ORIGINAL ARTICLE 
doi: 10.1111/j. 1558-5646.2008.00368.X 
EVOLUTIONARY RESPONSES TO 
ENVIRONMENTAL HETEROGENEITY IN 
CENTRAL AMERICAN ECHINOID LARVAE: 
PLASTIC VERSUS CONSTANT PHENOTYPES 
Justin S. McAlister 
Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599 
E-mail: jmcalis@clemson.edu 
Received November 25, 2007 
Accepted February 13, 2008 
Do changes in food resources lead to evolutionary changes in phenotypic plasticity or in different constant phenotypes? I addressed 
this question by studying plasticity of larval feeding arms for "geminate species pairs" in three echinoid genera. These closely related 
species were geographically isolated when the Panamanian Isthmus raised 2.8-3.1 million years ago, creating two different food 
level environments: high but variable food levels in the eastern Pacific versus chronically low food levels in the western Caribbean. 
I reared larvae of geminate species in different replicated food environments for 10 days postfertilization, collected morphological 
measurements of individual arm and body lengths, and calculated degrees of plasticity of relative arm length for each species. 
In contrast to previous studies with temperate echinoids, there was no significant plasticity of arm length in either the Pacific or 
Caribbean species considered here. Caribbean species, however, had significantly longer relative arm lengths than Pacific species, 
regardless of food levels. These results suggest that historical changes in food levels have led to the evolution of constant rather 
than plastic differences between Pacific and Caribbean echinoids. The evolution of plasticity may be limited by the timing of 
reproduction or by egg size in this system. 
KEY WORDS: Constant phenotype, Diadema, echinoid, Echinometra, Eucidaris, evolution, geminate species pairs, larvae, pheno- 
typic plasticity. 
The expression of a phenotype is intricately associated with the 
environment in which an organism resides. In some cases, the phe- 
notype expressed by a given genotype can be influenced by envi- 
ronmental conditions, a phenomenon known as phenotypic plas- 
ticity (Bradshaw 1965; Stearns 1989). Alternatively, a genotype 
can produce the same phenotype across environments, indicating 
that the expression of the phenotype is constant. In heterogeneous 
environments, phenotypic plasticity may allow an organism to 
maximize fitness (Gotthard and Nylin 1995); given appropriate 
genetic variability for plasticity and predictable environmental 
cues in a population, adaptive phenotypic plasticity is expected 
to evolve (Via et al. 1995). Conversely, expression of a constant 
phenotype is expected to confer high fitness and to evolve in en- 
vironments with low heterogeneity and constant environmental 
characteristics. 
The association between environmental changes and the ex- 
pression of plasticity has been studied at several levels of evo- 
lutionary inquiry. Researchers have documented plasticity in re- 
sponse to different environments in many taxa (Boidron-Metairon 
1988; Fenaux et al. 1988; Hart and Scheibling 1988; see reviews 
by Robinson and Wilson 1994; Skulason and Smith 1995; Smith 
and Skulason 1996; West-Eberhard 2003). In addition, studies 
have demonstrated variation in the degree of plasticity among 
populations (or species associations) in response to the degree of 
variation in the environment (DeBenedictis 1974; Kaitala 1991; 
Blouin 1992; Leips and Travis 1994; Buchholz and Hayes 2000; 
? 2008 The Author(s). Journal compilation ? 2008 The Society for the Study of Evolution. 
Evolution 
JUSTIN S. McALISTER 
Leips et al. 2000; Hayes 2002; Langerhans et al.2003; Reinikainen 
and Repka 2003; Morey and Reznick 2004; Stauffer and Van 
Snik Gray 2004). However, few studies explore whether historical 
changes in environments are associated with the evolution of phe- 
notypic plasticity or of different constant phenotypes (see Morey 
and Reznick 2004 for one example). What remains unknown in 
many systems are the relationships and times of divergence among 
different species, and how they have adapted to unique habi- 
tats since separation. These unknown variables make this level 
of evolutionary inquiry of greatest interest because no research 
has demonstrated an association between historical environmen- 
tal changes and the repeated evolution of plastic or constant phe- 
notypes. A comparison of phenotypic expression between close 
relatives that occupy habitats with different patterns of resource 
availability would therefore provide a crucial empirical test of 
the environmental factors underlying the evolution of alternative 
mechanisms for the expression of a phenotype. 
A comparison of this type is provided by "geminate species 
pairs," formed when previously continuous species were sepa- 
rated before or during the raising of the Panamanian Isthmus 
2.8-3.1 million years ago (Keigwin 1982; Duque-Caro 1990). 
Geminate species pairs occur in multiple phyla (Jordan 1908), 
and although their time of divergence is variable (Knowlton et al. 
1993; Knowlton and Weigt 1998; Marko and Jackson 2001), they 
have been evolving in isolation for at least 3 million years since 
the final rise of the Isthmus (Coates and Obando 1996). The rise 
of the Isthmus also separated the tropical western Atlantic (the 
western Caribbean Sea) and tropical eastern Pacific oceans, pro- 
ducing two environments that are markedly different with regard 
to productivity, which equates to food for plankton-feeding or- 
ganisms. The eastern Pacific is characterized by strong, seasonal 
upwelling that produces variable yet predictably high phytoplank- 
ton food levels, whereas the western Caribbean experiences little 
upwelling, has low primary production, and is thus constantly nu- 
trient poor and low in phytoplankton food (Glynn 1982; Keigwin 
1982). Transisthmian geminate species offer a unique, replicated 
natural research system (Moran 2004) that can be used to address 
the evolution of adaptive phenotypic plasticity in response to the 
heterogeneity of food resource levels. 
Morphological phenotypic plasticity in response to food level 
has been demonstrated in planktotrophic pluteus larvae from 
several species in the echinoderm class Echinoidea (Boidron- 
Metairon 1988; Strathmann et al. 1992; Hart and Strathmann 
1994). These larvae depend on exogenous phytoplankton food, 
and, in response to low food availability, larvae increase the length 
of the ciliated band used for collecting food by growing longer 
larval arms; plasticity of ciliated band length is correlated with 
lengthening of skeletal arm rods in echinoplutei. Increased cili- 
ated band length enhances larval ability to capture phytoplankton, 
and increases in ciliated band length under low food conditions 
have been demonstrated to be adaptive because larvae with longer 
ciliated bands have greater maximum clearance rates (Hart and 
Strathmann 1994). In addition, by increasing ciliated band length, 
the larval surface-to-volume ratio increases, which could increase 
intake of dissolved organic matter (Manahan et al. 1983). For 
this reason, plasticity in arm length has been used as a measure 
of larval feeding history in the field (Strathmann et al. 1992). A 
recent study demonstrates genetic variation of larval arm length 
plasticity in response to food limitation in the echinoid Lytechinus 
variegatus (J. S. McAlister, unpubl. data). 
Here I examine the evolution of phenotypic plasticity of lar- 
val feeding structures in response to differences in environmental 
heterogeneity for planktotrophic larvae of the echinoid geminate 
species pairs found off the coasts of Panama. For each geminate 
pair, one species lives in the highly productive but variable east- 
ern Pacific, whereas the other inhabits the minimally productive 
and constant western Caribbean. Three sets of hypotheses can be 
made regarding the effect of food level heterogeneity on plastic- 
ity of larval arm length. First, the "Plasticity" Hypothesis posits 
that all species will exhibit some degree of phenotypic plasticity 
of larval arm length. This expectation can be justified by the fact 
that plasticity of larval arm length has been demonstrated in a 
large number of echinoid species in which it has been examined 
(Boidron-Metairon 1988; Hart and Scheibling, 1988; Strathmann 
et al. 1992; Hart and Strathmann 1994; Sewell et al. 2004; Reitzel 
and Heyland 2007). 
Second, the "Differential Plasticity" Hypothesis posits that 
larvae evolving in the western Caribbean, which has constant 
low phytoplankton food levels, will exhibit low to no degrees 
of phenotypic plasticity of arm length. Conversely, larvae evolv- 
ing in the eastern Pacific, characterized by variable phytoplankton 
food levels, will exhibit greater degrees of phenotypic plasticity of 
arm length. In support of this hypothesis, larval echinoid species 
from tropical or subtropical waters with low food levels show 
minimal plasticity (Boidron-Metairon 1988; Eckert 1995; Reitzel 
and Heyland 2007), whereas species from cold temperate waters 
with more variable food levels show greater degrees of plastic- 
ity (Boidron-Metairon 1988; Hart and Scheibling 1988). None of 
these studies are comparative or examined many taxa however. 
Third, the "Constant Differences" Hypothesis posits that lar- 
vae evolving under constantly low food levels, characteristic of the 
western Caribbean, will grow longer arms relative to body length 
than larvae evolving in the variable food levels of the eastern 
Pacific. If phenotypic plasticity confers a benefit only in hetero- 
geneous environments, then there may be no benefit of plastic- 
ity for larvae evolving in the homogeneous environment of the 
Caribbean. A better evolutionary strategy for resource acquisition 
may be to evolve longer arms under all conditions, especially if 
there is a cost of phenotypic plasticity (DeWitt et al. 1998). The 
number of examples in the literature is too small to thoroughly 
EVOLUTION 2008 
EVOLUTIONARY RESPONSE TO ENVIRONMENT HETEROGENEITY 
test the patterns described by these hypotheses, nor have these 
ideas been tested in a rigorous phylogenetic context. My results 
indicate that historical changes in food availability can lead to 
the repeated evolution of differences in the expression of con- 
stant phenotypes between species, and suggest that the evolution 
of phenotypic plasticity may hinge in part on selection for other 
life-history characteristics associated with resource acquisition, 
for example, egg size. 
Materials and Methods 
I investigated whether heterogeneity of food level is correlated 
with the expression of plastic and/or constant larval arm length by 
studying three geminate pairs of marine sea urchins in the genera 
Diadema, Echinometra, and Eucidaris. These species are found in 
coral reef habitats off the Caribbean and Pacific coasts of the Re- 
public of Panama (Lessios 1979, 1981; Bermingham and Lessios 
1993; McCartney et al. 2000). I performed two sets of experiments 
over the course of two summer field seasons in Panama. The first 
set of experiments examined larval morphological plasticity un- 
der two different food levels in two true geminate pairs, Diadema 
antillarum in the Caribbean with D. mexicanum in the Pacific and 
Eucidaris tribuloides in the Caribbean with Eu. thouarsi in the Pa- 
cific; genetic divergence among these species pairs is pegged to 
the final closure of the Central American Seaway approximately 
2.8-3.1 million years ago (Lessios et al. 1999, 2001). In addi- 
tion, I included in this experiment the Echinometra complex: Ec. 
lucunter and sister taxa Ec. viridis in the Caribbean with Ec. van- 
brunti in the Pacific. The most recent common ancestor of Ec. 
lucunter and Ec. viridis is thought to be the geminate partner of 
Ec. vanbrunti, diverging approximately 3.1 million years ago; Ec. 
lucunter and Ec. viridis diverged approximately 1.27-1.62 mil- 
lion years ago (McCartney et al. 2000). Although these three pair- 
ings are not the only echinoid geminates, they represent the gen- 
era with planktotrophic larvae that are most easily collected and 
spawned, and were therefore most amenable to this analysis. A 
second set of experiments examined the effects of food limita- 
tion on growth of Ec. vanbrunti and Ec. viridis larvae reared in 
one of five different food levels, including satiating and starvation 
conditions. 
Adults of the sea urchins D. mexicanum, Ec. vanbrunti, and 
Eu. thouarsi were collected from the Pacific Ocean in June and 
July 2005 by SCUBA from populations located in waters off 
Isla Taboguilla near Panama City, Panama (see Fig. 1). Pacific 
species were placed in coolers filled with seawater and trans- 
ported by boat to the Smithsonian Tropical Research Institute's 
(STRI) Naos Island Laboratories (Naos) near Panama City. Adults 
of their geminate species counterparts, D. antillarum,Ec. lucunter, 
Ec. viridis, and Eu. tribuloides were collected from the Caribbean 
Sea by snorkel in the vicinity of STRI's Galeta Marine Labo- 
ratory near Colon, Panama (see Fig. 1). Caribbean species were 
placed in disposable plastic containers (3^1 urchins per container) 
filled with a small amount of seawater. The containers holding 
Caribbean urchins were stacked in a cooler and transported by 
vehicle to Naos. All species were maintained in flow-through 
seawater aquaria at Naos; at this facility, effluent from aquaria 
containing Caribbean species is treated with bleach before dis- 
charge into Pacific coastal waters. 
LARVAL CULTURE 
Gametes were obtained from adult urchins by injecting approxi- 
mately 1 mL of 0.5M KC1 through the peristomium into the body 
x                 Caribbean Sea 
N 
}--_                                                    Galera Marine lab ^^^ 
-$- 
^jiCotori     c? %^ 
Jj&t  Jtinoma City 
.)            Panama J ftoos l5tond tubs V, 
Gulf of Chlnqul. \       ~*              \. Bay of Panama          \ 
60 km                                       ?^     _f~~ 
Pacific Ocean 
Figure 1. Map of the Republic of Panama indicating the locations of the Smithsonian Tropical Research Institute's Galeta Ma- 
rine Laboratory on the Caribbean coast and the Naos Island Laboratories on the Pacific coast (modified from map available at 
http://www.enchantedlearning.com). Adult urchins used to obtain gametes and produce larvae for this study were collected in wa- 
ters in the immediate vicinity of Galeta Marine Laboratory and at Isla Taboguilla, located approximately 10 km offshore from Naos Island 
Laboratories. 
EVOLUTION 2008 
JUSTIN S. McALISTER 
Table 1. Initial mean (?1SE) egg diameters (bold text; units = micrometers) and volumes (normal text; units = nanoliters) for females 
of each species used to produce different larval families. Values were calculated using 25 eggs from each female. 
Species Ocean 
Female used for each Full-sibling Family 
1 2 3 4 Average 
D. antillarum C 74.56 (0.45) N/A N/A N/A 74.56 (0.45) 
0.22 (0.00) 0.22 (0.00) 
D. mexicanum P 64.96 (0.34) 64.8 (0.45) 68.16 (0.19) N/A 65.97 (0.27) 
0.14(0.00) 0.14(0.00) 0.17 (0.00) 0.15 (0.00) 
Ec. lucunter C 83.2 (0.47) 82.08 (0.40) 79.84 (0.66) 87.28 (0.56) 83.1 (0.38) 
0.30 (0.00) 0.29 (0.00) 0.27 (0.00) 0.35 (0.00) 0.30 (0.00) 
Ec. Viridis c 90.8 (0.41) 90.08 (0.38) 89.44 (0.45) N/A 90.11 (0.25) 
0.39 (0.00) 0.38 (0.00) 0.38 (0.00) 0.38 (0.00) 
Ec. vanbrunti p 67.84 (0.19) 67.76 (0.29) 69.76 (0.43) N/A 68.46 (0.21) 
0.16(0.00) 0.16(0.00) 0.18 (0.00) 0.17 (0.00) 
Eu. tribuloides c 92.64 (0.58) 92.16 (0.70) 93.44 (0.40) N/A 92.74 (0.34) 
0.42 (0.00) 0.41 (0.00) 0.43 (0.00) 0.42 (0.00) 
Eu. thouarsi p 86.08 (0.40) N/A N/A N/A 86.08 (0.40) 
0.33 (0.00) 0.33 (0.00) 
cavity. Eggs were collected and washed once in 0.45-|xm filtered 
seawater and sperm were collected by mouth pipette and kept on 
ice until use. Full-sibling larval families of all species were estab- 
lished by separately fertilizing eggs from one female with sperm 
from one male. Four separate full-sibling families were estab- 
lished for Ec. lucunter. Three separate full-sibling families were 
established for D. mexicanum, Ec. vanbrunti, Ec. viridis, and Eu. 
tribuloides. Due to the difficulty in finding reproductively mature 
adult females, one full-sibling family was established for both D. 
antillarum and Eu. thouarsi. Initial mean (?1 SE) egg diameters 
(means of 25 eggs each) and egg volumes (assuming a sphere) for 
females from each species are given in Table 1. 
Fertilized embryos and larvae of each species were reared in 
one of two replicated food environments (5 and 1 algal cells/^l). 
Each food level was then replicated among three cultures. Each 
larval culture was fed the unicellular alga Dunaliella tertiolecta 
(UTEX Algal Supply, Austin, TX) daily, starting at 48 h (all 
ages reported are postfertilization). All cultures were reared in 
0.45-(jim filtered seawater in 1-1 plastic tri-pour beakers at den- 
sities of 1 larva per mL and water was changed every day. The 
cultures were maintained in a recirculating water bath held at 28?C 
and were continually stirred at approximately 10 strokes per min 
with acrylic paddles to homogenize food and to keep larvae in 
suspension (Strathmann, 1987). Dunaliella tertiolecta was cul- 
tured at room temperature in microwaved 0.45-|xm filtered sea- 
water enriched with a modified Guillard's f/2 medium (Florida 
Aqua Farms, Inc., Dade City, FL). Algae were separated from the 
growth medium by centrifugation and then resuspended in fresh 
0.45-(jim filtered seawater before use. 
MEASURES OF PHENOTYPE 
On days 2, 3, 4, 5, 6, 8, and 10 approximately 10 larvae were 
removed from each culture. Larvae were placed on a glass slide, 
immobilized with a dilute (<10%) solution of buffered formalin 
in seawater, and covered with a glass cover slip raised on clay 
feet. Three-dimensional Cartesian coordinates were recorded of 
multiple morphological features for five larvae from each culture 
(Fig. 2). These landmarks included the tip and base of each an- 
terolateral, postoral, posterolateral, and posterodorsal arm rod, the 
posterior tip of the larva, and the tip of the oral hood (i.e., the mid- 
point of the soft-tissue that stretches between the pair of anterolat- 
eral arms). To collect data from each larva, I used a camera lucida 
(drawing tube) and a digitizing tablet (Hyperpen 12000U, Aiptek 
Inc., Irvine, CA) to capture x and y coordinates of morphologi- 
cal landmarks. Simultaneously, I obtained z coordinates from a 
rotary encoder (U.S. Digital, Vancouver, WA) coupled to the fine 
focus knob of a Wild M-20 compound microscope (McEdward 
1985). Using these 3-D Cartesian coordinates, I geometrically 
reconstructed individual arm and body lengths (measured in mil- 
limeters) for each larva. Because the postoral arms were the first 
arm pair to develop in all species used in this study, and were the 
most prominent arms at all developmental stages when I collected 
measurements, my analysis focuses on plasticity in their summed 
length (sum of postoral arms). 
STATISTICAL ANALYSIS 
Analysis of variance (PROC MIXED: SAS Institute, Gary, NC) 
tests were conducted (1) across all species and geminate pairs 
("ocean analysis") and (2) for each geminate species pairing 
EVOLUTION 2008 
EVOLUTIONARY RESPONSE TO ENVIRONMENT HETEROGENEITY 
Diadema antiHarum      Eucidaris fribuloides Echinometra viridis 
100 (im 
Figure 2. Low-fed Diadema antiHarum, Echinometra viridis, and Eucidaris thouarsi larvae at 10 days of development postfertilization. 
Morphological characters that I measured on days 2, 3, 4, 5, 6, 8, and 10: PO, postoral arm; BL, body length at midline. All larvae are 
displayed at the same magnification; scale bar represents 100 microns. 
("paired species analyses"), using the natural log-corrected sum 
of the postoral arm lengths (arm length) as the response variable 
in all statistical models. For the ocean analysis, I tested for the 
effect of variation among ocean, genus, family, day of develop- 
ment (day), food level (food), and culture replicate (culture) on 
arm length. The statistical model included the following interac- 
tion terms: ocean with food, ocean with day, day with food, ocean 
with genus, genus with food, and the three-way interactions of 
ocean by day by food and ocean by genus by food. Ocean, genus, 
day, food, and the interaction terms were coded as fixed effects 
and family and culture as random effects. The factor culture was 
nested within ocean, family, and food. 
For the paired species analyses, I tested for the effect of 
variation among species, family, day, food, and culture on arm 
length. The model included terms to account for variation due 
to the interaction of species with food, day with food, species 
with day, and the three-way interaction of species by day by food. 
Species, day, food, and the interaction terms were coded as fixed 
effects and family and culture as random effects. The factor cul- 
ture was nested within species, family, and food. The following 
paired species analyses were conducted: D. antillarum-D. mex- 
icanum; Ec. vanbrunti-Ec. lucunter; Ec. vanbrunti-Ec. viridis; 
and Eu. thouarsi-Eu. tribuloides. 
In both the ocean analysis and the paired species analyses, 
day was coded as a repeated measure with culture as the subject; 
the type of covariance structure of the R matrix was specified 
as Compound Symmetry (CS). Degrees of freedom were calcu- 
lated using the DDFM = BW (Between-Within) option in PROC 
MIXED. Natural log-corrected midline body length (body length) 
was included in all models as a quantitative covariate. I compared 
models both with and without the body length interaction terms 
and used the models (no interaction terms) that provided the better 
fit to the data using Akaike's information criteria (AIC) (Littell 
et al. 1996). 
TEST OF FOOD LIMITATION 
I conducted a second experiment to test the effects on larval de- 
velopment of food levels lower than 1 algal cell/fjil. Adult Ec. 
vanbrunti and Ec. viridis sea urchins were collected in August 
2006 from the same respective Pacific and Caribbean field sites 
as described for the 2005 study (see above). Transportation of adult 
urchins to Naos and their maintenance in flow-through seawater 
aquaria were similar for this experiment. Gametes were obtained 
from adult urchins by peristomial injection of 0.5 M KC1. Fer- 
tilizations were conducted by combining eggs from seven female 
with sperm from four male Ec. viridis, and in a separate container, 
eggs from two female with sperm from four male Ec. vanbrunti. 
Initial mean (?1 SE) egg diameters (means of 10 eggs each) for 
Ec. vanbrunti females were 70.17 (?0.45) and for Ec. viridis fe- 
males were 86.53 (?0.35). Assuming a sphere, mean egg volumes 
(?1 SE) were 0.18 (?0.00) and 0.34 (?0.00) nl, respectively. 
Fertilized embryos and larvae of each species were reared 
in one of five replicated food environments (High?5, Low?1, 
Half?0.5, Limit?0.1, and Zero?0 algal cells/pJ). Each food 
level was then replicated among three cultures. Larval cultures 
were fed the unicellular algal), tertiolecta (UTEX Algal Supply, 
Austin, TX) daily, starting at 48 h. All cultures were reared in 
0.45-p.m filtered seawater in 1-1 plastic tri-pour beakers at densi- 
ties of 1 larva per mL and water was changed every day. Larval 
cultures were maintained in the same manner (i.e., placed in a 
recirculating water bath held at 28?C, etc.), and D. tertiolecta cul- 
tures were reared and dispensed to larvae as described for the 2005 
experiment. 
For this second experiment, measures of phenotype were col- 
lected on days 2, 3, 4, 6, and 8. Analyses of variance (PROC 
MIXED: SAS Institute, Gary, NC) test were conducted between 
the two species and five food levels using the natural log-corrected 
sum of the postoral arm lengths (arm length) and/or midline body 
length (body length) as the response variables in the statistical 
EVOLUTION 2008 
JUSTIN S. McALISTER 
models. I tested for the effect of variation among species, day, 
food, and culture on arm length in one analysis of variance 
(ANOVA) and on body length in a separate ANOVA. The sta- 
tistical models included terms to account for variation due to the 
interaction of species with food, day with food, species with day 
and the three-way interaction of species by day by food. Species, 
day, food, and the interaction terms were coded as fixed effects 
and culture as a random effect. The factor culture was nested 
within species and food. An analysis of variance (PROC MIXED) 
was also conducted between the two species and only the high 
and zero food levels using arm length as the response variable. 
Body length was included in the models testing for differences 
in arm length as a known quantitative covariate. In all statistical 
models for the food limitation experiment, day was coded as a 
repeated measure with culture as the subject and the covariance 
structure of the R matrix was specified as Compound Symmetry 
(CS). Degrees of freedom were calculated using the DDFM = 
BW (Between-Within) option in PROC MIXED. 
Results 
ANOVA among larvae from all Pacific and all Caribbean species 
(the ocean analysis) fed High (5 algal cells/(JLI) or Low (1 algal 
cell/|xl) food levels in the 2005 experiment using arm length as 
the response variable detected significant effects due to genus, 
ocean, day, body length, and the interactions of ocean with day 
and ocean with genus (Table 2). There was no effect due to food, 
the interactions of ocean with food, day with food, genus with 
food, or to the three-way interactions of ocean by day by food and 
ocean by genus by food. The least square mean (? 1 SE: units = 
In mm) estimate of arm length corrected for body length was 
Table 2. Analysis of Variance (ANOVA) results for all Caribbean 
versus all Pacific species larvae. Dependent variable is the natu- 
ral log of the sum of postoral arm lengths. Natural log-corrected 
midline body length was included in the model as a known quan- 
titative covariate. 
Effect df: N, D F Value Pr>F 
Genus 2,40 1247.93 <0.0001 
Ocean 1,38 942 0.0032 
Day 6,228 467.05 <0.0001 
Food 1,38 0.33 0.5686 
Ocean x Food 1,38 0.57 0.4556 
Ocean x Day 6,228 10.90 <0.0001 
Day x Food 6,228 1.18 0.3156 
Ocean x Genus 2,40 397.93 <0.0001 
Genus x Food 2,40 3.13 0.0547 
Ocean x Day x Food 6,228 0.51 0.8025 
Ocean x Genus x Food 2,40 3.01 0.0608 
In (body length) 1,3347 732.62 <0.0001 
-0.5542 ? 0.062 (t38 = -8.96; P < 0.0001) for the Caribbean 
and -0.6314 ? 0.063 (t3g = -10.03; P < 0.0001) for the Pacific. 
Longer absolute arm lengths were expressed by Caribbean 
species of the genera Echinometra (Fig. 3A) and Diadema 
(Fig. 4A) over all developmental days postfertilization as com- 
pared to their Pacific geminate counterparts. Conversely, longer 
absolute arm lengths were expressed by the Pacific Eu. thouarsi 
through Day 6 (Fig. 5A); between Day 6 and Day 10, the Caribbean 
Eu. tribuloides exhibited an increase in absolute arm length 
(Fig. 5A). Longer relative arm to body lengths were expressed 
E 
< 
Genus Echinometra 
High: E. vanbrunti (Pacific) 
Low: E vanbrunti 
High: E viridis (Caribbean) 
Low: E. viridis 
High: E lucunter (Caribbean) 
Low: E. lucunter 
10 
Time (days) 
B 
Genus Echinometra 
High: E vanbrunti (Pacific) 
? Low: E. vanbrunti 
? High: E viridis (Caribbean) 
? Low: E. viridis 
? High: E lucunter (Caribbean) 
? Low: E. lucunter 
-1.6 -1.4 -1.2 
In (Body Length) 
-1.0 
Figure 3. (A) Mean (?1SE) natural log-corrected summed length 
of Postoral arms for High-food (filled symbols) and Low-food (open 
symbols) larvae from the genus Echinometra over time. (B) Mean 
(?1SE) natural log-corrected summed length of Postoral arms ver- 
sus mean (?1 SE) natural log corrected Body Length at midline for 
High-food (filled symbols) and Low-food (open symbols) larvae 
from the genus Echinometra. In both A and B, circle symbols indi- 
cate values for Echinometra vanbrunti, triangle symbols indicate 
values for Echinometra viridis, and square symbols indicate values 
for Echinometra lucunter. Units are In millimeters. 
EVOLUTION 2008 
EVOLUTIONARY RESPONSE TO ENVIRONMENT HETEROGENEITY 
0.4 
0.2 
0.0 
|>   -0-2 
-I    -0.4 
E 
<    -0.6 
ra 
?   4.8 
| -1.0 
-1.2 
-1.4 
-1.6 
Genus Diadema 
High: D. mexicanum (Pacific) 
Low: D. mexicanum 
High: D. antillarum (Caribbean) 
Low: D. antillarum 
4 6 
Time (days) 
10 
High: D. mexicanum (Pacific) 
Low: D. mexicanum 
High: D. antillarum (Caribbean) 
Low: D. antillarum 
-1.8 -1.6 
In (Body Length) 
Figure 4. (A) Mean (?1SE) natural log corrected summed length 
of Postoral arms for High-food (filled symbols) and Low-food (open 
symbols) larvae from the genus Diadema over time. (B) Mean 
(?1SE) natural log-corrected summed length of Postoral arms ver- 
sus mean (?1SE) natural log-corrected Body Length at midline for 
High-food (filled symbols) and Low-food (open symbols) larvae 
from the genus Diadema. In both A and B, circle symbols indicate 
values for Diadema mexicanum and triangle symbols indicate val- 
ues for Diadema antillarum. Units are In millimeters. 
?   -0.5 - 
E 
< 
E 
< 
Genus Eucidaris 
B 
High: E thouarsi (Pacific) 
Low: E. thouarsi 
High: E tribuloides (Caribbean) 
Low: E tribuloides 
10 
Time (days) 
Genus Eucidaris 
High: E thouarsi (Pacific) 
Low: E thouarsi 
High: E tribuloides (Caribbean) 
Low: E tribuloides 
-1.4 -1.3 -1.2 -1.1 -1.0 -0.9 -0.8 
In (Body Length) 
Figure 5. (A) Mean (?1SE) natural log-corrected summed length 
of Postoral arms for High-food (filled symbols) and Low-food (open 
symbols) larvae from the genus Eucidaris over time. (B) Mean 
(?1SE) natural log-corrected summed length of Postoral arms ver- 
sus mean (?1SE) natural log-corrected Body Length at midline for 
High-food (filled symbols) and Low-food (open symbols) larvae 
from the genus Eucidaris. In both (A) and (B), circle symbols in- 
dicate values for Eucidaris thouarsi and triangle symbols indicate 
values for Eucidaris tribuloides. Units are In millimeters. 
by both Caribbean Echinometra species (Fig. 3B) compared to 
the Pacific species over all days. A similar pattern was exhibited 
by the Caribbean D. antillarum as compared to the Pacific D. mex- 
icanum after approximately 4-5 days of development (Fig. 4B). 
Trajectories of arm to body length for both Eucidaris species indi- 
cate that larvae of the Caribbean Eu. tribuloides have larger bodies 
than the Pacific Eu. thouarsi throughout the period of measure- 
ment (Fig. 5B). The distinct arm length relative to body length 
growth patterns expressed by both Eucidaris species, as com- 
pared to the other species used in this study (Figs. 3B, 4B, and 
5B), may reflect the fact that over the time frame of this study 
neither of these species projected distinct anterolateral arms with 
rigid structural elements from the oral hood (the soft-tissue area 
between the anterolateral arms; see Fig. 2). The anterolateral arms 
help to lengthen and support the larval bodies in most species, pro- 
viding for more accurate linear body measurements. The bodies 
of Eucidaris larvae tended to curl inwards as they grew larger, 
even while alive and before slide preparation procedures were 
conducted. This slight curling of the body affected measurements 
of Eucidaris sp. body lengths, making them artificially shorter. 
Paired-species ANOVAs using arm to body length ratio as 
the response variable between larvae fed High (5 algal cells/fjil) 
or Low (1 algal cell/pJ) food levels in the 2005 experiment sup- 
port the visual interpretations of Figures 3-5 and detected the 
EVOLUTION 2008 
JUSTIN S. McALISTER 
Table 3. Analysis of Variance (ANOVA) results for larvae from geminate species pairs, that is, separate tests between a given Caribbean 
species versus its Pacific species geminate. Dependent variable in each model is the natural log of the sum of postoral arm lengths. Natural 
log-corrected midline body length was included in each model as a known quantitative covariate. Effect abbreviations: S, species; D, day; 
F, food; InBL, natural log of body length. Test abbreviations: df, degrees of freedom; n, numerator; d, denominator. 
Species pairs tested: Analysis of Variance 
E. viridis vs. 
E. vanbrunti 
E. lucunter vs. 
E. vanbrunti 
D. antillarum vs. 
D. mexicanum 
E. tribuloides vs. 
E. thouarsi 
Effect df: n, d F value Pr>F df: n, d F value Pr>F df: n, d F value Pr>F df: n, d F value Pr>F 
S 1,32 153.41 < 0.0001 1,38 105.35 <0.0001 1,20 215.43 < 0.0001 1,20 17.13 0.0005 
D 6, 185 153.97 < 0.0001 6,221 197.03 <0.0001 6, 109 312.59 < 0.0001 5,77 466.66 <0.0001 
F 1,32 0.12 0.7353 1,38 0.08 0.7723 1,20 0.24 0.6319 1,20 1.01 0.3267 
S xF 1,32 0.22 0.6395 1,38 0.19 0.6660 1,20 0.67 0.4218 1,20 0.60 0.4470 
S xD 6, 185 58.32 < 0.0001 6,221 15.40 <0.0001 6, 109 10.24 < 0.0001 5,77 21.50 <0.0001 
D x F 6, 185 1.65 0.1365 6,221 1.90 0.0813 6, 109 0.16 0.9864 5,77 1.95 0.0960 
S x D x F 6, 185 1.71 0.1201 6,221 1.59 0.1512 6, 109 0.59 0.7412 5,77 0.76 0.5819 
InBL 1, 1151 1234.13 < 0.0001 1, 1351 1104.55 <0.0001 1,722 199.09 < 0.0001 1,558 4344 <0.0001 
following patterns (see Table 3 for values). The ANOVAs be- 
tween larvae of Ec. vanbrunti and Ec. viridis, Ec. vanbrunti and 
Ec. lucunter, D. mexicanum and D. antillarum, and Eu. thouarsi 
and Eu. tribuloides detected significant effects of species, day, 
body length, and species with day within each analysis. In each 
analysis, there was no effect due to food, species with food, day 
with food, or to the three-way interaction of species by day by 
food. Visual inspection of the arm to body length trajectories for 
each species support this result (Figs. 3B, 4B, and 5B). The least 
square mean (?1 SE : units = In mm) estimates of arm length 
to body length ratio for species from each analysis are given in 
Table 4. 
FOOD LIMITATION ANALYSIS 
Caribbean Ec. viridis larvae did not respond significantly to limit- 
ing food conditions; arm to body length trajectories are compara- 
ble across all five food treatments (Fig. 6A). The growth of larval 
arms relative to body in Pacific Ec. vanbrunti larvae was affected 
by food concentrations lower than 1.0 algal cell/|xl; trajectories 
for the lower food treatments do not extend as far as for the higher 
food treatments (Fig. 6B). This result suggests that food is limit- 
ing for Pacific species but not for Caribbean species. In support of 
this finding, the ANOVA between Ec. vanbrunti and Ec. viridis 
larvae fed High (5 algal cells/\iA), Low (1 algal cell/^l), Half (0.5 
algal cell/|xl), Limit (0.1 algal cell/jxl), or Zero (0 algal cell/|xl) 
food levels in the 2006 experiment using body length as the re- 
sponse variable detected significant effects of species (F12o = 
165.19, P < 0.0001), day (F4>74 = 572.49, P < 0.0001), food 
(F1>20 = 6.50, P = 0.0016), species with day (F4,74 = 13.70, 
P < 0.0001), day with food (Fi6,74 = 4.76, P < 0.0001), and the 
three-way interaction of species by day by food (F1(5j74 = 2.68, 
P = 0.0022). There was no effect due to species with food (F42o = 
2.30, f = 0.0938). 
Low food levels did not induce statistically significant phe- 
notypically plastic responses in larvae from any food treatment 
lower than or equal to 1.0 algal cell/pJ; there was no effect due 
Table 4. Least square mean estimates (units=ln mm) from each of the analyses of variance (ANOVAs) between oceans (Table 2) and 
geminate species pairs (see Table 3). 
Analysis Effect Ocean/Species Estimate (?1SE) df t Value Pr>|t| 
Ocean Analysis Ocean Caribbean -0.5542 ? 0.062 38 -8.96 <0.0001 
Pacific -0.6314 ?0.063 38 -10.03 <0.0001 
Species Ec. viridis -0.2575 (?0.056) 32 -4.59 <0.0001 
Ec. vanbrunti -0.5902 (?0.056) 32 -10.51 <0.0001 
Paired Species Analysis Species Ec. lucunter -0.3552 (?0.066) 38 -5.35 <0.0001 
Ec. vanbrunti -0.5846 (?0.067) 38 -8.68 <0.0001 
Species D. antillarum -0.3468 (?0.1219) 20 -285 0.0010 
D. mexicanum -0.6222 (?0.1209) 20 -5.15 <0.0001 
Species Eu. tribuloides -0.9604 (?0.046) 20 -20.70 <0.0001 
Eu. thouarsi -0.7705 (?0.058) 20 -13.27 <0.0001 
EVOLUTION 2008 
EVOLUTIONARY RESPONSE TO ENVIRONMENT HETEROGENEITY 
E    -0.2 
< 
E. viridis: Caribbean 
? High: 5 
? Half 0.5 
? Limit: 0.1 
? Zero: 0.0 
-1.4 -1.2 
In (Body Length) 
-1.6 -1.4 
In (Body Length) 
Figure 6. Mean (?1SE) natural log-corrected summed length of 
Postoral arms for High-food (5 algal cells/jxl: filled circle symbols). 
Low-food (1 algal cell/fj I: open circle symbols). Half-food (0.5 algal 
cell/|xl: filled triangle symbols). Limit-food (0.1 algal cell/jxl: open 
triangle symbols), and Zero-food (0.0 algal cell/jxl : filled square 
symbols) larvae versus mean (?1SE) natural log-corrected Body 
Length at midline. In (A), values for Caribbean Echinometra viridis 
larvae are indicated. In (B), values for Pacific Echinometra van- 
brunti larvae are indicated. Larvae from both species were reared 
in these food treatments during the subsequent food-limitation 
experiment conducted in 2006. Units are In millimeters. 
to food in the ANOVA using arm length as the response variable. 
This ANOVA was structured the same as the ANOVA for body 
length described above, although including body length as a quan- 
titative covariate, and detected significant effects of species, day, 
body length, and the interactions of species with day, species with 
food, day with food, and the three-way interaction of species by 
day by food (see Table 5). A smaller ANOVA between Ec. van- 
brunti and Ec. viridis larvae fed High (5 algal cells/pJ) or Zero (0 
algal cell/fjil) food levels using arm length as the response vari- 
able and body length as a covariate detected significant effects of 
Table 5. Analysis of Variance (ANOVA) results for Caribbean Echi- 
nometra viridis versus Pacific Echinometra vanbrunti larvae fed 
either High (5.0 algal cells/jul). Low (1.0 algal cell/^l), Solow (0.5 
algal cell/|xl), Limit (0.1 algal cell/^l), or Zero (0.0 algal cell/fji I) food 
levels. Dependent variable is the natural log of the sum of pos- 
toral arm lengths. Natural log-corrected midline body length was 
included in the model as a known quantitative covariate. 
Effect df: N, D F value Pr>F 
Species 1,20 306.12 <0.0001 
Day 4,74 320.78 <0.0001 
Food 4,20 1.84 0.1605 
Species x Food 4,20 3.69 0.0209 
Species x Day 4,74 21.34 <0.0001 
Day x Food 16,74 6.24 <0.0001 
Species x Day x Food 16,74 4.60 <0.0001 
In (Body Length) 1,649 308.07 <0.0001 
species (Fi,s = 78.97, P < 0.0001), day (F4,29 = 147.66, P < 
0.0001), body length (Fij256 = 79.42, P < 0.0001), the interac- 
tions of species with day (F429 = 5.69, P = 0.0017), species with 
food (Fljg = 6.50, P = 0.0342), day with food (F4>29 = 16.77, 
P = < 0.0001), and the three-way interaction of species by day 
by food (F4,29 = 9.84, P = < 0.0001). There was no effect due to 
food (Fh% = 3.89, P = 0.0842). 
Discussion 
In the introduction, I posed three hypotheses regarding how dif- 
ferences in the heterogeneity of phytoplankton food levels may 
influence the evolution and expression of larval arm length. The 
results presented in this study do not support the two plastic- 
ity hypotheses; however they do support the constant differences 
hypothesis. 
CONSTANT DIFFERENCES 
The results from the "ocean" analysis (Tables 2 and 4) indicated 
that larvae from the Caribbean had longer arms relative to body 
length than larvae from the Pacific. Similarly, the results from 
the "paired species" analyses of variance indicated that larvae 
from Caribbean species in the genera Diadema and Echinometra 
had longer arms relative to body length than their Pacific gem- 
inate counterparts. There were significant effects due to species 
on postoral arm length corrected for body length in each of these 
"paired species" analyses of variance (Table 3) and the least square 
mean estimates were greater for each Caribbean species than for 
each Pacific species, within each respective comparison (Table 4). 
However, the result from the "paired species" analysis of variance 
between the Caribbean and Pacific Eucidaris species indicated the 
opposite; Eu. thouarsi from the Pacific had longer arms relative 
to body length than Eu. tribuloides from the Caribbean (Tables 3 
EVOLUTION 200S 
JUSTIN S. McALISTER 
and 4). The result for Eucidaris may reflect the fact that only 
one Eu. thouarsi family was used in the analysis, as opposed to 
three Eu. tribuloides families. I had considerable difficulty obtain- 
ing mature gametes from E. thouarsi during the time I conducted 
the experiments (mid-June through early September in 2005 and 
August of 2006); I injected over 120 individuals with 0.5 M KC1 
to induce spawning and obtained mature gametes from only one 
male and one female. 
The significant difference in relative larval arm length de- 
tected across ocean basins when incorporating all species (the 
large "ocean" analysis of variance) must be interpreted carefully 
(Table 2). Analysis of variance incorporates the magnitude as well 
as the direction of differences between categories; therefore it is 
possible that a large, directional difference in one (or more) gem- 
inate species pairings could have lead to the overall significant 
difference detected across ocean basins. I conducted the paired 
species analyses to account for this possibility and to aid in the in- 
terpretation of this result. Note that three of the four species pairs 
I examined exhibited the same pattern: longer relative arm lengths 
for the Caribbean species in each separate pairing of Echinome- 
tra sp. and with the Diadema (Table 3). Eucidaris was the only 
genus that showed the opposite pattern, perhaps influenced in part 
due to the body length measurement issue mentioned in Results 
(above). A signed-rank test would aid in the interpretation of the 
overall pattern, however there are not enough easily collectable 
echinoid geminate species pairs with feeding larvae (i.e., at least 
six independent pairs) in this system to perform this type of test. 
The results from the "ocean" analysis and the "paired species" 
analyses for Diadema and Echinometra support the Constant Dif- 
ferences Hypothesis; larvae evolving in the constantly low phyto- 
plankton food levels of the western Caribbean grew longer arms 
relative to body length than larvae evolving in the variable food 
levels of the eastern Pacific. In an environment with little to no het- 
erogeneity in food resources, as characterized by the Caribbean, an 
appropriate evolutionary strategy for resource acquisition may be 
to express constantly long arms relative to body length. Express- 
ing a constant long arm phenotype may produce a better return (in 
terms of exogenous energy acquisition) on the investment in long 
arms (in terms of materials to produce and metabolism to main- 
tain) than expressing a plastic arm length phenotype. If pheno- 
typic plasticity confers a benefit in heterogeneous environments, 
as characterized by the Pacific, then there may be no benefit from 
plasticity of arm length for larvae evolving in the homogeneous 
environment of the Caribbean; a cost of plasticity may also con- 
strain the expression of arm length plasticity in a homogeneous 
environment. 
PHENOTYPIC PLASTICITY 
Contrary to the published findings of several researchers using 
various, diverse echinoid species (Boidron-Metairon 1988; Hart 
and Scheibling 1988; Strathmann et al. 1992; Hart and Strathmann 
1994; Sewell et al. 2004; Reitzel and Heyland 2007), none of the 
species I reared in this study exhibited phenotypic plasticity of 
larval arm length. There was no significant effect due to food on 
postoral arm length corrected for body length detected in either 
the "ocean" analysis of variance (Table 2) or any of the "paired 
species" analyses of variance (Table 3). This surprising finding 
begs the question as to why there was no, or minimal, that is, no 
statistically significant, phenotypic plasticity of larval arm length 
exhibited by any of the seven species reared in this study? 
The simplest explanation for this result may be that the low 
food level I used (1.0 algal cell/|xl) was not low enough to induce 
a phenotypically plastic response. In other words, this food level 
may not have been representative of a food limiting condition for 
these larvae; however, this food level falls within the range of 
"low" food levels used in other studies demonstrating arm length 
plasticity in larval echinoids (2 algal cells/jxl: Miner 2005; Reitzel 
and Heyland 2007; ~1.3 algal cells/fjul: Boidron-Metairon 1988; 
0.6 algal cells/fjul: Sewell et al. 2004; 0.5 algal cells/|jd: McAlister 
2007; 0.3 cells/^1: Hart and Strathmann 1994). I chose 1.0 algal 
cell/|xl as a low food treatment because lower food levels have 
been demonstrated to result in stalled larval development in some 
invertebrate species (Pechenik et al. 1984; Eckert 1995; Herrera 
et al. 1996). The results from the food limitation experiment I 
conducted in 2006 (the second experiment described above) us- 
ing Ec. viridis and Ec. vanbrunti indicated that these species did 
not express plasticity of larval arm length at food treatments lower 
than 1.0 algal cell/|xl; there was no significant effect due to food on 
postoral arm length corrected for body length detected by the anal- 
ysis of variance among the five different food treatments (Table 5). 
There was no effect on larval body length with decreasing food 
ration on Caribbean Ec. viridis (see Fig. 6A). However, the two 
lowest food rations (0.1 algal cell/fjil and 0.0 algal cell/fjil) did 
limit development of larval body length in Pacific Ec. vanbrunti 
(see Fig. 6B). The results from the food limitation experiment 
suggest that the lack of measurable levels of phenotypic plasticity 
of arm length within this system (i.e., all of the species used in the 
2005 experiment) is a true finding. As mentioned, these results 
run counter to the published findings of plasticity from multi- 
ple other echinoderm species (Boidron-Metairon 1988; Hart and 
Scheibling, 1988; Strathmann et al. 1992; Hart and Strathmann 
1994; Sewell et al. 2004; Reitzel and Heyland 2007). These re- 
sults do not preclude the fact that there may be additional species 
that are similarly nonplastic; other negative findings may have no 
record of publication. 
None of the species examined in this study demonstrated 
phenotypic plasticity of larval arm length; the results indicated 
that there are no differences in degree of plasticity across all 
species and do not support either the plasticity or differential 
plasticity hypotheses. The results do suggest that despite the well 
10 EVOLUTION 2008 
EVOLUTIONARY RESPONSE TO ENVIRONMENT HETEROGENEITY 
documented and historical differences in productivity between 
the eastern Caribbean and western Pacific (Glynn 1982; Keigwin 
1982), there may be less difference in the variability of food re- 
sources between these environments, on a scale that is relevant to 
larvae. Additionally, when differences in egg size across the gem- 
inate pairs are considered, the lack of plasticity in these species 
suggests that selection may have acted on other life-history char- 
acteristics to account for differences in the levels of exogenous 
phytoplankton food. Alternatively, the possibility exists that a dif- 
ferent measure of plasticity (e.g., arm length relative to juvenile 
rudiment length) might detect significant plasticity in response to 
food and/or divergence in plasticity across the Isthmus. This com- 
parison remains for future studies because the larvae cultured in 
this study were only reared for 10 days, that is, not long enough for 
all species in each food treatment to develop juvenile rudiments 
consistently. Although the negative finding of a lack of plasticity 
is the main strength of this study, interpreting this result, and the 
interesting trends in relative arm length, as evolutionary responses 
must be tempered, however, by the fact that we do not know, and 
cannot determine in this system, what the ancestral conditions 
were with regard to plasticity, relative arm length, or egg size. I 
discuss these possibilities, concerns, and the collective results of 
my experiments below. 
PHYLOGENETIC CONSIDERATIONS: IMPLICATIONS 
FOR THE EVOLUTION OF PLASTICITY 
Recent phylogenetic evidence for echinoderm classes supports 
two plausible phylogenies: echinoids + holothuroids as a sister 
group to asteroids + ophiuroids and/or echinoids + holothuroids 
as a sister group to ophiuroids, which combined form a sister group 
to asteroids (Littlewood et al. 1997; Janies 2001). Within the echi- 
noids, this study examines plasticity in species representing the 
oldest lineage (Order Cidaroida) and second oldest lineage (Order 
Diadematoida) that still have feeding larvae and in a more recent 
lineage (Order Echinoida) (Giese et al. 1991). Although plasticity 
has been observed in echinoid species from other recent lineages 
(reviewed in Sewell et al. 2004), no other studies have examined 
any taxa in the two oldest lineages. The lack of plasticity in the 
Eucidaris sp. and Diadema sp. examined in this study suggests 
that phenotypic plasticity may have only evolved in more derived 
echinoids, and thus a lack of plasticity may be the ancestral condi- 
tion. Alternatively, data from studies on asteroids (George 1994, 
1999) and ophiuroids (Podolsky and McAlister 2005) indicate that 
the species examined from these groups exhibit phenotypic plas- 
ticity of feeding structures, whereas data from one holothuroid 
species showed no difference in body form between well-fed and 
poorly fed larvae (Strathmann et al. 1994), which may indicate 
that plasticity is ancestral. The number of species examined for 
groups other than the echinoids are extremely limited however 
and do not provide strong support for either plasticity or a lack of 
plasticity as the ancestral condition. 
ENVIRONMENTAL VARIATION IN RESOURCE LEVELS: 
LOCAL AND LATITUDINAL CONSIDERATIONS 
Tropical coastal marine ecosystems are commonly oligotrophic 
with patchy food resources (Koblentz-Mishke et al. 1970; Mackas 
et al. 1985) for planktonic larvae. Alternatively, levels of primary 
productivity in temperate coastal ecosystems can cycle between 
low levels in winter and large peaks during spring and summer al- 
gal blooms (Lalli and Parsons 1993). Values of chlorophyll a con- 
centration, a measure of phytoplankton concentration, for coastal 
waters suggest that larvae, regardless of ecosystem, are usually 
food-limited to some degree (Paulay et al. 1985), although com- 
parison among ecosystems is crude because of different assem- 
blages of algal species and lack of information about natural di- 
etary preferences. Published chlorophyll a concentrations are less 
and in some areas approximately one order of magnitude lower 
in tropical (0.01 to 0.35 (Jig/L for Moorea, Society Islands; Ri- 
card, 1981; 0.19 to 0.52 |JLg/L in waters of the Great Barrier Reef; 
Lucas, 1982; 0.59 (Jig/L during the rainy season and 1.48 during 
the dry season of upwelling in the Bay of Panama, and 0.41 dur- 
ing the rainy season and 0.36 during the dry season at San Bias 
Point in the Caribbean; calculated from values reported in mg per 
m3 by D'Croz and Robertson 1997) than in temperate ecosys- 
tems (<1 ug/L in winter to >15 |JLg/L in spring blooms off the 
Washington and Oregon coasts; Richards 1950; Anderson 1964; 
Harrison et al. 1983; and 1.3 to 3.8 |xg/L in August in Long Island 
Sound; Whitledge and Wirick 1983). These values suggest that 
larvae of tropical species may be food-limited to a greater degree 
than larvae of temperate species. 
Faced with constant low food levels, the tropical plank- 
totrophic larvae from the Caribbean species examined in this study 
may have evolved to express a constant long larval arm length phe- 
notype instead of plasticity of arm length. In tropical habitats with 
widespread resource patchiness, expressing a constant long arm 
length phenotype likely increases the food gathering capability of 
a given larva. Conversely, plasticity of arm length may be an evo- 
lutionary strategy that results in greater food gathering capability 
for larvae in temperate habitats. Matched against the patterns of 
ecosystem productivity, plasticity of arm length in pluteus larvae 
has been demonstrated primarily in temperate species. Some of 
the highest magnitudes of larval arm length plasticity are recorded 
for species from cold temperate waters (Boidron-Metairon 1988; 
Hart and Scheibling 1988; Sewell et al. 2004) whereas some of 
the lowest are recorded for species from warm tropical or subtrop- 
ical waters (Boidron-Metairon 1988; Eckert 1995; Podolsky and 
McAlister 2005). There are certainly exceptions to the observation 
that the magnitude of plasticity varies with the productivity of an 
EVOLUTION 2008 11 
JUSTIN S. McALISTER 
ecosystem (e.g., Fenaux et al. 1994 detected plasticity in Paracen- 
trotus lividus from nutrient poor Mediterranean waters), however 
the general pattern suggests that there may be a latitudinal gra- 
dient in phenotypic plasticity of larval feeding structures. Com- 
parative studies of plasticity in multiple populations of species 
whose ranges span tropical and temperate ecosystems would help 
to discern this pattern. To my knowledge there are limited studies 
that compare plasticity among multiple populations (see Bertram 
and Strathmann 1998 for one example) of any species of marine 
invertebrate larvae. 
Similar to the Caribbean species, the tropical Pacific species 
larvae in this system may not have evolved to express pheno- 
typic plasticity because they may only experience low resource 
levels. Despite the well-documented annual heterogeneity of re- 
source levels, some Pacific echinoids (e.g., Diadema mexicanum 
and Ec. vanbrunti) do not release their eggs during the period 
of the year with peak phytoplankton production (Lessios 1981). 
Consequently, larval settlement tends to occur before the period of 
seasonal upwelling (Lessios 1981). Reproduction during the off- 
season, with respect to phytoplankton production, suggests that 
these species may not be taking advantage of the higher resource 
levels during upwelling. However, timing their reproduction to 
avoid upwelling may mitigate the effects on duration of the larval 
period that could result from the lower water temperatures dur- 
ing upwelling (Thorson 1950; Glynn 1972; Hinegardner 1975; 
Lessios 1981). Species evolving in this habitat may time their re- 
production and the duration of larval development to guarantee 
that larvae reach metamorphosis before upwelling. Furthermore, 
the upwelling period is characterized not only by high nutrient 
levels and lower water temperatures, but also by strong offshore 
transport (Smayda 1966; D'Croz and Robertson 1997). Larvae 
that are transported offshore may not be able to find suitable sites 
for postmetamorphic settlement (Lessios 1981). Reaching meta- 
morphosis before upwelling would increase the probability that 
larval settlement occurs near shore. 
Alternatively, there may be finer-scale, localized heterogene- 
ity in food levels within each respective ocean basin. For example, 
intensity of upwelling varies along the Pacific coast of Central 
America (Wyrtki 1967; Legeckis 1985; McCreary et al. 1989). 
The adult urchins collected from the Pacific in this study came 
from the Bay of Panama, which has localized high levels of nu- 
trient upwelling (D'Croz and O'Dea 2007). Other areas along the 
Pacific coast of Panama have lower levels of nutrient upwelling, 
for example, the Gulf of Chiriqui (D'Croz and O'Dea 2007). 
Within-ocean basin differences in the heterogeneity of food re- 
sources may affect the evolution of plasticity if there are high 
levels of larval exchange and genetic mixing among populations 
from different locales. Spatially heterogeneous environments with 
a high degree of patchiness are thought to select for the evolu- 
tion of phenotypic plasticity (Levins 1968). However, in light of 
the timing of reproduction, selection for small egg size in Pa- 
cific species and the constraints that low endogenous energetic 
resources may have on the expression of plasticity (see below), 
and the possibility of high levels of larval exchange among Pacific 
locales, selection may have favored a generalist fixed arm length 
strategy for resource acquisition, instead of a phenotypically 
plastic one. 
SELECTION ON LIFE-HISTORY CHARACTERS: 
THE CONFOUNDING ASPECT OF EGG SIZE 
A discussion of the evolution of phenotypic plasticity or pheno- 
typic fixation of feeding structures in this system must consider 
the documented differences in egg size between Caribbean and 
Pacific species. Egg size has long been considered an important 
component of the life histories of marine organisms (Thorson 
1950; Vance 1973; Christiansen and Fenchel 1979; Strathmann 
1985; Jaeckle 1995; Levitan 2000; Moran 2004; Allen 2005). 
In the Panamanian Isthmus system egg size is larger in many 
Caribbean species than in their Pacific geminates. Lessios (1990) 
has shown that members of geminate pair echinoids found in the 
western Caribbean have larger egg sizes than their eastern Pacific 
counterparts due to changes in productivity following the rise of 
the Isthmus of Panama. The results from the current study show 
the same pattern (see Table 1) in a subset of the species examined 
by Lessios (1990). A similar pattern has been demonstrated for 
bryozoans (Jackson and Herrera 1999) and arcid bivalves (Moran 
2004). This pattern supports theoretical models that predict that the 
greater endogenous resources found in large eggs, which represent 
an increased maternal investment per ovum, evolve in response 
to a poor larval feeding environment, as found in the western 
Caribbean (Vance 1973; Lessios 1990; Levitan 2000). Conversely, 
small egg sizes in the eastern Pacific likely represent an evolution- 
ary response to high levels of oceanic productivity (Lessios 1990; 
Moran 2004). 
An investigation of the effects of egg size on the expression 
of phenotypic plasticity in the Panamanian echinoid system would 
be ideal. However, the arguments for the expression of plasticity 
as an evolutionary response to historical heterogeneity in food re- 
source levels and to a reduction in egg size are confounded in the 
Panamanian system, that is, within each geminate pair, the species 
with smaller egg size inhabits the heterogeneous environment of 
the eastern Pacific. Results from a recent study I conducted us- 
ing echinoid species in the genus Strongylocentrotus that differ in 
egg size (McAlister 2007) indicate, however, that large egg size 
is associated with the expression of greater degrees of phenotypic 
plasticity and of longer arm relative to body lengths than small 
egg size. In light of the results obtained in the current study, the 
expression of longer arm relative to body lengths in the Caribbean 
species may reflect the fact that these species develop from a 
larger egg than their Pacific counterparts. Caribbean species may 
12 EVOLUTION 2008 
EVOLUTIONARY RESPONSE TO ENVIRONMENT HETEROGENEITY 
have obtained a greater benefit, in terms of fitness, by having 
experienced selection for larger initial endogenous energetic re- 
serves, that is, larger egg size, than for phenotypic plasticity of 
exogenous food collection structures. The result of a greater de- 
gree of phenotypic plasticity in the larger-egged Strongylocentro- 
tus species does not match the results obtained in the current study. 
This may be due to the fact that Strongylocentrotus is a temperate 
genus and the larger-egged species (S.franciscanus) examined by 
McAlister (2007) develops from an egg that is larger in size than 
any of the tropical species examined in the Panamanian system. 
Further research on larval growth and egg composition/quality 
using different populations of each Panamanian system species 
evolving in areas of different productivity may help to elucidate 
the patterns found in this study. 
ACKNOWLEDGMENTS 
I owe considerable debts of gratitude to H. Lessios for supporting this 
research in his laboratory and to J. Kingsolver and A. Moran for intel- 
lectual support developing the ideas presented by this research and for 
helpful comments on multiple drafts of the manuscript. Thanks as well 
to P. Marko for the initial idea and to B. Podolsky for early discussions 
of the topic. Thanks to J. Mate, A. Calderon, R. Collin, and E. Ochoa for 
their contributions to the completion of this research. Thanks to the staff 
of the Smithsonian Tropical Research Institute (STRI), in particular the 
Naos Island Laboratories and the Galeta Marine Laboratory. Funding to 
JSM was provided by a STRI Short Term Fellowship, a STRI Supple- 
mental Research Award, the Exploration Fund of the Explorer's Club, a 
Fellowship for Graduate Student Travel from the Society for Integrative 
and Comparative Biology, the American Museum of Natural History's 
Lerner-Gray Fund for Marine Research, and the Graduate Student Op- 
portunity Fund of the Graduate School and the H. V. Wilson Fund for 
Summer Field Research of the Department of Biology of the University 
of North Carolina at Chapel Hill. 
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Associate Editor: D. Ayre 
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