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Current Zoology 61 (4): 739–748, 2015

Received Mar. 18, 2015; accepted May 27, 2015.

Corresponding author. E-mail: [email protected]

© 2015 Current Zoology

Color change in a marine isopod is adaptive in reducing predation

Kristin M.HULTGREN*, Hannah MITTELSTAEDT

Department of Biology, Seattle University, 901 12th ave., Seattle WA 98122

Abstract Although background matching is a common form of camouflage across a wide diversity of animals, there has been

surprisingly little experimental work testing the fitness consequences of this camouflage strategy, especially in marine ecosys-

tems. In this study, we tested whether color camouflage enhances survival of the intertidal marine isopod Pentidotea (Idotea) wosnesenskii, quantified patterns of camouflage in different algal habitats, and examined how algal diet affected color change and

growth using laboratory assays. In the field, isopods collected from two differently colored algal habitats (the brown alga Fucus distichus and the red alga Odonthalia floccosa) matched the color of their respective algal habitats, and also differed significantly

in body size: smaller red isopods were found on red algae, while larger brown isopods were found on brown algae. Predation ex-

periments demonstrated these color differences had fitness benefits: brown isopods that matched their brown algae habitats sur-

vived at higher rates than red unmatched isopods. Surprisingly, despite the propensity of isopods to match their algal habitats, al-

gal diet had no effect on color change in color change experiments. Instead, isopods in all treatments turned browner, matching

the color of the algal habitat that many isopods are found on as adults. In summary, our data supported our hypothesis that back-

ground matching serves an adaptive function in reducing predation, with important evolutionary implications for explaining the

wide variation in color change mechanisms in idoteid isopods [Current Zoology 61 (4): 739–748, 2015].

Keywords Isopod, Pentidotea wosnesenskii, Camouflage, Crypsis, Background matching, Color change

One of the most common observations about the nat-ural world is that many animals vary in coloration or patterning (Cott, 1940). Color variation is especially prevalent in marine organisms, many of which match the host or habitat on which they occur (Lee and Gil-christ, 1972; Bauer, 1981; Jormalainen et al., 1995; Palma and Steneck, 2001; Hultgren and Stachowicz, 2008; Mäthger et al., 2008; Bush et al., 2010; Cournoy-er and Cohen, 2011; Zylinski et al., 2011). As Tinbergen (1963) pointed out, one can take two different approa-ches to explaining this color variation: a proximal ap-proach (“how” variation in color occurs, in terms of mechanism) and an ultimate approach (“why” color variation occurs, in terms of fitness).

From a proximal perspective, several processes— operating on different time scales—can drive intraspe-cific color variation. First, different individuals of the same species can have permanent color or pattern mor-phs, i.e. polymorphism (Reimchen, 1979; Bauer, 1981; Jormalainen et al., 1995). For example, the shrimps Heptacarpus pictus and H. paludicola occur in five dif-ferent morphs that vary in coloration (transparent or colored) and patterning (bands, stripes, and spots; Bauer,

1981). Second, on slower timescales (days to months), an individual may change color or pattern with the sea-son (Cott, 1940), through ontogeny (Booth, 1990; Pal-ma and Steneck, 2001, Todd et al., 2006, 2009), or due to habitat shifts (Lee and Gilchrist, 1972; Iampietro, 1999; Hultgren and Stachowicz, 2008). Many species that undergo ontogenetic color changes are cryptic as juveniles and lose cryptic coloration upon maturity (Cott, 1940; Booth, 1990). For example, the crab Cancer ir-roratus is polymorphic as a juvenile to match polyc-hromatic juvenile habitats, but molts into a rather drab adult (Palma and Steneck, 2001). Color change is di-rectly linked to shifts in habitat for some species (e.g. crustaceans) that are able to sequester pigments from their habitats into their cuticles and change color upon molting (Iampietro, 1999; Hultgren and Stachowicz, 2008). Other crustaceans are able to modify dietary pig-ments into cuticle pigments for coloration when moving among habitats (Lee, 1966b; 1966c; Lee and Gilchrist, 1972). Finally, on much shorter timescales (millise-conds to days), many species are able to change color or pattern using chromatophores, iridophores, melano-phores, and leucophores (Lee and Gilchrist, 1972; Bauer,

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740 Current Zoology Vol. 61 No. 4

1981; Wilmer et al., 1989; Hanlon, 2007; Barbosa et al., 2008; Mäthger et al., 2008; Hanlon et al., 2009; Stuart- Fox and Moussalli, 2009; Cournoyer and Cohen, 2011; Zylinski et al., 2011; Stevens et al., 2013, 2014). Chro-matophore-based color change involves sensory detec-tion of the environment, and adjustment of color or pat-terning by dispersion or concentration of pigment with-in chromatophore cells, which are typically under neural or endocrine control (summarized in Stuart-Fox and Moussalli, 2011). For example, cuttlefish and other ce-phalopods can detect complex visual backgrounds using their keen vision, and adapt their own coloration (and body texture, in some cases) to match their background in seconds using extremely rapid chromatophore changes (summarized in Hanlon, 2007; Hanlon et al., 2009). The littoral isopod Ligia oceanica uses melanophores to change color on a circadian rhythm (to adjust both ca-mouflage and body temperature), but can also make fine-scale adjustments to color on the scale of minutes (Wilmer et al., 1989). Some species use multiple pro-cesses to change color; for example, the marine isopod Idotea baltica occurs in six different pattern morphs over the Atlantic, but can change their base cuticle color with pigments between different habitats (Jormalainen et al., 1995).

Color changes in animals often facilitate background matching (sometimes known as phenotype-habitat mat-ching), a specific form of camouflage defined as colors or patterns in an animal that match a random sample of that animals’ habitat (Stevens and Merilaita, 2009). From an ultimate perspective, background matching is thought to be adaptive in reducing predation, and is generally thought to result from differential predation of different color morphs on different habitats and/or ha-bitat selection by different color morphs for different habitats (Reimchen, 1979; Hacker and Madin, 1991; Jormalainen et al., 1995; Merilaita and Jormalainen, 1997; Palma and Steneck, 2001; Bush et al., 2010; Hultgren and Stachowicz, 2010). Alternately, some an-imals visually detect their backgrounds and can adapt their body patterns using chromatophores over variable time scales (seconds to days; Hanlon, 2007; Mäthger et al., 2008; Zylinski et al., 2011; Stevens et al., 2014). It is also possible that background matching could result from animals consuming their plant or algal hosts and directly sequestering pigments into their cuticle, but could have little adaptive value in terms of reducing predation.

Examining whether background matching can reduce predation is typically tested using two approaches. First,

visual modeling studies test whether prey can be de-tected by predators with different visual systems (Stuart- Fox and Moussalli, 2009; Cournoyer and Cohen, 2011; Stevens et al., 2014) or whether prey are well-matched to their environment (Mäthger et al., 2008; Zylinski et al., 2011). Second, field or laboratory experimental stu-dies typically test whether camouflaged individuals are attacked by predators less than non-camouflaged indi-viduals, either in the field (Hultgren and Stachowicz, 2008) or in the lab (Jormalainen et al., 1995; Merilaita, 2001; Manríquez et al., 2008). However, despite the widespread occurrence of background matching across a wide range of animals, experimental tests of whether this strategy is adaptive in reducing predation are rela-tively rare, especially in marine systems (Stevens and Merilaita, 2009).

Isopods in the genus Pentidotea are primarily found in intertidal and shallow subtidal habitats in temperate areas of the eastern Pacific, and typically spend some or all of their life cycle living on algae, often on brown laminarian algal species (Brusca and Wallerstein, 1979; Merilaita, 2001). Predation by fishes is a major source of mortality for idoteid isopods (Lee and Gilchrist, 1972; Wallerstein and Brusca, 1982), and increased fish diversity and abundance in the subtropical east Pacific is thought to be a major factor determining the southern range limits of these isopods, which do not occur below the Tropic of Cancer (Brusca and Wallerstein, 1979; Wallerstein and Brusca, 1982). Reported fish predators (ascertained by gut content analyses) include Gibbonsia elegans, G. metzi, Xererpes fucorum, Micrometrus mi-nimus, Sebastes atrovirens, and Hyperprosopon argen-teum, among others (Mitchell, 1953; Hobson and Chess, 1976). While little is generally known about the visual abilities of these fishes, studies on surfperch (including Micrometrus minimus) suggest they have two retinal cones and are potentially dichromats with some poten-tial for color vision (Cummings and Partridge, 2001). Many idoteid isopods are well-matched to the color of their algal habitat, which generally also serves as a food source (Menzies, 1950; Lee and Gilchrist, 1972; Ru-esink, 2000; Zimmer et al., 2002; Van Alstyne et al., 2006; Carr et al., 2010). In the eastern Pacific, color change in this group has been most thoroughly studied by Welton Lee, who conducted numerous studies on the physiology and ecology of color change and pigmenta-tion in many species, including Pentidotea (Idotea) re-secata (Lee and Gilchrist, 1972), P. (Idotea) monte-reyensis (Lee, 1966a, 1966b, 1972), and Idotea granu-losa (Lee, 1966c). In all three species, color is deter-


HULTGREN KM, MITTELSTAEDT H: Isopod color change 741

mined by some combination of chromatophores and pigmentation of various layers of the cuticle and epi-dermis. However, the three species differ in their ability to change color, based on experiments in which differ-ent colored individuals were fed different colored algal diets. P. resecata appears to have no capability for color change, and color variation (brown or green) is thought to be genetically determined (e.g., color polymorphism; Lee and Gilchrist, 1972). Conversely, P. montereyensis can readily change color to match a new algal habitat, using chromatophores and cuticular pigments (Lee, 1966a; Lee and Gilchrist, 1972). Interestingly, detailed biochemical studies indicate that diet-derived algal pig-ments play no direct role in isopod coloration (i.e., iso-pods are not directly sequestering dietary pigments into their cuticle). Rather, isopods may be modifying algal pigments before incorporating them into their cuticle for coloration (Lee, 1966c; 1966b; Lee and Gilchrist, 1972). Despite extensive work on the proximal mechanisms of color change in Pentidodea, no experimental studies have directly examined the ultimate fitness conse-quences—whether background matching is adaptive in reducing predation in eastern Pacific idoteid isopods (for work on the northern Atlantic species I. baltica, see (Jormalainen et al., 1995; Merilaita, 2001).

In this study, we investigated color variation in the marine intertidal isopod Pentidotea wosnesenskii, using both ultimate and proximal approaches. First, we inves-tigated the fitness consequences of color variation by testing whether background matching occurred in this species, and whether it reduced predation in field te-thering experiments. If color variation in the field was adaptive as camouflage, we would expect i) a correla-tion between algal habitat color and isopod color, and ii) lower predation of color-matched isopods (relative to color-mismatched isopods). Second, from a proximal perspective, we used color change experiments to ex-amine whether algal diet/habitat affects color change. Under this hypothesis, we would expect isopods fed a diet of brown algae (Fucus sp.) would become browner, and isopods fed a diet of red algae (Odonthalia sp.) would become more red. Furthermore, if isopods sub-stantially changed color in different diet treatments be-fore they molted, it would suggest they could use chro-matophores to adjust their color; if color changes oc-curred primarily after molting, this would suggest cu-ticle pigmentation was more important in determining color. Alternatively, isopods may not change color at all, or isopods may change color in ways unrelated to diet or habitat. Finally, since consuming different algal ha-

bitats likely has effects on isopod growth (as well as on color change), we measured growth rates of adult and juvenile P. wosnesenskii on diets of brown algae and red algae.

1 Materials and Methods

1.1 Natural history Pentidotea wosnesenskii is found in intertidal habi-

tats in protected and open rocky coasts, ranging from eastern Russia to the Gulf of California (Brusca and Wallerstein, 1979; Ruesink, 2000; Zimmer et al., 2002; Van Alstyne et al., 2006; Carr et al., 2010). P. wosne-senskii—like other members of the genus Pentidotea— generally prefers living on various species of brown algae as habitat and food source (Brusca and Wallerstein, 1979). Across its range, it has also been reported from the green algae Ulva spp., the brown algae Fucus disti-chus spp. (Fig. 1), the angiosperm Zostera marina, the red algae Odonthalia floccosa, and occasionally under rocks (Menzies, 1950; Lee and Gilchrist, 1972; Van Alstyne, 1988; Ruesink, 2000; Zimmer et al., 2002; Van Alstyne et al., 2006; Carr et al., 2010). 1.2 Field color and size surveys

All field surveys were done at Alki Point in West Seattle, WA (45.57° N, 122.42°W). Seattle is located in the Salish Sea, a marginal sea of the Pacific Ocean that includes the Strait of Georgia, the Strait of Juan de Fuca, and Puget Sound. We collected isopods from two habi-tats: the brown algae Fucus distichus (hereafter Fucus)

Fig. 1 The isopod Pentidotea wosnesenskii on the brown alga Fucus distichus (A), and the red alga Odonthalia floc-cosa (B) Photographed in the field.



and the red algae Odonthalia floccosa (hereafter Odon-thalia). We focused on these two habitats based both on previous literature documenting dietary preferences of P. wosnesenskii in the Salish Sea region (Van Alstyne, 1988; Ruesink, 2000), and on our own preliminary inter-tidal and subtidal seagrass surveys at the study site and at other sites in this region (Whidbey Island, 48.231°N, 122.767°W; Redondo Beach: 47.349°N, 122.325°W; Hultgren, unpublished data). We measured body size from isopods collected in these two habitats in June 2014 (Fucus: n = 25 isopods, Odonthalia: n = 30), and collected body size and color data from isopods col-lected in May 2015 (Fucus: n = 22, Odonthalia: n = 23). During the 2015 survey, we also collected individual samples of each algal habitat (n = 12 individuals from each species) to quantify habitat coloration.

We measured body size (total length in mm) of all isopods using dial calipers. To measure color, individual isopods or algae were digitally photographed in a con-tainer of seawater (2 cm depth) on a 60% gray-scale photography board (Delta, Dallas, Tex.) fitted with red and green color swatch standards (Ace Hardware) under consistent camera height (30 cm). Using ImageJ (Ab-ramoff et al., 2004), we quantified color from these photographs using a method modified from Hultgren and Stachowicz (2008). Briefly, we selected the entire dorsal surface of the isopod, and initially measured red, blue, and green color channel values using the color histogram function on ImageJ. Because natural light conditions varied in the field, we also measured red, blue, and green channel values of the color swatch stan-dards in each isopod photo, and used them as a cova-riate in an ANCOVA analysis. As preliminary analyses of field color data indicated that green color channel values varied the most between isopods collected in different habitats, we used variation in the green color channel (hereafter “color”) to quantify color. In general, color values also corresponded to lightness, such that isopods with higher color values were lighter in colora-tion (hereafter referred to as “brown”), and lower color values corresponded to darker isopods (hereafter re-ferred to as “red”). As this method is potentially biased towards human visual perception, and because cameras may have non-linear responses to light that affects their color values (Stevens et al., 2007), we also tested whether color variation we quantified using our method was adaptive in the field (e.g. for predator avoidance; see Tethering experiments).

Quantifying the match of animals to the environment has typically been done by comparing the spectral ref-

lectance of animals and their background using a spec-trometer (Mäthger et al., 2008; Cournoyer and Cohen, 2011), or by various forms of digital image analysis (Hultgren and Stachowicz, 2008; Zylinski et al., 2011; Stevens et al., 2013, 2014). We quantified differences in color between isopods from the two habitats, and the match of isopods to their two algal habitats, using an ANCOVA run on JMP (Cary, NC). The original model included two treatment effects—habitat (Fucus or Odonthalia), object (algae or isopod), and a habitat * object interaction—as well as a covariate (color value of the green color swatch standard). After log transforma-tion, an initial analysis indicated that there were no co-variate * treatment interactions, that residuals were normally distributed, and that variances were homoge-neous. We then proceeded with an ANCOVA with the covariate interaction term removed, and used Tukey post-hoc tests to measure pairwise differences in color between all four treatments (isopods from Fucus, Fucus habitat, isopods from Odonthalia, and Odonthalia habi-tat). We analyzed differences in isopod size as a func-tion of algal habitat (Fucus vs. Odonthalia) and sam-pling date (2014 and 2015). After log-transformation, size data were normally distributed and variances were homogeneous, so we used a GLM that included treat-ment effects and all interactions. Size and color data (and 95% confidence intervals) were back-transformed for all graphs. Effect sizes for all ANCOVA and GLM analyses (ETA-squared) were calculated by JMP. 1.3 Tethering experiments

We tested whether isopod color affected predation using field tethering experiments. In order to obtain size-matched isopods of different colors, we collected isopods from brown Fucus or red Odonthalia habitats and maintained them on these habitats in the lab until tethering experiments commenced (3–7 days). We created size-matched pairs of brown and red isopods by ensuring that isopods did not differ in size by more than 3 mm (mean size differential = 0.94 mm, or 4.5%). We then photographed each isopod pair prior to tethering for color analysis, and tested whether isopods in the two groups differed in color using the methods above. We then tethered each isopod by tying 2-lb clear fishing line around the body and securing the fishing line on the ventral side of the isopod with a drop of Super Glue. Both tethered isopods in a pair were attached to a cable tie (yielding tethers 10 cm in length), and attached to the base of an individual Fucus (~10–15 cm in length) along a 10-m transect running parallel to the shore at a tide height of -0.4 ft. at Alki Point (see section 1.2 for



coordinates). Isopods were naturally present in these Fucus habitats at this tide height during prior field col-lections. Isopod pairs were tethered 40‒50 cm apart, and we selected a tether length (10 cm) that would allow reasonable isopod movement, while trying to ensure isopods stayed on their Fucus habitat. For each pair, we marked different isopod tethers by making a small knot in the tether near the base of each brown isopod. Iso-pods missing from tethers were assumed to have been killed by predators, as isopods did not escape tethers or expire in preliminary laboratory tethering trials (n = 5 isopods, duration = 4 days). Although we acknowledge that tethering may artificially increase predation rates by preventing escape or migration of isopods (Peterson and Black, 1994), we were most interested in the rela-tive susceptibility of different colored isopods to preda-tion on Fucus, and assumed that tethering bias did not vary between different colored isopods.

After tethering in the field, we checked each isopod pair daily at low tide (between approximately 10–11 am) for three days. We ran two blocks of experiments (block 1: n = 30 pairs, start date 5-Aug-13; block 2: n = 16, start date 2-Sept-13) along two different transects of Fucus at the same tidal height (-0.4 ft). While checking block 1, we noted that one or both isopods in seven te-thered pairs were being consumed by sea anemones. Since visual predators such as fish are thought to be the primary predators of isopods (Mitchell, 1953; Wallers-tein and Brusca, 1982), and we were concerned that te-thering may have artificially increased predation by ses-sile anemones, we eliminated these paired replicates from the analysis. In block 2, to minimize any artifacts asso-ciated with sea anemone predation, we ensured that all isopods were tethered at least 20 cm away from any sea anemones, and found no evidence of anemone predation. To minimize any differences in predation rates between our two experimental blocks, we quantified survivor-ship of each replicate pair whenever at least one of the two isopods present were consumed as 0 = consumed, 1 = remaining; replicates in which both isopods were ea-ten (i.e., we did not know which one was consumed first) were scored as (0,0) and replicates in which neither in-dividual was eaten were scored as (1,1). There were no differences in effects of isopod color on predation rates between blocks, so we pooled data from the two blocks and used Fisher’s exact test to determine whether red (unmatched) isopods were consumed at a higher rate than brown (matched) isopods on brown Fucus habitat. 1.4 Color change and growth assays

We tested the abilities of isopods from two habitats

to change color when fed different diets in the laborato-ry using a 2 × 2 (habitat source x diet) design. All iso-pods were collected from Alki beach, Seattle in spring 2014. As in field surveys (Fig. 2B), isopods collected from Fucus were significantly larger than isopods from Odonthalia (Wilcoxon test, P < 0.0001; Fucus mean ± standard error = 18.0 ± 5.2 mm, Odonthalia = 10.4 ± 2.0 mm, data not figured), and we did not control for size (i.e., we did not adjust these size differences be-tween habitats) in our color change experiment. Isopods were maintained in individual ~ 1 L clear containers with small holes (to allow water circulation while li-miting isopod escape) in indoor seawater tanks main-tained at 13°C, and fitted with multiple air stones for circulation. As closely related species of isopods (P. montereyensis) only change color when exposed to light (Lee, 1965; 1966a), our experimental tanks were ex-posed to natural sunlight for the duration of the experi-ment (April–June 2014). Prior to starting the experiment, isopods were maintained on the same habitat source they were collected on (Fucus or Odonthalia) until they were randomly assigned to either a Fucus diet treatment (n = 15 isopods collected from Fucus, n = 20 from Odonthalia) or Odonthalia diet treatment (n = 15 from Fucus, n = 20 from Odonthalia).

We initially photographed isopods to quantify “start” color using methods described above, and supplied iso-pods with enough algae to cover the bottom of their container (~100 g), such that the algae effectively served as diet and habitat. Isopods were then checked approximately daily for molts, measured weekly with dial calipers, and photographed weekly to quantify any color variation that occurred between molts. For each individual isopod, we calculated three color values: start color, molt color (photographs taken after the isopod molted), and premolt color (mean color from all photos taken after the experiment started, but before the isopod molted). Significant differences between start color and premolt color would indicate use of chromatophores to change color, while start—molt color differences or premolt—molt color differences would indicate cuticu-lar pigments were used for color change.

We estimated relative growth rate of isopods in each of the 4 treatments using the formula ln(TLm - TLs

-1) * (days to molt)-1, where TLs = start total length, TLm = total length after one molt. For isopods that molted twice during the experiment, we used the mean growth rate over 2 molts; to minimize effects of prior diet on growth, we did not use isopods that molted within the first 6 days of the experiment (approximately 25% of



the mean time to molt) in the analysis. As data were normally distributed, we analyzed growth data using a 2-way GLM with habitat source (Fucus or Odonthalia), algal treatment (Fucus or Odonthalia), and all interac-tions as factors using JMP 5.1 (SAS Institute).

As data were normally distributed and variance was similar among treatments, we used two parametric ana-lyses to measure color differences across the four treat-ments. First, we analyzed color at different time points (start, premolt, and molt color) using three separate ANCOVA analyses. In each analysis, we included the main effects and interactions (habitat source and diet treatment) and the covariate (standard color value), and initially included all main effect * covariate interactions. Since there were no significant interactions between the covariate and any of the main effects or main effect interactions, we removed these interaction terms for the final analysis. Second, we examined color change over time using a repeated-measures ANOVA. We first re-moved the effect of the covariate by calculating the re-siduals from a model of isopod color with the covariate (standard color value) as the main effect. We then used these residuals in a univariate repeated-measures ANO-VA with the main effects (habitat source and algal treatment), a time effect (start, premolt, or molt), and interactions between the main effects and time.

2 Results

2.1 Field color and size surveys For analyses of isopod and habitat color, our final

ANCOVA model (F2, 61 = 28.71, P < 0.0001, effect size = 0.485) included a significant effect of habitat (Fucus or Odonthalia: P < 0.0001) and a non-significant effect of the covariate (green standard color value: P = 0.2735). When we compared all four treatment groups, there were significant differences in color between iso-pods collected on different habitats (P < 0.05, Tukey test); isopods from Odonthalia (red algae) had signifi-cantly darker (more red) color values than isopods from Fucus (Fig. 2A). Similarly, Fucus algal habitat was sig-nificantly lighter in color than Odonthalia (P < 0.05), but there were no significant differences in color be-tween isopods collected on a habitat and the color of the habitat itself. That is, isopods collected on Fucus did not differ significantly in color from Fucus algae, and iso-pods collected from Odonthalia did not differ signifi-cantly in coloration from Odonthalia algae (Fig. 2A, P > 0.05). For body size, our final model was signifi-cant (F3,96 =18.47, P < 0.0001, effect size = 0.366) and included significant effects of habitat source (P <

0.0001), date (P = 0.029), and a habitat * date interac-tion (P = 0.025). Isopods from Fucus did not differ sig-nificantly in size between survey dates (P > 0.05, Tukey test), but isopods collected from Odonthalia in 2015 were significantly larger than isopods collected in 2014 (P < 0.05, Fig 2B). However, isopods collected from Odonthalia were always significantly smaller than iso-pods collected from Fucus, across both sampling dates (P < 0.05, Tukey test, Fig. 2B). 2.2 Tethering experiments

Isopod color significantly affected survivorship of te-thered isopods on brown Fucus. Isopod color was signi-ficantly different between brown and red treatments (ANCOVA, F2, 101 = 18.92, P < 0.001, data not figured), and brown isopods had significantly higher survivorship than red isopods (Fisher’s exact test, n = 46 pairs, P = 0.0164, Fig. 3). Paired isopods from the two color treatments did not vary in size (Wilcoxon signed-rank test, Test statistic = 22.00, 2-tailed P = 0.801, data not figured), but isopod size in the tethering experiment (overall mean ± standard deviation = 2.09 + 0.31) was slightly but significantly larger (t-test, P < 0.001) than

Fig. 2 Isopod and algae color (A), and isopod total body length (B) in the field Isopods were collected from either brown algae (Fucus distichus, light gray bars) or red algae (Odonthalia floccosa, dark gray bars). Error bars indicate 95% confidence intervals; means sharing the same letters are not significantly different according to Tukey-Kramer post hoc tests (A, ANCOVA; B, GLM). Numbers at the base of each bar indi-cate n-value.



isopod sizes recorded from Fucus in the field (1.62 ± 0.41 cm). 2.3 Growth rates

Isopods across all four habitat source * algal treat-ment combinations differed significantly in growth rate (F3,55 = 20.624, P < 0.0001, effect size = 0.529, Fig. 4A), and there were significant effects of both habitat source (F1,55 = 24.713, P < 0.0001) and algal treatment (F1,55 = 31.23, P < 0.0001) on growth rate. Individuals from both habitat sources had higher growth rates on Fucus diets relative to Odonthalia (P < 0.05, Tukey test), and adult isopods from Fucus, when fed diets of Odonthalia, had significantly lower growth rates than any other treatment combination (P < 0.05, Tukey test, Fig. 4A). 2.4 Color change

When isopod color among the four different treat-ments was examined at different time points in the color change experiment (ANCOVA analyses), there were significant treatment effects only for isopod start color (ANCOVA full model: F2, 66 = 11.12, P < 0.0001, effect size = 0.252). Specifically, there were significant effects of habitat source on isopod start color (F1,66 = 20.216, P < 0.0001); individuals collected from Fucus were browner in color than individuals from red algae (Fig. 4B), as seen in field experiments. Otherwise, there were no significant effects of algal treatment nor any algal treatment * habitat source interactions (ANCOVA, P > 0.129). There were no significant isopod color differ-ences among treatments in the full model in either pre-molt color (ANCOVA, F4, 62 = 1.46, P = 0.223, effect size = 0.086) or in molt color time points (ANCOVA, F4,52 = 0.834, P = 0.510, effect size = 0.603, Fig. 4B).

Fig. 3 Survivorship of brown (matched) isopods vs red (contrasting) isopods (n = 46 pairs) tethered on brown algae Fucus distichus P-value indicates significant differences in survivorship (Fisher’s exact test).

Fig. 4 Mean growth rate (A) and color at different time points (B) in laboratory experiments Error bars indicate standard error. For (A), means sharing the same letters are not significantly different (Tukey-Kramer post-hoc tests). In (B), statistical differences between treatment groups at each individual timepoint (ANCOVA) are given in boxes; ns indicates no significant differences between any treatment groups (Tukey-Kramer post-hoc tests). Lower-case letters in (B) indicate significant differences in color (treatments pooled) between different experimental time points (repeated-measures ANOVA).

Isopods in all treatments became lighter (browner)

over time: in the repeated measures analysis, there were significant effects of time (F2,187 = 41.256, P < 0.0001); pooled across all treatments, isopod molt color was sig-nificantly lighter than start color and premolt color (Tukey Test, P < 0.05), while start values and premolt color values did not significantly differ (P > 0.05, Fig. 4B). In this analysis, there were no significant interac-tions between diet and time (P = 0.8708). However, there was a significant time * habitat source interaction (F2,187 = 3.09, P = 0.047), as isopods from different ha-bitat sources were significantly different in color only at



the starting timepoint (when the experiment began), but not at subsequent timepoints.

3 Discussion

In this study, we found strong support for our hypo-thesis that color variation is adaptive as camouflage in the isopod Pentidotea wosnesenskii. In field tethering experiments, on a background of brown Fucus, brown isopods had higher survivorship than red isopods (Fig. 3). In field surveys, isopods collected from red Odon-thalia were significantly more red than individuals from brown Fucus, and both color forms matched their algae well (Fig. 2A). In terms of how color change occurs, algal diet had no apparent effect on color change; iso-pods in all diet treatments turned browner (lighter in color) upon molting, such that there were no distin-guishable differences in color among isopods in any of the treatments by the end of the experiment. Across all treatments, there were no consistent or significant dif-ferences between start color and premolt color, sug-gesting color changes detected by our method were likely due primarily to changes in cuticle coloration rather than chromatophore changes.

Our finding that algal diet had no effect on color change in P. wosnesenskii is unexpected, but not entire-ly surprising. Although many species of closely related isopod (P. resecata, P. montereyensis, Idotea granulosa) readily change color to match their algal habitats in la-boratory color change experiments, physiological ana-lyses demonstrate that the pigments used for coloration in these isopods are not the same pigments found in the algae, suggesting these species may metabolize or oth-erwise modify algal pigments before incorporating them into their carapace (Lee, 1966a; 1966b; 1966c; Lee and Gilchrist, 1972). Indeed, early experiments suggest iso-pods may be able to sense their habitat color, and adjust their dietary pigments to match their habitat (Lee, 1965). As P. wosnesenskii do not appear to be directly seques-tering algal pigments into their carapace, we propose two potential hypotheses to explain why they turned brown across all treatments. First, it is possible that all isopods were responding to a shared component of our experiment, such as the stress of captivity or light avai-lability. We exposed isopods to natural sunlight, as ex-periments on related species showed color change was not possible in the absence of light (Lee, 1965); howev-er, it is possible they may experience darker or more filtered light conditions in the field, e.g. during high tides. However, preliminary color change experiments conducted under shade cloth-filtered sunlight (P. wos-

nesenskii collected from Fucus and fed Fucus or Odon-thalia, n = 22–23) showed similar patterns (all isopods turned browner after molting; Hultgren, unpublished data, 2013). Second, it is possible that P. wosnesenskii makes an ontogenetic habitat shift from red algae to brown algae as they grow, and ontogenetic color chan-ges from red to brown (irrespective of diet) have evo-lved to mirror this habitat shift. Other crustacean spe-cies undergo ontogenetic changes in coloration (Booth, 1990; Todd et al., 2006, 2009); for example, juveniles of Cancer irroratus are polymorphic in color and cryptic against their cobblestoned juvenile habitats, but molt into drab brown adults (Palma and Steneck, 2001). On-togenetic habitat shifts have been documented in other isopod species that use color camouflage; for example, Idotea baltica in the Baltic sea live on green algae (Cladophora) as juveniles, migrate to brown algae (Fu-cus vesicolosus) as adults, and change their base cuticle color in the process (Jormalainen et al., 1995). Our field surveys provide some support for the idea of an onto-genetic habitat shift: isopods on red Odonthalia were significantly smaller than isopods on brown Fucus (Fig. 2B), and we never found very large individuals on Odo-nthalia (maximum size on Fucus = 27 mm, maximum size on Odonthalia = 16.9 mm). Differences in growth rates on different algal diets also provide some support for the idea of an ontogenetic habitat shift. One theory for why ontogenetic habitat shifts occur is that small individuals experience lower growth (but also lowered mortality) in juvenile habitats, but migrate to habitats where they can grow faster (but also experience higher rates of predation) once they reach a size where they are less susceptible to predation (Werner and Gilliam, 1984). In this scenario, growth rates should be higher on the adult habitat (in this case, Fucus), and adult individuals would have very low growth rates on juvenile habitats (Odonthalia), since they would rarely migrate back to those habitats. In our study, isopods from both habitat sources (small isopods from Odonthalia and large iso-pods from Fucus) grew significantly faster in the Fucus diet treatments, while adult isopods from Fucus—when fed Odonthalia (juvenile habitat)—had the slowest growth rates of any treatment (Fig. 4A). Alternatively, it is possible that Odonthalia may just be a lower-quality habitat for isopods, which could explain low growth rates on this algae and lower overall size on this habitat in field surveys. Explicit testing of the ontogenetic ha-bitat shift hypothesis would require more extensive field surveys of size variation in this isopod across its geo-graphic range, as well as experimental tests of relative



predation risk in Fucus vs. Odonthalia habitats. Regardless of the proximal mechanisms driving vari-

ation in color, background matching in P. wosnesenskii reduced predation in our experiments, and is ultimately important in terms of fitness (Fig. 3). Color-based visual crypsis is likely the most effective against visual preda-tors such as fishes, and fish are indeed thought to be the primary predators of idoteid isopods (Mitchell, 1953; Lee and Gilchrist, 1972; Hobson and Chess, 1976; Wal-lerstein and Brusca, 1982). It is also possible that higher survivorship of brown isopods on brown algae occur if fish predators are inherently more attracted to red iso-pods, regardless of habitat; additional tethering experi-ments, either on algae-free substrates or on red algae (Odonthalia) habitats, are needed to examine this alter-nate hypothesis. Additionally, non-visual predators such as crabs (Cancer spp.) are also thought to prey on iso-pods and other small crustaceans (Stevens et al. 1982), and the relative importance of these predators relative to visual predators (fish and birds) may vary.

Individual isopod behavior also could have affected tethering experiments. It is important to note that dif-ferent portions of Fucus plants showed some variation in color, with the distal growing tips being slightly ligh-ter in coloration than the older stipes, and it is possible that different-colored isopods could have chosen dif-ferent microhabitats on an individual Fucus alga (within the constraints of their 10-cm tethers), which could have affected relative predation rates. Microhabitat partition-ing has been documented in the isopod Idotea baltica inhabiting Fucus vesiculosus in the Baltic Sea (Merilai-ta and Jormalainen, 1997). Finally, although we did not find direct evidence for color change via chromato-phores in P. wosnesenskii (i.e., premolt color was not si-gnificantly different from start color), many other close-ly related isopods can change their base color with chro-matophores, including P. montereyensis (Lee, 1972) and I. baltica (Merilaita, 2001). It is possible that subtle chromatophore-driven changes in coloration in P. wos-nesenskii, not detectable by our color measurement te-chniques, can enable partial matching of isopods to dif-ferent algal habitats on shorter time scales.

Although camouflage is recognized as one of the most widespread anti-predator adaptations in animals (Cott, 1940; Stevens and Merilaita, 2009), explicit test-ing of the ultimate fitness benefits of camouflage— reducing predation—is relatively rare, especially in ma-rine systems. As such, this study is an important contri-bution to the detailed literature on color change in other closely related eastern Pacific isopods (Lee, 1966a; 1966c;

Lee and Gilchrist, 1972). For P. wosnesenskii, additional biochemical analyses are needed to examine the pig-mentary components of coloration, and whether isopods are modifying dietary pigments during color change. A more detailed understanding of how color change is achieved in P. wosnesenskii could add to a growing body of knowledge about the wide variability of color change mechanisms in closely related idoteid isopods, from color polymorphism (Pentidotea resecata, e.g. Lee and Gilchrist, 1972) to modification of diet-derived pigments to match perceived background color (P. mon-tereyensis, e.g. Lee 1965, 1966a), to color change pri-marily via chromatophores (Idotea metallica, summa-rized in Lee and Gilchrist, 1972). From a broader evolu-tionary perspective, examining these comparative data in a phylogenetic context could ultimately enable an understanding of the evolution of color change in this common group of crustaceans.

Acknowledgements K.H. and H.M. were funded by a grant from the M.J. Murdock Charitable Trust, administered through Seattle University. We would also like to thank the MaST Marine Lab (Highline Community College) and Rus Higley for assistance in preliminary trials of this experiment. Isopod collection and experiments were conducted under Washington State Scientific Collection permit # 12-264. Tish Con-way-Cranos assisted in algal identifications, and Duane Saf-ford helped with set-up of the laboratory color change expe-riment. This manuscript was greatly improved by the com-ments of Martin Stevens and two anonymous reviewers.

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