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Distribution patterns of zoochlorellae and zooxanthellae hosted by two Pacific northeast anemones, Anthopleura elegantissima and a. xanthogrammica.

Introduction

Obligate associations with unicellular photosynthetic dinoflagellates allow corals to thrive in nutrient-impoverished tropical oceans (Muscatine, 1990). The widespread phenomena of coral reef bleaching and the cascading community shifts known to follow such events (e.g., Knowlton, 2001) have driven a large effort to tease out the effects of environmental variability on the disruption of coral-algal symbioses. One mechanism enabling certain symbiotic cnidarians to withstand changing environmental conditions is the formation of associations with multiple algal partners that have different physiologies (Buddemeier and Fautin, 1993). Although tropical symbioses have received the majority of research effort, endosymbiotic associations also occur in temperate systems but are often facultative in nature (Muller-Parker and Davy, 2001). Even so, these symbioses can contribute significantly to local primary productivity, and in spite of high nutrient concentrations and availability of particulate food in temperate systems, algal symbionts can provide a significant portion of their host's energy budget (Muller-Parker and Davy, 2001).

Model organisms for studies aiming to determine the role of physical differences in environment on the disruption and productivity of symbioses involve two northeast Pacific anemones (Cnidaria, Anthozoa)--Anthopleura elegantissima Brandt, 1835, and A. xanthogrammica Brandt, 1835--and two photosynthetic symbionts (Saunders and Muller-Parker, 1997; Muller-Parker and Davy, 2001; Mitchelmore et al., 2002). The two anemones host both green algae (zoochlorellae: ZC) and brown dinoflagellates (zooxanthellae: ZX), which allows for baseline comparisons in distributions of the two symbionts. Although the genetic affiliation of the A. xanthogrammica symbionts remains to be determined, ZC hosted by A. elegantissima have recently been placed in the class Trebouxiphyceae (Lewis and Muller-Parker, 2004). In addition, LaJeunesse and Trench (2000) identified two brown dinoflagellate species from A. elegantissima as Symbiodinium californium (restricted southern latitudes) and S. muscatinei nom. nud. (California, Oregon, and Washington). Comparative studies of symbiont distribution in the two Anthopleura species are possible because both anemones are abundant intertidally, are easily identified in the field, and can be manipulated in experimental studies (e.g., Bates, 2000; Secord and Muller-Parker, 2005). Furthermore, ZC and ZX are readily distinguished under a compound light microscope on the basis of size and color (ZC are [approximately equal to]6-8 [micro]m and bright green, ZX are [approximately equal to] 10-15 [micro]m and olive brown: e.g., Secord and Augustine, 2000; Bergschneider and Muller-Parker, 2008). Thus, anemones can be categorized into algal types with relative ease (e.g., O'Brien and Wyttenbach, 1980; Saunders and Muller-Parker, 1997; Secord and Muller-Parker, 2005) by the ratios of the two symbionts that they contain: green and brown types are dominated by ZC and ZX, respectively, or both symbionts (mixed) can be abundant within one host. In low light conditions, algae-free Anthopleura spp. also occur, and these anemones are typically white.

An impressive number of laboratory studies have focused on quantifying the respective photobiology and physiological tolerances of ZC and ZX isolated from Anthopleura elegantissima; temperature and irradiance have emerged as key factors governing the symbiosis (e.g., McCloskey et al., 1996; Verde and McCloskey, 2001, 2002, 2007; Muller-Parker et al., 2007; Bergschneider and Muller-Parker, 2008). For instance, elevated seawater temperatures (>20 [degrees]C) resulted in lower densities and chlorophyll content of in hospite ZC, while similar changes were not observed for ZX (Saunders and Muller-Parker, 1997; Verde and McCloskey, 2001). Furthermore, zoochlorellate anemones were light-saturated at lower irradiances than were zooxanthellate anemones (Verde and McCloskey, 2002). High light intensity also reduced the content of chlorophyll in ZC and increased the content of carotenoids, which serve a photoprotective function, in ZX (Verde and McCloskey, 2002). Thus, ZC are predicted to occur in hosts from habitats with low temperature and low irradiance, while ZX are more tolerant of a range of temperature and light conditions.

Environmental parameters likely play an important role in regulating the distribution and population dynamics of symbionts (Rowan et al., 1997). Indeed, results from field surveys and transplant experiments along natural environmental gradients at various scales are consistent with laboratory studies indicating that temperature and light are key factors driving distributions of the two algal symbionts in both Anthopleura species. At the individual scale, tissue sections of mixed A. elegantissima revealed that ZC and ZX are relatively abundant in the body column and tentacles, respectively (Dingman, 1998, as cited in Bergschneider and Muller-Parker, 2008). This distribution may reflect in hospite light gradients. Algal type also varies predictably with shore height: green algal types are restricted to low shore positions and shaded habitats, while brown anemones dominate higher shore regions where temperature and irradiance are relatively high (McCloskey et al., 1996; Secord and Augustine, 2000; Bates, 2000). Furthermore, algal type relates to small-scale light gradients. For example, individuals of A. elegantissima at the entrances of two northern Washington caves were brown; green types were limited to mid-cave locations that were shaded; and algae-free anemones occupied dark locations at the back of the cave (Secord and Muller-Parker, 2005). At larger scales, ZX were the exclusive symbionts in southern populations of Anthopleura spp. (south of 35[degrees]N) where climate conditions are relatively warm and irradiance is high, while ZC increased in abundance northward (from 35 to 48[degrees]N) at latitudes typified by cooler environmental temperatures and lower irradiances (Secord and Augustine, 2000).

Although environmental conditions may regulate algal complement, Secord and Augustine (2000) presented compelling evidence that latitudinal distribution of ZC and ZX varies between the two Anthopleura species: ZC extend farther south in A. xanthogrammica than in A. elegantissima. They suggested two explanations that are based on differences in environmental conditions experienced by each anemone for the observation that A. xanthogrammica hosts ZC at latitudes where sympatric A. elegantissima hosts ZX. First, A. xanthogrammica reaches larger sizes than A. elegantissima (Sebens, 1981b, 1982c), and is consequently buffered from thermal change (Dingman, 1998, as cited in Bergschneider and Muller-Parker, 2008). Second, A. xanthogrammica is more abundant at lower shore heights (Hand, 1955) where temperature and irradiance are relatively low.

Our goal was to quantify the algal type of A. elegantissima and A. xanthogrammica in a number of rocky shore microhabitats. In doing so, we hoped to determine whether the distribution of the two algal symbionts responds similarly to physical regime in both host species and thus to contribute to the body of research characterizing environmental influences on algal-cnidarian symbioses. To eliminate the confounding effects of microhabitat variation due to differences in host size or location, we sampled similarly sized polyps of each species that were located in close proximity in tidepools, aerially exposed in rock crevices, on the underside of rock ledges, and along natural light gradients in intertidal caves. To explain host-specific differences in algal complement that emerged from our field surveys, we hypothesized that the growth rates of the symbionts might vary between the Anthopleura species. As a first step in addressing this possibility, we tested for differences in the division rates of algae in both anemones exposed to different light regimes.

Materials and Methods

Anemone algal type

To estimate percentages of each symbiont type, we sampled anemone tissues for microscopic analyses of algal abundance at beaches near Port Renfrew (48[degrees]32'00"N, 124[degrees]26'50"W), Bamfield (48[degrees]50'0"N, 125[degrees]9'0"W), and Tofino (49[degrees]7'60"N, 125[degrees]54'0"W) on Vancouver Island (west coast of Canada: Fig. 1). Because algal ratios can vary in different tissues (ZX and ZC are relatively abundant in the tentacles and body column, respectively; Dingman, 1998, as cited in Bergschneider and Muller-Parker, 2008), all samples included tentacles (n = 1 to 4), and at Port Renfrew and Tofino, a portion of the mid-body column (about 1 [cm.sup.3]) was also excised by dissection for algal counts. Samples were stored at 4 [degrees]C while in the field, and upon return to the laboratory, were frozen at -4 [degrees]C until analysis. Tissues were then added to 10-20 [micro]1 of distilled [H.sub.2]O in an Eppendorf tube, homogenized with a glass rod, and viewed under a light microscope at 250 or 400X on a stage hemocytometer or a glass slide (with eight sampling points marked a priori). ZX and ZC were visually scored: at least 100 cells of the most abundant symbiont species and the number of cells of the less abundant symbiont were counted at the eight random positions. The entire slide and 1 to 3 additional slides were then examined to ensure that the counts were representative (> 1000 cells). Anemones were then classed into four "algal types" (following convention, in terms of percentage of ZX of total symbiont counts): (1) white = symbiont cells absent or rare (an arbitrary cutoff of <10 ZX cells per sample); (2) green = <10% ZX; (3) mixed = 10% to 90% ZX; and (4) brown = >90% ZX. Note that algal type is based on body-column and/or tentacle tissues and is not necessarily representative of the entire anemone.

Inter-specific patterns in algal type

To compare the algal types of the two Anthopleura species in the same location, we sampled pairs of A. elegantissima and A. xanthogrammica that were similar in size (within [approximately equal to] 1 cm oral diameter) and separated by less than 5 cm. A. elegantissima reproduces asexually by fission (Sebens, 1982b) and can occur in extensive aggregates, whereas A. xanthogrammica is solitary and reaches much larger sizes (Hand, 1955; Sebens, 1981b, 1982c). Thus, we targeted adult A. elegantissima (reproductive size: 1.4 cm: Sebens, 1981a) and juvenile A. xanthogrammica (reproductive size: >6.5 cm). Potential pairs of anemones were identified visually and then measured with calipers (with the exception of specimens from Bamfield crevices) to confirm that the polyps were within 1 cm in body-column diameter. Similarly sized pairs of these two species were difficult to find, so random sampling was not possible. Instead, we searched over low-tide windows until our target sample size was reached. To minimize the possibility of repeatedly sampling individuals from identical clones of A. elegantissima, we spaced each collection by several meters (with the exception of cave sampling, see below). To ensure the generality of evident patterns, we also surveyed multiple sites on Vancouver Island (Fig. 1).

[FIGURE 1 OMITTED]

Shore heights were determined using survey techniques relative to the Canadian Hydrographic Service chart datum (Forbes et al., 2002), which approximates the lowest astronomical tide. Data were collected at sample sites in four microhabitats: tidepools, crevices, under rock ledges, and in caves.

Tidepools. Neighboring specimens of the two species were sampled, as determined by availability, in tidepools ranging from 0.5 to 1.5 m (shore height) at Port Renfrew (Botanical Beach, n = 8 pairs, January 2008, tentacle and body-column tissues); Bamfield (Scott's Bay, Dixon Island, Brady's Beach, Sandford Island, and Benson Island, n = 33 pairs, October--November 2005, tentacle tissues); and Tofino (Chesterman's Beach, n = 30 pairs, February 2008, tentacle and body-column tissues). Data for the three sampling locations and different sampling methods (tentacles versus tentacles plus body-column tissue) were pooled because of similarity (Fisher exact probability test: P = 0.096).

Crevices. Paired specimens of each Anthopleura species were located in rock crevices exposed to light near Scott's Bay (Bamfield: n = 45 pairs, November 2007, tentacle tissues) and Chesterman's Beach (Tofino: n = 30 pairs, January 2008, tentacle and body-column tissues) between 0.5 and 1.5 m (shore heights). Data for the two sampling locations and tissue types were pooled because of similarity (Fisher exact probability test, P = 0.55).

Ledges. Tentacles and body-column tissues were sampled from anemone pairs located in shaded locations: the underside of overhanging rock shelves, at Chesterman's Beach (Tofino: n = 30 pairs).

Caves. Two caves, one near Brady's Beach (Bamfield: July 2007) and the other near Chesterman's Beach (Tofino: February 2008), were selected because of the presence of both anemone species, a relatively uniform tidal height (< 0.5 m range), accessibility at low tide, and a visible declination in light intensity (cave entrances experienced full light while the back of each cave was dark). We elected not to measure change in irradiance with distance into each cave, as point measurements of light in caves can be misleading: we observed patches of sunlight at the back of the each cave depending on the daily and seasonal orientation of the Sun. Anemones were sampled at 1-m intervals along transects (length 20 m at Bamfield, 13 m at Tofino) positioned from the cave entrance to the darkest point in the cave (shore height [approximately equal to]1.0 m for both caves). Specimens of Anthopleura xanthogrammica larger than 7.0 cm in body-column diameter were present in the Bamfield cave within 0.5 m of either side of the transect line and were sampled opportunistically (n = 13 individuals) to verify the generality of patterns in algal type.

Mitotic index (MI)

As a first step in addressing whether symbiont growth rate varies between Anthopleura elegantissima and A. xanthogrammica, we conducted an experiment and a field survey to compare the MI of ZX isolated from the two hosts. Although we intended to include ZC in this work, we were not able to find enough green or mixed A. elegantissima for experiments (in spite of screening 300 specimens).

To quantify MI, tentacles were excised, chilled, homogenized, and viewed under a microscope as described for "Anemone algal type" (above). We counted the total ZX and those with a division furrow within the field of view for eight random positions per slide (total number ZX counted ranged from 100 to 800). MI was calculated by averaging the percentage of cells with division furrows (as in Wilkerson et al., 1983).

Laboratory. Anthopleura elegantissima and A. xanthogrammica (n = 20 for each species, split evenly between two treatments) were haphazardly collected from a moderately exposed rocky outcrop near Scott's Bay (Bamfield). Each anemone was removed from the substrate with a dull knife and placed in a bucket containing seawater for transport. Once in the laboratory, each individual was positioned on a petri dish (that had been roughed with sandpaper) and placed in a seawater flow table under ambient light and temperature conditions until re-attachment occurred. To determine the effect of light intensity on the MI of ZX, animals were held for 26 days in a low temperature diurnal illumination incubator (model: 2015) at 12 [degrees]C on a 14:10 h (light/dark) photoperiod in four aquaria under two irradiance conditions: (1) 100%: four 40 W fluorescent tubes delivering 80 [micro] mol quanta [m.sup.-2][s.sup.-1] measured using a LICOR photometer (model LI-1000) and (2) 50%: irradiance reduced by using window screening as a neutral density filter. Every 3 days, seawater was refreshed and anemones were fed 1 small (<2 cm) mussel. Prior to the start of the experiment and at its completion, tentacle samples were collected (at [approximately equal to] 1000 h) and frozen (-4 [degrees]C) until microscope analysis of MI.

Field. Tentacles were excised from pairs of Anthopleura elegantissima and A. xanthogrammica at low ([approximately equal to]0.8 m: n = 17) and high ([approximately equal to] 1.8 m: n = 15) shore heights (16 pairs at each shore height). To minimize any differences in MI due to the timing of sampling, tentacles were collected simultaneously from pairs of the two species on a mid-morning (1100 h) low tide (see "Anemone algal type" for a description of sampling methods). Samples were stored on ice until return to the laboratory, where MI was quantified. Samples that returned less than 100 ZX cells per tissue sample were excluded, leaving 13 and 9 pairs from low and high shore, respectively. As a consequence, our sample size and statistical power were reduced. Thus, we first tested for significant differences in the high- versus low-shore samples for each species using a two-sample Student's t-test; and because P > 0.05 in both cases, we pooled data from both shore heights.

Statistics

All statistics were conducted in NCSS 2007 software (NCSS, Kaysville, Utah); specific analyses and results are reported in the Results section where appropriate, [alpha] = 0.05 in all cases.

Results

Inter-specific patterns in algal type

Comparing the algal type of tentacle and body-columns tissues collected from similarly sized neighboring Anthopleura species in three habitats revealed consistent patterns (Fig. 2). Zoochlorellae (ZC) were rarely hosted by A. elegantissima on the outer west coast of Vancouver Island at Port Renfrew, Bamfield, and Tofino sites. We did not observe the green algal type in tidepools (Fig. 2a), rock crevices (Fig. 2b), or shaded on the underside of rock ledges (Fig. 2c) (n = 177). In fact, more than 95% (pooled across all habitats) of symbiotic A. elegantissima were brown (the remaining were mixed), while A. xanthogrammica displayed the range of algal types in locations exposed to sunlight: green (55%), brown (30%), and mixed (15%) (Fig. 2a, b: note similarity in percent algal type in tidepools and crevices). In shaded areas on the underside of ledges more than 99% of A. xanthogrammica were classed as green, while neighboring A. elegantissima were brown (55%), white (30%), or mixed (14%) (Fig. 2). Overall, body-column diameter (mean [+ or -] 1 SD) for Anthopleura elegantissima and A.xanthogrammica (pooled across all localities and habitats, with the exception of specimens from Bamfield crevices) was, respectively: 4.4 [+ or -] 1.0 and 4.7 [+ or -]1.2 cm, and did not differ significantly between the two species (Wilcoxon signed-rank test for differences in medians: Z = 1.42, P = 0.16).

[FIGURE 2 OMITTED]

At both cave entrances, anemones harbored symbionts (Fig. 3). As found in tidepools and rock crevices (Fig 2a, b), Anthopleura xanthogrammica (tentacles: Bamfield; tentacles + body column, Tofino) hosted primarily green symbionts, but A. elegantissima tended to be brown (although a few mixed and green A. elegantissima did occur in both caves) (Fig. 3). There was a sharp transition from symbiotic to algae-free (white) A. xanthogrammica in both caves (see dotted lines on Fig. 3). In comparison, the distributions of symbiotic and algae-free A. elegantissima overlapped. A. elegantissima displayed white, brown, and mixed algal types at distances closer to the cave entrance in areas where specimens of A. xanthogrammica were green (a similar pattern was observed under rock ledges: see Fig. 2c). Thus, white A. elegantissima occurred nearer to the cave entrance than did white A. xanthogrammica. Although we could not find similarly sized anemones in the Bamfield cave--A. elegantissima (mean [+ or -] 1 SD: 1.3 [+ or -] 0.4 cm) were significantly smaller than A. xanthogrammica (3.1 [+ or -] 1.1 cm); two-sample t test: P = 0.001--patterns in algal type were consistent in the two caves (results of a two-sample t test indicated that anemone diameter in the Tofino cave did not differ between the two species: A. elegantissima, 3.7 [+ or -] 1.0 cm; A. xanthogrammica, 4.0 [+ or -] 0.9 cm). Furthermore, 13 large A. xanthogrammica (7.0 to 10.0 cm) displayed a distribution similar to that of small specimens: green algal types were observed at 0.8, 1.3, 3.5, 3.8, 3.9, 6.2, 7.5, and 9.1 m, while white algal types were found at 10.7, 11.4, 12.4, 16.5, and 18.6 m.

[FIGURE 3 OMITTED]

Mitotic index (MI)

Light intensity significantly impacted MI in anemones exposed to 80 (100%) and 40 (50%) [micro]mol quanta [m.sup.-2] [s.sup.-1] for 26 days in a laboratory experiment (two-way ANOVA results for temperature: F = 18.71, P = 0.00014) (Fig. 4). Although the MI values did not differ significantly in the tentacles of the two species, the interaction between species and light was significant (two-way ANOVA results for species X light: F = 10.06, P = 0.0033). The MI of zooxanthellae (ZX) from A. xanthogrammica was more responsive to light and was slightly higher at 100% and lower at 50% irradiance than ZX from A. elegantissima: MI values (mean [+ or -] 1 SD) for post-treatment A. xanthogrammica at 100% and 50% irradiance were respectively 7.3% [+ or -] 1.3% and 3.9% [+ or -] 0.9% versus 6.2% [+ or -] 1.3% and 5.6% [+ or -] 1.4% for A. elegantissima.

[FIGURE 4 OMITTED]

Results from a field survey comparing the MI of ZX isolated from the tentacles of the two Anthopleura species support laboratory findings. Mean MI ([+ or -] 1 SD) of ZX in 22 neighboring Anthopleura pairs was significantly higher in A. xanthogrammica (5.9% [+ or -] 2.0%) than in A. elegantissima (4.6% [+ or -] 1.7%) (data were pooled from the two shore transects at 0.8 and 1.8 m: paired t test, t = 2.038, df = 21, P = 0.027).

Discussion

Inter-specific patterns in algal type

This study compared the algal type of two Anthopleura species on the outer west coast of Vancouver Island, British Columbia (Canada), by sampling similarly sized specimens of each species that were located close together, and presumably, in similar environmental conditions. Emergent patterns were consistent among the Port Renfrew, Bamfield, and Tofino sampling locations and for the different tissues samples (tentacles: Bamfield; tentacles plus body column: Tofino and Port Renfrew) (Fig. 2). The results of this study corroborate previous studies (e.g., McCloskey et al., 1996; Bates, 2000; Secord and Augustine, 2000) documenting shifts in the relative abundance of the two symbiont types with gradients in light or thermal regime. Analysis of the algae present in the tentacles and body column indicated that zoochlorellae (ZC) were only rarely hosted by Anthopleura elegantissima; zooxanthellae (ZX) were the dominant symbiont in tidepool and crevice habitats exposed to sunlight between the shore heights of 0.5 and 1.5 m (> 98% were brown; n = 147). In comparison, A. xanthogrammica from the same locations hosted both symbionts exclusively, as well as mixed symbiont populations. The ZC-A. elegantissima symbiosis was also restricted in low irradiance settings. On the underside of rock ledges and in intertidal caves, symbiotic individuals of A. xanthogrammica were green while A. elegantissima were usually brown and, more rarely, mixed. In shaded locations, A. elegantissima was much more likely to occur without symbionts (e.g., under ledges, 35% of pairs were white A. elegantissima and green A. xanthogrammica).

The low representation of green algal type Anthopleura elegantissima (< 1%) in an array of microhabitats (shore heights down to 0.5 m and shaded locations) was unexpected, as previous studies from northern Washington locations indicate that green anemones can be abundant in the low intertidal (< 0.6 m mean lower low water) and in locations shaded from solar radiation (e.g., McCloskey et al., 1996; Shick et al., 2002; Secord and Muller-Parker, 2005). While Secord and Augustine (2000) found that A. xanthogrammica (typically > 8 cm body-column diameter) harbored significant numbers of ZC in Oregon and northern California, A. elegantissima (typically 1 to 3 cm) collected from the same localities, but not necessarily the same microhabitats, was primarily brown. The results of this study, in concert with the findings of Secord and Augustine (2000), indicate that A. xanthogrammica is more likely than A. elegantissima to host ZC and suggest that the patterns reported herein may be applicable to a variety of latitudes.

Because we sampled polyps that were the same size in similar microhabitats, the observed host-specific differences in algal type cannot be readily explained by extrinsic environmental factors. However, the two Anthopleura species differ in a number of life-history and biological traits (e.g., Sebens, 1981a, 1982a, 1983) that may influence the intrinsic conditions experienced by the symbionts. For instance, we compared adult-sized A. elegantissima (cloned: Sebens, 1982b) with juvenile-sized A. xanthogrammica (Sebens, 1982a), and it is possible that algal type might differ with life-stage (Abrego et al., 2009). Even so, tentacle samples from juvenile and adult A. xanthogrammica from the Bam-field cave displayed similar patterns in algal type. A. elegantissima (Bamfield cave [approximately equal to] 1.0 cm vs. Tofino cave >3 cm) also displayed the same algal types irrespective of size. Furthermore, species-specific differences in motility likely did not confound our results, as both adult A. elegantissima and juvenile A. xanthogrammica are motile (Sebens, 1982a, 1983) and symbiotic anemones of both species are phototaxic (A. elegantissima, Pearse, 1974; A. xanthogrammica, K. Sebens, unpubl. data, as cited in Sebens, 1982a). We also obtained consistent patterns in algal type for each anemone species with different sampling methods, among replicate sampling locations and habitat types, and for a range of shore heights. Therefore, future studies should test for mechanisms driving the different distribution patterns of symbionts hosted by the two Anthopleura species, as discussed below (Broader implications).

Mitotic index (MI)

The paired sampling technique indicates that MI is relatively responsive in Anthopleura xanthogrammica in comparison to A. elegantissima, at the low irradiance levels used in experiments and during aerial exposure at low tide. For instance. ZX from A. xanthogrammica specimens exposed to artificial light displayed a slightly higher MI at 80 [micro]mol [m.sup.-2][s.sup.-1] than A. elegantissima specimens (respectively, 7.5 vs. 6.8) and a relatively lower MI at 40 [micro]mol[m.sup.-2][s.sup.-1] (3.8 vs. 5.8). Although the duration of cytokinesis was not measured and MI shows a diel pattern in magnitude (Verde and McCloskey, 1996; Fitt, 2000) (we sampled tentacles for MI quantification during mid-morning hours only), the greater variation in the MI of ZX from A. xanthogrammica suggests that the growth rates of the symbionts might vary between the two hosts in the same environmental conditions. While factors such as contraction and expansion of tentacles (Pearse, 1974; Shick and Dykens, 1984) and attachment of debris to the body column (Dykens and Shick, 1984) may also be species-specific and modify the light reaching the algae, specimens included in the laboratory experiments remained open (except after feeding) and were free of external debris. Further studies are therefore required to substantiate whether algal growth rates differ between the Anthopleura species, and if so, to determine the driving mechanism.

The MI values quantified from ZX in the tentacles of both Anthopleura species are higher than those from studies conducted on A. elegantissima from Washington (e.g., 2.9%: Wilkerson et al. 1983; 1.2% to 1.7%: McCloskey et al, 1996). Because we quantified MI from between 100-and 800-cell samples (1000-cell samples are standard), it is possible that we over-estimated MI. However, Bergschneider and Muller-Parker (2008) also measured MI of ZX hosted by A. elegantissima in autumn (October) and reported similar values: [approximately equal to]4%. Another possible explanation for our elevated MI values relates to nutritional state and ambient nutrient levels which can influence a number of zooxanthellar traits, including rates of cell division (e.g., Wilkerson et al., 1983; Cook et al., 1988; Davy et al. 2006). In this study, laboratory animals were fed frequently (every 3 days), and productivity on the ocean shelf off the coast of Vancouver Island is consistently higher than in Washington waters (Sackmann et al., 2004).

Broader implications

The Anthopleura elegantissima symbiosis was dominated by ZX in a variety of microhabitats with different thermal and light regimes (e.g., intertidal tidepools and caves) and rarely occurred as mixed or white types, while A. xantho-grammica in the same conditions hosted dense populations of ZC in tentacle and body-column tissues. A number of underlying mechanisms might limit the ZC-A. elegantissima symbiosis; we discuss five of them. First, anemones with different algal types may display differential survival. Augustine and Muller-Parker (1998) observed that an intertidal sculpin species preferred brown A. elegantissima. Second, the transmission of ZC in A. elegantissima might be less likely than the transmission of ZX. How ZC are transmitted and whether associations with each symbiont type can be re-established from an environmental pool is currently unknown, although Schwartz et al. (2002) suggested that ZX within egested material from anemones provide aposymbiotic A. elegantissima larvae with a source of ZX that are taken up into the endodermal cells. Third, the different symbiont types may be selective of host species. For instance, Voolstra et al. (2009) recently demonstrated that successful symbionts elicit almost no change in their coral host's transcriptome and rarely occurred as mixed or white types, supporting the hypothesis that competent coral symbionts do not trigger recognition and rejection by their host. Fourth, the two Anthopleura species may host ZC and ZX entities with different physiological capabilities (the genetic affiliation of the A. xanthogrammica symbionts remains to be determined). Fifth, the two symbiont types might perform differently in the two Anthopleura hosts, irrespective of environmental regime. Indeed, Lewis et al. (2004) demonstrated shifts in the relative abundance of variants of Symbiodinium clade B following initial infection. Several factors might influence symbiont performance. For example, hosts can regulate symbiont division via mechanisms such as nutrient provisioning (e.g., Muscatine and Pool, 1979) or selective expulsion of dividing cells (e.g., Baghdasarian and Muscatine, 2000). It is also feasible that the intrinsic milieu of each anemone may influence the competitiveness of each symbiont differentially.

However, the occurrence of mixed Anthopleura elegantissima in low-irradiance locations where photosynthesis by algal symbionts is restricted (Muller-Parker and Davy, 2001) calls into question what factors limit ZC from overgrowing ZX in environments where co-occurring green A. xanthogrammica are abundant. The influence of light intensity on the MI of ZX from the tentacles of A. elegantissima was also less apparent than for ZX from A. xanthogrammica. In combination, these results suggest that once the symbiosis has been established, algal growth rates may differ in the two host species. Host selectivity or intrinsic differences in the interaction between host and symbiont may influence the ability of ZC to competitively exclude ZX (as mentioned above) and offers a feasible explanation for our data.

Several lines of evidence also suggest that ZX may be a more beneficial symbiont type than ZC and provide an adaptive explanation for why Anthopleura elegantissima might selectively foster ZX over ZC. For example, a model of symbiont carbon flux based on data from the intact A. elegantissima symbiosis indicates that, compared to ZC, ZX have greater potential to translocate photosynthetic carbon to their host in all seasons (Verde and McCloskey, 2007). Isotope data provide additional evidence that ZX contribute more to their host's nutrition than ZC, likely because the productivity of ZX is higher (Bergschneider and MullerParker, 2008). Verde and McCloskey (2007) point out that the higher contribution of ZX to animal respiration should provide a selective advantage to the host if carbon-limitation is an issue. However, whether the two host species are carbon-limited and how differences in their respective diets might influence the symbiosis is currently unknown. While juvenile Anthopleura xanthogrammica, like A. elegantissima, probably feed on small prey items such as zooplankton (e.g., Sebens, 1981b), adult A. xanthogrammica feed primarily on detached mussels (Sebens, 1982c). The two hosts also depend to a different extent on their symbionts: estimates of (14) C translocation in green specimens of A. xanthogrammica and A. elegantissima are (respectively) 4% and 31% (Muscatine, 1971; O'Brien, 1980; Engebretson and Muller-Parker, 1999). It is also possible that ZC may provide additional organic compounds, such as amino acids (Minnick, 1984, as cited in Verde and McCloskey, 2007). How the respective diets and reliance of each anemone host on the two symbionts for nutrition might relate to differences in the relative abundance of each symbiont is currently unknown and underscores the importance of comparative studies aiming to quantify the costs and benefits to hosting ZC and ZX.

Our inability to differentiate among the various scenarios that potentially govern the distribution of ZX and ZC in each anemone species points to a need for further studies on this system to establish the genetic diversity of symbionts hosted by Anthopleura and whether these genetic entities have similiar physiological capabilities in both host species. Nevertheless, collectively, the lack of flexibility in algal type and MI imply that mechanisms other than extrinsic environmental parameters restrict the ZC-Anthopleura elegantissima symbiosis. In contrast, environmental parameters may play a relatively important role in the A. xanthogrammica algal symbioses. For instance, A. xanthogrammica individuals are green in shaded microhabitats, but in locations exposed to light (tidepools and rock crevices), brown algal types are common. Furthermore, A. xanthogrammica commonly hosts mixed symbiont populations ([approximately equal to]15%), a finding that is suggestive of environmental control, as evolutionary theory predicts that hosting populations of competing symbiont genotypes will be relatively costly (Frank, 1996). Secord and Augustine (2000) also showed that the relative abundance of ZX and ZC is more responsive to the effects of latitude and tidal height in A. xanthogrammica than in A. elegantissima. While research has focused on A. elegantissima as a model organism to tease out the effects of temperature and light on the in hospite and in vitro photobiology and physiology of the two symbiont species (e.g., Saunders and Muller-Parker, 1997; Verde and McCloskey 2001, 2002, 2007; Bergschneider and Muller-Parker, 2008), A. xanthogrammica, because of its greater flexibility in algal type, may provide additional insights into environmental regulation of the intact symbiosis.

In summary, the two Anthopleura species display differences in the distributions of ZX and ZC, as well as in the MI values of their tentacle ZX. These differences may reflect host-specific differences in the relative importance of extrinsic environmental regulation of symbiont type. Our results highlight the importance of comparative approaches to further our understanding of how the respective physiological capabilities of each symbiont, environmental conditions, and the host's biology play out in influencing host-symbiont interactions, and ultimately, in determining the distribution patterns of ZX and ZC in Anthopleura spp.

Acknowledgments

We are grateful to the Bamfield Marine Sciences Centre for hosting this study as part of their University Programme to promote undergraduate research. Detailed comments from two anonymous reviewers and S. Davy greatly improved the manuscript. Technical assistance and intellectual input from J. Anticamara, C. Bergstrom, B. Cameron, and T. Macdonald are especially appreciated. L. Bird, T. Bird, R. Eustace, M. Lloyd, J. Marcus, and V. Vergara helped with fieldwork. A. and J. Gower and P. and J. Hutchings provided accommodation to A. Bates during sampling at Tofino locations.

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Received 15 January 2009; accepted 19 April 2010.

*To whom correspondence should be addressed. E-mail: bates. amanda@gmail.com

Abbreviations: MI, mitotic index; ZC, zoochlorellae; ZX, zooxanthellae.

AMANDA E. BATES*, LILY MCLEAN,PATRICK LAING, LISA A. RAEBURN, AND CRYSTAL HARE

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Author:Bates, Amanda E.; McLean, Lily; Laing, Patrick; Raeburn, Lisa A.; Hare, Crystal
Publication:The Biological Bulletin
Article Type:Report
Geographic Code:1CANA
Date:Jun 1, 2010
Words:7049
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