained any mycosporine-2 glycine. A lower level of mycosporine-2 glycine was also seen in a naturally bleached clonemate of one of the specimens of A. elegantissima from Santa Cruz (Fig. 4), as well as in naturally ap osymbiotic individuals of A. elegantissima from Bodega Bay (Stochaj et al., 1994) and Santa Barbara, California (Banaszak and Trench, 1995). The correspondence between the zooxanthellate condition and high levels of mycosporine-2 glycine was not absolute, however, because the levels of this MAA were relatively low in zooxanthellate specimens of A. xanthogrammica, and all specimens of all Anthopleura species contained some mycosporine-2 glycine (Fig. 28 and Fig. 4).

The situation is further complicated by the finding of Banaszak and Trench (1995) that S. californium isolated from A. elegantissima did not produce any MAAs in culture, nor indeed did these authors detect mycosporine-2 glycine in any species of zooxanthella cultured from diverse symbioses (Banaszak et al., 2000). However, the mobile phase they used (25% aqueous, acidic methanol) would not have resolved mycosporine-2 glycine from porphyra-3 34, even if it were present (Stochaj et al., 1994); and we (J. M. Shick and C. Ferrier-Pages, unpubl. results) subsequently have found mycosporine-2 glycine in zooxanthellae cultured from the scleractinian coral Galaxea fascicularis.

The failure of S. californium to produce MAAs in culture is also strong evidence that zooxanthellae are not the source of the MAAs found in those symbiotic specimens of A. elegantissima from California that harbor this alga (Banaszak and Trench, 1995), nor perhaps in the sympatric A. sola and A. xanthogrammica, assuming that they also include S. californium. Because the anemones from Santa Cruz probably also harbor S. muscatinei (LaJeunesse and Trench, 2000), MAAs might originate with this species of zooxanthella; but as already mentioned, based on its phylo-types it is doubtful that S. muscatinei can produce MAAs. Consider also that zooxanthellae cultured from a symbiosis may not reflect the entire complement of algal genotypes present in hospite (Santos et al., 2001); thus, although S. californium in culture does not produce MAAs (Banaszak et al., 2000), the host anemones might contain other phylo-types of Symbiodinium that do. Zooxanthellae might also provide an essential precursor, such as deoxygadusol, t hat the anemones use to synthesize MAAs (Shick and Dunlap, 2002).

Even if the endosymbiotic algae are not the source of the MAAs in Anthopleura species, might their presence stimulate the accumulation of MAAs (from whatever source) in the host anemone? Aposymbiotic and zooxanthellate anemones differ qualitatively in their expression of proteins (Weis and Levine, 1996; Stochaj and Grossman, 1997) induced by the algae, and the presence of zooxanthellae enhances the synthesis of carbonic anhydrase by the host in A. elegantissima (Weis and Reynolds, 1999). Might the presence of algal endosymbionts also affect the expression of host enzymes involved in the biosynthesis or modification of MAAs (especially mycosporine-2 glycine)? The specific enzymes involved in the metabolism of MAAs have not been identified to test this possibility.

Role of solar radiation in determining the concentrations of MAAs in Anthopleura spp.

Are there environmental correlates that are consistent with a sunscreening role of the compounds? Again, the evidence is equivocal, which is not surprising in light of previous experiments showing that varying the exposure of the anemones to UVR did not affect the concentrations of MAAs in zooxanthellate or aposymbiotic individuals of A. elegantissitna (Scelfo, 1988; Stochaj et al., 1994; Banaszak and Trench, 1995). Nevertheless, concentrations of MAAs are lowest in A. artemisia (Fig. 4), which typically inhabits sheltered microhabitats, such as deep holes in rocks and rocks buried in sand, so that only its oral disc is visible (Hand, 1955; Pearse and Francis, 2000). Might constitutively low concentrations of MAAs reflect the restriction of this species to sheltered microhabitats? A. sola and large individuals of A. xanthogrammica exhibit the next-lowest concentrations of MAAs. These are low intertidal species that extend well into the subtidal or occur in tidepools and thus experience lower solar irradiances than does the characteristically intertidal A. elegantissima, which typically is exposed to direct sunlight during aerial exposure (Pearse, 1974; Shick and Dykens, 1984), and which has the highest concentrations of MAAs. Although low by comparison with the concentrations of MAAs in tropical corals ([approximately equal to] 150-1000 nmol MAA [mg.sup.-1] protein), those in Anthopleura species (Fig. 5:10-20 [micro]mol MAA [g.sup.-1] dry weight, equivalent to about 15-30 nmol MAA [mg.sup.-1] protein) are the same as in temperate red macroalgae ([approximately equal to] 10 [micro]mol MAA [g.sup.-1] dry weight: Karsten et al., 1998).

Several uncertainties temper this seemingly clear environmental correlation. Anemones from Fidalgo Island have concentrations of MAAs as high as those in specimens of A. elegantissima at Santa Cruz (Fig. 5). This is true even though the Fidalgo Island anemones live at a latitude 11[degrees] higher, and thus have lower daily and seasonal exposures to solar UVR (Madronich, 1993) than the Santa Cruz specimens (although the coastal fog in California in summer, and the early-morning timing of summer low tides, may diminish the dose of UVR there). Concentrations of MAAs in small specimens of A. xanthogrammica are just as high as those in A. elegantissima; however, small specimens of A. xanthogrammica settle in mussel beds, where ours were collected, and move to the low intertidal only as they grow in size. Thus, their relatively high levels of MAAs are not unexpected. We do not know whether concentrations in the former specimens changed while they were held in shallow outdoor aquaria for 3 months before analysis, a lthough the aforementioned experiments with A. elegantissima suggest that they would not. Also, the concentration of MAAs in large specimens of A. xanthogrammica may be low compared with smaller specimens, because large individuals may have disproportionally greater amounts of extracellular mesoglea (Shick, 1991) relative to the mass of cells in which the MAAs are located, leading to a low mass-specific concentration of MAAs. The same may apply to large specimens of A. sola, as compared with small specimens of A. elegantissima. Finally, concentrations of MAAs in aposymbiotic individuals of A. elegantissima held in darkness in the laboratory for more than 1 year are just as high as or higher than in zooxanthellate specimens, as are those in a naturally bleached clonemate of the latter (Fig. 4; also see Stochaj et al., 1994; and Banaszak and Trench, 1995). Thus, proximal environmental effects as determinants of MAA concentrations seem less important than these four species' evolutionary histories, including bro ad interspecific differences in habitat.

Other possible physiological functions of MAAs

MAAs function as osmolytes in cyanobacteria (Oren, 1997; Portwich and Garcia-Pichel, 1999), but this role does not seem important in sea anemones, because in A. elegantissima, the concentration of MAAs is only 3% of that of free amino acids (FAAs), the principal organic intracellular osmotic effectors here (Shick, 1991; Shick and Dunlap, 2002).

Next, FAAs have been implicated as "host factors" that induce the release of photosynthate from the endosymbiotic algae (Gates et al., 1995), and taurine in particular has been studied as such by Wang and Douglas (1997). Might mycosporine-taurine or other MAAs also have such a regulatory role in these symbioses, as proposed by Gates et al. (1995)? The concentrations of MAAs are lowest in the asymbiotic A. artemisia (Fig. 4 and Fig. 5) and are higher in the symbiotic species, and mycosporine-2 glycine varies with the proportion of zooxanthellae in A. elegantissima (Fig. 2B); these facts are consistent with a role of MAAs as a host factor. A high concentration of MAAs in aposymbiotic individuals of A. elegantissima does not necessarily argue against such a role because aposymbiosis is a secondary, environmentally imposed condition that would not per se affect the inherent capacity of the host to accumulate MAAs from exogenous sources. Nevertheless, it remains to be established whether mycosporine-taurine, mycos porine-2 glycine, or any other MAA at physiologically relevant concentrations effects the release of photosynthate by zooxanthellae or zoochlorellae.

Finally, because specimens of A. elegantissima harboring these different algae have identical complements of MAAs, these compounds are unlikely to account for the differential palatability of zoochlorellate and zooxanthellate specimens to a predatory fish (Augustine and Muller-Parker, 1998), and both types of anemones are readily eaten by a predatory nudibranch (Seavy and Muller-Parker, 2002). In short, a clear physiological (osmolyte or host factor) or defensive (sunscreening or feeding deterrent) role for MAAs that would explain their patterns of occurrence among species of Anthopleura remains to be elucidated.

The role of dietary MAAs

Our experiments unequivocally show that A. elegantissima can absorb MAAs from its food and retain them for at least 8 days (Fig. 8). Not only does this result support a possible trophic origin of MAAs in sea anemones, but it also may explain local and temporal differences in the complement of minor and trace MAAs (cf. Stochaj et al., 1994, and Banaszak and Trench, 1995), including the unusual and surprising occurrence of usujirene in specimens of A. elegantissima from Bodega Bay used in our feeding experiment. The possibility that unpredictable variations in diet can affect the complement of MAAs in a population must be borne in mind when interpreting qualitative field surveys of MAAs in diverse animal taxa, particularly if the data are intended to delineate broad phylogenetic patterns (Karentz, 2001). This seems particularly relevant in polyphagous opportunists such as sea anemones (Shick, 1991). Indeed, such stochastic variability in the minor MAAs (e.g., we had never seen usujirene in diverse seasonal coll ections of A. elegantissima from Bodega Bay dating back to 1988) prompted us to use only the four MAAs that occur in all specimens of all species in constructing the phylogeny (although this obscured the qualitative differences between A. artemisia and the other three species: Fig. 4). Thus, like these compounds in cyanobacteria (Karsten and Garcia-Pichel, 1996), dinoflagellates (Carreto et al., 2001), and cultured zooxanthellae (Banaszak et al., 2000; LaJeunesse, 2001), the predominant MAAs in Anthopleura spp. do appear to be useful phylogenetic characters.

Determining the diet and ration in sea anemones is tedious work, but it has been done in A. elegantissima (Sebens, 1981; Zamer, 1986). To establish the relative importance of various food items in affecting the MAAs present in the anemones in nature would require long-term studies of the MAAs in their natural diet for comparison with their own time-averaged complement of MAAs. A single sample of the phytoplankton, zooplankton, and particulate detritus >25 [micro]m diameter in the water column above the population of A. elegantissima at Bodega Bay (J.M. Shick, unpubl. data) revealed the presence of most major and minor MAAs found in the anemones (including usujirene). The exceptions were mycosporine-glycine:valine and mycosporinetaurine, the latter being the predominant MAA that apparently is unique to anemones in this genus. Thus, mycosporine-taurine may be produced by the anemones themselves, perhaps by modifying other MAAs, as seems to be the case for certain MAAs and their precursors in corals (Shick et al ., 1999), holothuroid echinoderms (Dunlap and Shick, 1998), fishes (Mason et al., 1998), and red macroalgae (Franklin et al., 1999).

As we have noted elsewhere (Stochaj et al., 1994), taurine is by far the most concentrated component of the free amino acid poo1 in Anthopleura species. Although the biosynthetic origin of mycosporine-taurine remains enigmatic, it may result from the modification of a pre-existing (dietary) MAA or deoxygadusol by the substitution of taurine in the chromophore conjugation. Thus, the anemones may be exploiting the ready availability of this amino acid to synthesize mycosporine-taurine, which not only absorbs the shortest, most damaging UV wavelengths (309 nm) of any MAA, but which moreover is a moderate antioxidant (Shick et al., 1996; Shick and Dunlap, 2002). This is an important consideration for a phototrophic symbiosis, in which the metazoan host experiences hyperoxia (Dykens and Shick, 1982), because this hyperoxia, together with exposure of the animal to solar UVR, leads to an enhanced flux of reactive oxygen species (Dykens et al., 1992).

We deliberately collected the specimens from Santa Cruz in a habitat where all four species of Anthopleura are present in close proximity, to ensure that all of them potentially have access to the same prey. Although Sebens (1981) documented size-related differences between A. elegantissima and A. xanthogrammica in the prey that they capture, he also found considerable overlap between these species in their major food items. Therefore, if the MAA complements of these anemones are affected by their diets, their disparate MAA spectra are surprising, unless the heavier reliance of A. xanthogrammica on mussels (Mytilus spp.), which are rich in shinorine (Chioccara et al., 1979, 1985; J. M. Shick, unpubl. data), enhances the concentration of this MAA in A. xanthogrammica.

A. artemisia consistently contained an MAA that we never saw in any of the other species--mycosporine-glycine:valine. This MAA is common in Antarctic phytoplankton and the krill that eat them (Karentz et al., 1991; Dunlap et al., 1995; Newman et al., 2000); this substance has also been found in a red-tide dinoflagellate (Vernet and Whitehead, 1996) and in particulate organic matter (Whitehead and Vernet, 2000) in California coastal waters. Thus, mycosporine-glycine:valine probably is present in the plankton available to all of these Anthopteura species (although it was not in the assemblage of particulate material sampled at Bodega Bay), and its restriction to A. artemisia underscores the biochemical distinction of this species from its sympatric congeners. This finding of a unique MAA, together with the absence of palythine and palythene from this species alone of the four (Fig. 4), reinforces the phylogenetic conclusion of Geller and Walton (2001) that A. artemisia is not closely related to the other three species, but belongs to a western Pacific clade of the genus Anthopleura.

Diet does not explain the MAA complement in aposymbiotic specimens of A. elegantissima maintained in the laboratory. Stochaj et al. (1994) suggested a dietary source of MAAs in laboratory-maintained aposymbiotic specimens, but the UV-absorbing compounds in the squid fed to the anemones in that study subsequently proved not to be MAAs, and the MAA complement after month-long feeding on this diet was the same as in freshly collected specimens (J.M. Shick, unpubl. data). Aposymbiotic individuals maintained in the laboratory in Santa Cruz for more than 1 year and occasionally fed previously frozen adult brine shrimps (Artemia sp.) had the same MAA complement, although in different proportions, as zooxanthellate individuals fresh from the field (Fig. 6). Those brine shrimps were not analyzed for MAAs, but results for other samples of Artemia indicate that they probably contained principally shinorine, along with some asterina-330 and porphyra-334 (Grant et al., 1985), and possibly mycosporine-2 glycine (J.M. Shick , unpubl. data); Artemia tissues also contain gadusols (Grant et al., 1985), likely precursors of MAAs (Shick and Dunlap, 2002). The diet may provide the gadusols and certain MAAs that the sea anemones ultimately convert into the suites of MAAs that characterize the several species.

De novo biosynthesis of MAAs by cnidarians?

As we have noted here and elsewhere (Shick and Dunlap, 2002), the same major MAAs occur in aposymbiotic and zooxanthellate individuals of A. elegantissima, and controlled diets fail to modify this pattern; moreover, zooxanthellae freshly isolated from A. elegantissima lack MAAs, and cultured S. californium apparently cannot synthesize MAAs (Banaszak and Trench, 1995). These considerations all suggest that the anemones themselves make these compounds. The tentative finding that scleractinian corals can synthesize aromatic amino acids (Fitzgerald and Szmant, 1997) may also point to the unexpected presence of the shikimic acid pathway in these metazoans. But like Fitzgerald and Szmant (1997), we might equivocate, suggesting that like aromatic amino acids, the MAAs originate in bacteria associated with the anemones, although only one marine bacterium has ever been reported to produce MAAs (Arai et al., 1992). Alternatively, the algal endosymbionts and the food might be sources of deoxygadusol, which the animal ho st might use to produce MAAs. Testing these possibilities, and looking more rigorously in cnidarians for, especially, the early steps of the shikimic acid pathway (including the postulated branchpoint leading to MAAs via deoxygadusol), might clarify the otherwise unexplained presence of certain MAAs in these metazoans.

In summary, the complements of the MAAs in four species of Anthopleura seem more closely associated with their phylogenetic position than with other factors. Several lines of evidence indicate that the algal endosymbionts do not contribute the MAAs seen in the symbiotic anemones, although in A. elegantissima the level of mycosporine-2 glycine does seem to be affected by the predominance of zooxanthellae. The successful culturing of zoochlorellae and of Symbiodinium muscatinei would facilitate the analysis of their MAA complements and do much to resolve the uncertainty about their contributions to the suite of MAAs in the symbiosis. Identifying the enzymes involved in the biosynthesis of primary MAAs, and in the secondary structural modification of these, remains a major unresolved problem. Its solution would allow us to study the phylogenetic distribution of the enzymes in concert with the occurrence of particular MAAs, and would also permit us to study the factors that affect the expression and regulation of these enzymes.

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Acknowledgments

We thank Dr. Gisele Muller-Parker for collecting the sea anemones in Washington, and Drs. James Clegg and Dennis Hedgecock for facilities and facilitation at the Bodega Marine Laboratory. We are grateful to Donald Mason, who first identified the MAAs present in lumpfish lenses, where the rare presence of usujirene suggested their use as a source of this dietary marker, and to Amy Carroll, who assisted in the laboratory. We appreciate Dr. Irv Kornfield's advice regarding the cluster analysis and his comments on the manuscript, and Francis Wihbey's assistance with producing the map. We thank the editors and two anonymous reviewers for their heroic efforts. This research was supported by U.S. National Science Foundation grant IBN-93 16426 to J.M. Shick and W.C. Dunlap, and in part by a grant from the National Geographic Society to J.M. Shick.

Received 3 April 2002; accepted 7 October 2002.

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J. Malcolm Shick (1), *, Walter C. Dunlap (2), John s. Pearse (3), and Vicki B. Pearse (3)

(1.) School of Marine Sciences, University of Maine, 5751 Murray Hall, Orono, Maine 04469-5751;

(2.) Environmental Biochemistry and Marine Biotechnology, Australian Institute of Marine Science, PMB No. 3, Townsville MC, Qld. 4810, Australia;

(3.) Institute of Marine Sciences, University of California, Santa Cruz, California 95064

* To whom correspondence should be addressed. E-mail: shick@maine.edu
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Author:Shick, J. Malcolm; Dunlap, Walter C.; Pearse, John S.; Pearse, Vicki B.
Publication:The Biological Bulletin
Geographic Code:100NA
Date:Dec 1, 2002
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