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Keywords:

  • adaptive divergence;
  • canalization;
  • ecomorphs;
  • ontogeny;
  • resource polymorphism

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Phenotypic plasticity is a developmental process that plays a role as a source of variation for evolution. Models of adaptive divergence make the prediction that increasing ecological specialization should be associated with lower levels of plasticity. We tested for differences in the magnitude, rate and trajectory of morphological plasticity in two lake populations of Arctic charr (Salvelinus alpinus) that exhibited variation in the degree of resource polymorphism. We reared offspring on diet treatments that mimicked benthic and pelagic prey. Offspring from the more divergent population had lower levels of morphological plasticity. Allometry influenced the rate of shape change over ontogeny, with differences in rate among ecomorphs being minimal when allometric variation was removed. However, plasticity in the spatial trajectory of development was extensive across ecomorphs, both with and without the inclusion of allometric variation, suggesting that different aspects of shape development can evolve independently.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Understanding the sources of variation for how novel, complex phenotypes evolve is an unresolved issue in biology (Pigliucci, 2008). Several authors have suggested that new variation may begin as environmentally induced phenotypic changes (West-Eberhard, 2003; Ghalambor et al., 2007; Young & Badyaev, 2007; Pfennig et al., 2010). This is plausible considering that most organisms and traits possess phenotypic plasticity, the ability of a genotype to produce different phenotypes in response to environmental cues (West-Eberhard, 2003, 2005; Pigliucci et al., 2006). Both classic theory and empirical data suggest that plasticity may improve an organism’s fitness under novel conditions (Baldwin, 1896; Schmalhausen, 1949; Day & McPhail, 1996, Parsons & Robinson, 2007; Ruehl & DeWitt, 2007). Given the extensive evidence that heritable variation in plastic responses exists in natural populations, selection should have the capacity to alter plasticity (Dewitt et al., 1998; Pigliucci & Murren, 2003; Pigliucci, 2005; Parsons & Robinson, 2006; Pigliucci et al., 2006; Pfennig et al., 2010).

Conceptual models of adaptive divergence have incorporated key roles for plasticity in general terms (Wimberger, 1994; Skúlason & Smith, 1995; Smith & Skúlason, 1996). For example, speciation via resource polymorphism, whereby sympatric ecomorphs exist within single populations that differ in trophic morphology, behaviour and life history in relation to habitat, is predicted to take place in the following temporal sequence: (i) invasion of a new or unexploited habitat; (ii) decreased intraspecific competition for resources due to rapid phenotypic shifts (primarily through phenotypic plasticity); (iii) divergent selection and evolution of more specialized and distinct morphological groups accompanied by reduced phenotypic plasticity; and (iv) reduced gene flow and the evolution of prezygotic reproductive isolation.

Current research on resource polymorphisms is typically focused on the external forces that generate diversity (i.e. selection), leaving an opportunity to extend this research to internal mechanisms, namely developmental changes and ontogenetic processes that occur during divergence. Therefore, plasticity, specifically ontogenetic plasticity, provides a gateway into a deeper understanding of the internal changes that occur during adaptation (Pigliucci, 1997; West-Eberhard, 2003; Woods, 2007; Parsons et al., 2010).

Changes in developmental rate and timing (heterochrony) could be responsible for the production of ecomorphs (Gould, 1977; Wake & Roth, 1989; Klingenberg, 1998). Heterochrony is a well-known phenomenon perceived as a special means of producing novelty by simple changes in development because an entire organism, not just isolated traits, can be affected by a single perturbation of timing or rate (Gould, 1977; Zelditch & Fink, 1996; Klingenberg, 1998). Similarly, changes in the patterning of morphospace during development (ontogenetic repatterning) could have an important influence on adaptation, but these have been considered to a much lesser degree in evolution (Zelditch & Fink, 1996; Webster & Zelditch, 2005). Currently, we are unaware of any studies that have tested whether heterochrony or ontogenetic repatterning can be environmentally induced even though it is reasonable to assume that developmental rates and trajectories of spatial patterning over ontogeny can be plastic.

Here, we determine the contribution of environmental and genetic effects to divergent phenotypes, but more importantly, we also delve deeper into the developmental processes that underlie phenotypic plasticity by taking an ontogenetic approach. An ontogenetic approach has the advantage of allowing us to test for heterochrony and ontogenetic repatterning, and their possible environmental induction. We quantified morphological variation over the early juvenile phase of development for resource polymorphic Arctic charr (Salvelinus alpinus) reared under different diets. This juvenile phase represents an important life history stage for determining habitat settlement (Sandlund et al., 1992).

Arctic charr colonized Iceland through a single invasion approximately 10 000 ybp and repeatedly diverged within lakes into sympatric ecomorphs along a benthic/limnetic gradient (Gíslason et al., 1999; Wilson et al., 2004). Benthic ecomorphs possess shortened deep body forms with blunt subterminal snouts, which are viewed as adaptations for foraging on benthic prey, whereas limnetic ecomorphs are fusiform and possess elongate gracile terminal mouths for foraging on zooplankton (Malmquist et al., 1992; Snorrason et al., 1994).

We investigated charr from two Icelandic lakes: Thingvallavatn (84 km2), a large lake, with a structurally rugged and furrowed lava bottom, and Vatnshlidarvatn, a relatively small (7 km2) and shallow lake with a simple muddy bottom. These lakes harbour contrasting levels of phenotypic divergence with Thingvallavatn possessing four discrete ecomorphs, whereas Vatnshlidarvatn possesses two subtly divergent ecomorphs (Jónsson & Skúlason, 2000; Parsons et al., 2010). Diet differences are especially prevalent in Thingvallavatn, being discrete and persistent throughout the year. In contrast, segregation of prey types among ecomorphs is seasonal in Vatnshlidarvatn and connected to periods of low prey availability (Snorrason et al., 1992; Jónsson & Skúlason, 2000).

The four Thingvallavatn ecomorphs consist of two benthic specialists; a small benthivore (SB), a large benthivore (LB) and two limnetic ecomorphs; one piscivore and a planktivore (PL). It is generally agreed that all ecomorphs descended from a single post-glacial recolonization of anadromous charr into Iceland, but that the ancestral relationships between ecomorphs remain unresolved (Volpe & Ferguson, 1996; Brunner et al., 2001; Wilson et al., 2004). Benthic ecomorphs are hypothesized to be paedomorphic as their subterminal mouth and blunt head, which characterizes embryos and juveniles of all charr, may represent the retention of juvenile characteristics to maturity (Skúlason et al., 1989). Among the benthivores, the LB ecomorph may be the most phenotypically derived from the putative anadromous ancestor as it maintains a subterminal mouth to a larger adult size (Godfrey & Sutherland, 1995). Further, the LB ecomorph spawns significantly earlier and within a more distinct period than other sympatric ecomorphs (Skúlason et al., 1999). Additionally, LB-like ecomorphs occur rarely in other charr polymorphisms in Iceland, with SB- and PL-like forms being relatively common (Kristjánsson, 2009). Finally, a prior analysis on this data set found that morphological variation is reduced more rapidly over ontogeny in LB ecomorphs relative to PL and SB ecomorphs, suggesting that LB ecomorphs face intense selection (Parsons et al., 2010).

The ecomorphs of Vatnshlidarvatn are referred to as brown (VB) and silver (VS) (Jónsson & Skúlason, 2000). Subtle divergence in shape related to swimming performance and manoeuvrability exists, with the VB ecomorph likely being more phenotypically derived as it possesses a benthic shape with a deeper body and more subterminal mouth. The ecomorphs show pronounced differences in life history traits such as growth, age and size at sexual maturation with the VB ecomorph being smaller. Although their diets overlap, the VB ecomorph has a more restricted diet than the VS ecomorph in early summer (Jónsson & Skúlason, 2000). The morphology of the VS ecomorph closely resembles that of neighbouring anadromous charr (K.J. Parsons, personal observation), which likely represents the ancestral phenotype given the post-glacial history of Iceland (Brunner et al., 2001). Lastly, a prior analysis has demonstrated that the VB ecomorph has rapid reductions in shape variation over ontogeny (like the LB ecomorph in Thingvallavatn) relative to the VS ecomorph (Parsons et al., 2010).

The varying degrees of specialization and phenotypic divergence among charr ecomorphs present an opportunity to contrast the levels and types of plasticity over ontogeny in direct relation to resource polymorphism theory. We tested an important component of the conceptual model of adaptive divergence via resource polymorphism (Smith & Skúlason, 1996) by hypothesizing that the degree of plasticity would be negatively associated with the magnitude of divergence among charr ecomorphs within and between populations. Within their respective lake populations, the LB and VB ecomorphs were hypothesized to be more derived. We therefore tested whether these ecomorphs showed reduced plasticity in developmental rates (heterochrony) and trajectory (ontogenetic repatterning) relative to other sympatric ecomorphs. At the population level, the diets and morphologies of the Thingvallavatn ecomorphs were more distinct than for the Vatnshlidarvatn ecomorphs. We therefore predicted that Thingvallavatn ecomorphs would be less morphologically plastic than those from Vatnshlidarvatn.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Ecomorph collection and rearing

Adult charr were collected from Thingvallavatn and Vatnshlidarvatn over several dates in 2003. Thingvallavatn charr ecomorphs spawn along the shoreline, with LB ecomorphs spawning from mid-July to mid-August and the PL ecomorph mid-October. In Vatnshlidarvatn, the charr spawn in two distinct areas of the lake, with the VB ecomorph spawning in early September and the VS ecomorph in mid-September.

Fish were collected with a series of gill nets with mesh sizes of 15–55 mm depending upon the ecomorph. Nets were set perpendicular to the shoreline and left for no more than 30 min to insure that the charr were still alive for transfer to the laboratory for spawning. Sex and the degree of sexual maturation were assessed in the field, with 15–30 individuals kept for each sex per ecomorph. Insufficient numbers of the piscivorous ecomorph were available for crosses, whereas the number of progeny that could be produced per family was too limited for the small benthic ecomorph to be included in our analysis.

Creation of families

Males and females from the same ecomorph were crossed to produce 2 full-sib families per ecomorph. We elected to analyse a smaller number of large families to facilitate genome scans to be included in a future study. Families were separately housed in a well-water-fed flow-through incubator at 5 °C (±1 °C) at Hólar University College, Skagafjördur, Iceland. When the progeny hatched (82–89 days post-fertilization), free embryos were moved to a larger container with a nylon window-screened bottom until they had absorbed their yolk and began accepting food exogenously. The progeny were then moved to another facility 30 km away in Sauðárkrókur, Iceland. Here, families were separately placed into large plastic buckets (35 cm deep × 29 cm diameter) that were continuously supplied with fresh well water. The fish were kept at 7 °C (±0.5 °C) for 26 days and fed 0.5 mm dry feed under continual light.

Rearing diets and experimental conditions

Twenty-six days after feeding started, the fish were large enough to be fed the experimental diets. Two replicate families per ecomorph were separately divided into two diet treatment groups, and the temperature was raised to 9 °C (±0.5 °C). Family sizes varied, especially among ecomorphs, so it was not possible to equalize densities (see below for a sub-experiment on density). We used 110 individuals per treatment for each of the LB and VS ecomorphs and 71–93 individuals for the PL and VB ecomorphs.

Both diets were a combination of dry commercial pellets, sheep liver and bloodworms (chironomid larvae). The same composition was used in both treatments, as nutritional differences affect morphology of fish (Wimberger, 1993). Dry food was fed continuously for 12 h a day via an automatic system. To reduce density-dependant effects, food quantity was adjusted monthly to provide 5% of total body weight daily in accordance with the number of fish and their average weight in a treatment group.

Diet treatments mimicked prey found in natural habitats. Food particle size was larger for ‘benthic’ treatments and administered at the bottom of the water column approximating the benthic prey used by LB and VB ecomorphs (Malmquist et al., 1992). Dry food (1 mm size for the first 90 days, 2.5 mm thereafter) was dropped from an automatic feeder into a funnel attached to a length of PVC pipe where it eventually emerged 3–5 cm from the bottom. Also, frozen sheep liver was grated and mixed with bloodworms after which the mixture was refrozen to the inside of a 10-cm-long piece of PVC pipe cut lengthwise to make a ‘U’ shape. The frozen pipe was then placed at the bottom of the tank in each benthic treatment group. Fish were fed this mixture one to two times a day until satiation and at least 6 h after the automatic feeder had ended its daily cycle.

The ‘limnetic’ treatment was intended to mimic a planktivorous diet and involved taking the same dried foods and reducing them to a powder-like consistency in a coffee grinder. Food was placed in the automatic feeder and dropped on the water surface and subsequently drawn into the water column. Also, frozen sheep liver and chironomids were mixed and grated to a very fine consistency. This food was then injected into the water column using a turkey baster, and the fish were fed until satiation once per day and at least 6 h after the automatic feeder had finished its cycle. Hereafter, a treatment group refers to a particular ecomorph reared on either a benthic or limnetic diet treatment.

Density

While food levels were adjusted in accordance with rearing density, it was still possible that shape was subject to density effects. To test for the effects of fish density, we reared a subset of two LB and VS families at three different densities of 50, 75 and 100 individuals. These families were divided and reared under both benthic and limnetic diet treatments to the 90-day period. Space and logistical demands did not allow for the VB and PL ecomorphs to be tested in this manner. Effects of density on shape (see below) were tested separately for each ecomorph with manova using the program repMeasMan (see: http://www3.canisius.edu/~sheets/).

Imaging and collection of morphological data

To measure morphology over ontogeny, fish were photographed on their left side using a Nikon coolpix 4500 camera (Nikon Corp., Tokyo, Japan) at 90 and 160 days after the start of diet treatments. At 90 days, charr were anaesthetized using phenoxyethanol (0.2 mL L−1 concentration). Once the fish were unconscious, they were marked using coloured visible implant elastomers (Northwest Marine Technologies, Inc., Shaw Island, WA, USA). Four elastomer colours were injected at six different locations on the fish allowing up to 60 individuals per family to be uniquely marked for later reidentification (Frederick, 1997). After marking, the fish were allowed to recover and were placed back into their treatment tanks until the 160-day sampling period.

Morphometric analysis

A geometric morphometric approach was used to quantify shape over ontogeny. Eighteen homologous landmarks (Fig. 1) were collected from digital photographs of individuals sampled at both 90 and 160 days using TPSdig2 (available at: http://life.bio.sunysb.edu/morph/). Cartesian coordinates of all landmark sets were then adjusted using a generalized Procrustes analysis (GPA) using the program CoordGen6 (available at: http://www3.canisius.edu/~sheets/morphsoft.html). This procedure centres each specimen onto a common centroid, scales all specimens to a common unit size and rotates each specimen to a common orientation, which minimizes the squared differences between corresponding landmarks (Rohlf & Slice, 1990; Adams et al., 2004).

image

Figure 1.  Depiction of the 18 landmarks used to quantify morphological variation in Icelandic Arctic charr (Salvelinus alpinus) for subsequent use in geometric morphometric analysis.

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While GPA standardizes size in shape data, it does not remove the effect that changes in size can have on shape (allometry). Therefore, to assess the influence of allometry, we performed all of our subsequent analyses twice, once without removing size-related shape variance and once after removing it. To remove allometric effects, we modelled our GPA-adjusted landmarks against ln centroid size using the program Standard 6 (available at: http://www3.canisius.edu/~sheets/morphsoft.html). This allowed us to assess ontogenetic shape changes as a function of time. Finally, to test for possible errors in landmark placement, a subset of 50 individuals were redigitized and the resulting partial warps scores (including the uniform component) of replicate measures were compared to the original data using discriminant function analysis.

Shape changes over ontogeny

Shape changes over ontogeny were assessed using both univariate and multivariate methods. Available multivariate methods were limited in their ability to assess the effects of families, which were nested within ecomorphs, and thus missed potential genetic effects at this level. However, these methods could account for genetic differences between ecomorphs, as well as diet, and ecomorph × diet interactions. We created a series of univariate mixed models to supplement a multivariate analysis by incorporating the random effect of family nested within ecomorphs. We used Procrustes distance, calculated as the distance of an individual from the mean of the group, as the metric to describe shape in the univariate models (Zelditch et al., 2004). Univariate models included a repeated measures analysis for each population, as well as stage-specific tests (measurements at either 90 or 160 days) that were implemented using the lmer package in the R programming language. Family effects were tested by comparing the fit of models with and without family as a factor using the Akaike Information Criterion approach as well as F-tests following Crawley (2007).

To determine which factors influenced multivariate morphological development over ontogeny, we applied a permutation-based repeated measures manova (Anderson, 2001). The advantage of this approach is that it does not rely on analytic models of F-distributions and their potential for multiple violations of assumptions as was the case for our multivariate data (analysis not shown). This approach has the other advantage of allowing the use of virtually any distance metric to compute the sum of squares (here all pairwise Procrustes distances). A balanced design (n = 46 chosen randomly for each treatment group) was used to allow for calculations of the variance explained by each factor.

We constructed separate manova models for each of the Thingvallavatn and Vatnshlidarvatn populations. This allowed us to determine the relative contribution of plasticity in each population, in line with the predictions of resource polymorphism theory (Skúlason & Smith, 1995). These models included ecomorph, diet and time, as well as their interactions as effects. All tests involved 1000 permutations and were conducted using RepMeasMan (see: http://www3.canisius.edu/~sheets/moremorph.html).

Stage-specific variation in ecomorph and diet effects

To determine the relative magnitude of ecomorph and diet effects at each of the 90- and 160-day sampling dates, we used stage-specific manovas on shape variables (sums of squared Procrustes distances). These were performed using the same permutation-based approach as the repeated measures models. All tests involved 1000 permutations and were conducted also using RepMeasMan.

Ecomorph and diet effects on developmental rates and trajectories

We tested whether ecomorph effects and diet could induce differences (i) in the rate of development (heterochrony); (ii) in the ontogenetic trajectories of shape (ontogenetic repatterning); and (iii) in both the rate of development and ontogenetic trajectory (Webster & Zelditch, 2005). This was done respectively by comparing both the shape of different ecomorphs reared on the same diet and the shape of the same ecomorph reared on different diets for each of 90 and 160 days.

Developmental rate was based on the morphometric distance between body shape at a given ontogenetic stage relative to the average specimen (of a treatment group at that stage of development), calculated as the partial Procrustes distance (Bookstein, 1996; Dryden & Mardia, 1998; Zelditch et al., 2006). Tests for differences in morphometric distance were performed with a Goodall’s F-test using 900 bootstrap replicates in the program TwoGroup6h (available at: http://www3.canisius.edu/~sheets/morphsoft.html).

We tested for differences in the ontogenetic trajectory of shape by comparing the growth angles of fish from the different treatment groups using VecCompare6 (available at: http://www3.canisius.edu/~sheets/morphsoft.html). This test is described in detail elsewhere (e.g. Zelditch et al., 2000, 2004; Frédérich et al., 2008). However, our approach differed slightly so that we could make comparisons between the data set that included allometric effects more comparable to the one with allometry removed. Usually this analysis is based on centroid size, but here a vector was comprised of regression coefficients of the shape variables (partial warps) on a discriminant function created by comparing the shape of a given treatment group at 90 and 160 days. The range of angles within such vectors was calculated using 900 bootstraps. This range was then compared with the observed between-group angle to determine whether it exceeds the 95% range of the within-group angles.

Visualization of morphological changes between diets and ontogenetic stages

To visualize changes in shape due to diet and ontogeny, we used a multistep approach. A discriminant function analysis using diet as a grouping variable was performed for each ecomorph at each stage of ontogeny. These analyses produced a canonical root score representing the shape variation due to diet for each individual at each stage of ontogeny. The root scores were then used as an independent variable in a multivariate regression that modelled their relationship to shape which was visualized as a deformation grid using the program tpsRegr (available at: http://life.bio.sunysb.edu/morph/).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

There were no detectable errors in the placement of landmarks. Our discriminant analysis of replicate landmark sets indicated no sampling error (all = 1). Selection under laboratory conditions was also unlikely as survivorship was over 90% for the duration of the experiment. Lastly, our sub-experiment on the effects of density on shape revealed no significant effects (all > 0.4). However, because this experiment did not include the PL and VB ecomorphs, some caution is needed for the interpretation of our results.

Shape changes over ontogeny

Our univariate models did not find significant family effects, and the fit of our models actually improved with the exclusion of family as a factor (results not shown). Univariate results for the remaining factors and interactions were very similar to multivariate results. Also, our findings were not due to allometric scaling as the removal of allometric variation caused only slight changes in the explained variance for all factors and did not change the incidences of significant effects. Thus, we report the multivariate results without the removal of allometric variation for the remainder of these manova results, although the analyses based on the adjusted data are included for comparison (Tables 1 and 2).

Table 1.   Parameters describing shape changes over ontogeny in a repeated measures manova. Shape changes are described for ecomorphs of Arctic charr from the Thingvallavatn and Vatnshlidarvatn lake populations. Results include tests conducted with and without allometric variation.
Source lakeModel parameterWith allometryAllometry removed
SSd.f.FPermutation of F valuePermutation of per cent variance explainedPer cent explainedSSd.f.FPermutation of F valuePermutation of per cent variance explainedPer cent explained
ThingvallavatnEcomorph0.0365142.690.0010.0019.370.0382146.720.00100.00110.07
Diet0.003013.560.0060.0190.780.003514.260.00500.0160.92
Time0.0108112.670.0010.0012.780.0106112.970.00100.0012.79
Ecomorph by diet0.002612.990.0300.0010.660.002112.600.02300.0010.56
Ecomorph by time0.0237127.670.0010.0016.080.0264132.310.00100.0016.96
Diet by time0.003614.250.0020.0010.930.003113.780.00500.0010.81
Ecomorph by diet by time0.001211.460.1040.0010.320.001311.560.09190.0010.34
Error0.3077360    0.2940360    
VatnshlidarvatnEcomorph0.0473148.090.0010.0019.780.0376140.810.00100.0018.59
Diet0.005615.670.0010.0021.150.005516.010.00100.0011.27
Time0.0554156.270.0010.00111.440.0441147.860.00100.00110.07
Ecomorph by diet0.005615.710.0020.0011.160.003814.170.00300.0010.88
Ecomorph by time0.009519.670.0010.0011.970.008819.600.00100.0012.02
Diet by time0.004714.810.0030.0010.980.004715.140.00200.0011.08
Ecomorph by diet by time0.001511.490.1230.0010.300.001511.590.11590.0010.33
Error0.3543360    0.3315360    
Table 2. manova parameters describing shape at stage-specific points in ontogeny (90 and 160 days of diet treatments) in ecomorphs of Arctic charr from Thingvallavatn and Vatnshlidarvatn lake populations.
SourceParameterWith allometric variation includedAllometric variation removed
SSd.f.FPermutation of F valuePermutation of per cent variance explainedPer cent explainedSSd.f.FPermutation of F valuePermutation of per cent variance explainedPer cent explained
Thingvallavatn 90 daysEcomorph0.0238124.160.00100.001011.590129.430.00100.001013.81
Diet0.002412.430.01700.03101.16012.470.02000.03601.16
Ecomorph by diet0.001911.890.06490.00100.91011.200.28470.00100.56
Error 180    0180    
Thingvallavatn 160 daysEcomorph0.0360150.280.00100.001021.050.0359153.990.00100.001022.17
Diet0.004115.720.00100.00202.390.004016.030.00100.00302.48
Ecomorph by diet0.002012.820.02200.00101.180.002313.460.01400.00101.42
Error 180     180    
Vatnshlidarvatn 90 daysEcomorph0.0201120.100.00100.00109.650.0173118.290.00100.00108.90
Diet0.003513.530.00500.00501.700.003613.750.00400.00401.82
Ecomorph by diet0.004614.560.00100.00102.190.003413.540.00200.00101.72
Error 180    0.1707180    
Vatnshlidarvatn 160 daysEcomorph0.0366138.090.00100.001016.740.0288132.590.00100.001014.67
Diet0.006616.870.00100.00103.020.006517.350.00100.00103.31
Ecomorph by diet0.002512.580.02600.00101.140.001912.180.05000.00100.98
Error0.0873180    0.1592180    

Morphological development differs between ecomorphs over ontogeny and is influenced by diet (Table 1). Both lake populations showed significant effects for all factors and their interactions within the repeated measures manova models (all < 0.05), except for the ecomorph–diet–time interaction. Although the ecomorph effect was similar in both lakes, the variation due to diet and time varied between them. Diet effects accounted for a relatively small proportion of the explained shape variation in both lakes. As predicted, charr from Thingvallavatn were less plastic over ontogeny than those from Vatnshlidarvatn (diet explains 0.8% vs. 1.2% respectively). Even when the total variation explained by diet and the factors it interacted with (diet × ecomorph, diet × time) are accounted for, the compiled values were small in both lakes, but in the predicted direction (2.4% for Thingvallavatn and 3.3% for Vatnshlidarvatn). Also, the effect of time was relatively small in Thingvallavatn (2.8%) relative to Vatnshlidarvatn (11.4%) indicating that the LB and PL ecomorphs showed more distinct ontogenetic changes. Indeed, ecomorph by time effects were stronger in Thingvallavatn (6.1%) relative to Vatnshlidarvatn (2.0%).

Stage-specific variation in ecomorph and diet effects

The effects of diet and ecomorph on morphology vary over specific stages of ontogeny in both lake populations (Table 2). The effects of ecomorph and diet were significant in all cases, and their explained variance increased over time. Notably, the interaction between ecomorph and diet was not significant at 90 days in Thingvallavatn, whereas in Vatnshlidarvatn, the explained variance of the ecomorph–diet interaction actually decreased over ontogeny. After 90 days of diet treatment, the ecomorph effect explained a larger amount of variation in Thingvallavatn (11.6%) compared with Vatnshlidarvatn (9.6%). Diet explained a small proportion of the variation in both lakes, with values of 1.2 and 1.7% for Thingvallavatn and Vatnshlidarvatn, respectively. Similarly, the effect of ecomorph × diet interactions explained a relatively low proportion of variation (2.2%) in Vatnshlidarvatn.

The explained variance of factors changed substantially between 90 and 160 days (Table 2). The ecomorph effect increased markedly over time in both lake populations (11.6–21.1% in Thingvallavatn and 9.7–16.7% in Vatnshlidarvatn). This suggests that the ecomorph effect was stronger in the more divergent Thingvallavatn population. The effect of diet also increased over time in both lake populations where it doubled in Thingvallavatn and almost so in Vatnshlidarvatn (Table 2). Interestingly, the influence of ecomorph × diet interactions decreased by almost half in Vatnshlidarvatn, suggesting that these interactions may play a more important role in the differentiation of ecomorphs at earlier stages.

Ecomorph effects on developmental rates and trajectories

Ecomorphs reared on the same diet showed significant differences in the rate of morphological development (Table 3). These differences were complex and depended upon diet and the allometric component of shape. With allometric variation included, the Thingvallavatn ecomorphs showed significant differences in rate when they were reared on the limnetic diet, whereas the Vatnshlidarvatn ecomorphs showed significant differences in the benthic diet (95% bootstrapped confidence intervals do not cross 0). However, when allometric effects were removed, all ecomorphs differed in developmental rate. This suggests (i) that heritable differences for developmental rate exist among ecomorphs and (ii) that certain environments may enhance these differences. Also, allometry may either enhance or mask differences in the rate of shape development depending upon the diet. Notably, the rates of morphological development in the Thingvallavatn ecomorphs were faster when fish were reared on ‘native’ diets (i.e. the partial Procrustes distance value for LB on benthic food was 0.0148 vs. 0.0118 on limnetic food; PL on limnetic food had a value of 0.0184 vs. 0.0128 on benthic food), suggesting that differences in developmental rate would be enhanced by conditions in the natural habitats of these divergent ecomorphs.

Table 3.   Differences between charr ecomorphs (LB = large benthivore, PL = planktivore, VB = brown, VS = silver) in the rate of shape change over development (90- to 160-day measurement periods) measured using partial Procrustes distances. Comparisons are equivalent to two separate common garden experiments, including benthic and limnetic treatments. Differences in the rate of shape change are reported for data including allometric variation and with allometric variation removed. Asterisks denote significant differences at α = 0.05.
ComparisonDietDifference in partial Procrustes distance between ecomorphs (with allometry)Difference in partial Procrustes distance between ecomorphs (allometry removed)
LB vs PLbenthic0.00200.0132*
Limnetic0.0066*0.0050*
VB vs VSBenthic0.0083*0.0071*
Limnetic0.00260.0125*

Ontogenetic trajectories of morphological development differed between ecomorphs reared on the same diet in both lakes (Table 4), indicating a heritable basis for ontogenetic repatterning that persisted across diet treatments. Allometric effects did not influence these results although the inclusion of allometric variation in the analysis revealed notably smaller differences in trajectory between Vatnshlidarvatn ecomorphs.

Table 4.   Vector angles representing ontogenetic repatterning between Icelandic Arctic charr ecomorphs (LB = large benthivore, PL = planktivore, VB = brown, VS = silver) reared under two sets of common diets mimicking benthic and limnetic diet treatments. Asterisks indicate significant differences in ontogenetic vector angles between ecomorphs. Asterisks denote significant differences at α = 0.05.
 With allometryAllometry removed
LB vs. PLVB vs. VSLB vs. PLVB vs. VS
Angle under benthic diet120.0°*59.2°*95.6°*113.6°*
Angle under limnetic diet111.6°*38.5°*115.3°*106.4°*

Diet effects on developmental rates and trajectories

When allometric variation was removed, there were no effects of diet on the rate of shape change, and given the above result that ecomorphs differed in rate, this suggested that rate differences were environmentally canalized among ecomorphs. With the inclusion of allometric variation, diet did however affect the rate of morphological development in the PL and VS ecomorphs which were hypothesized to be less derived (Table 5).

Table 5.   Differences in the rate of shape change due to diet over development (90- to 160-day measurement periods) in Arctic charr ecomorphs (LB = large benthivore, PL = planktivore, VB = brown, VS = silver) as measured using partial Procrustes distances. Diets included benthic and limnetic treatments. Differences in the rate of shape change are reported for data including allometric variation and with allometric variation removed. Asterisks denote significant differences at α = 0.05.
EcomorphWith allometryAllometry removed
Difference in partial Procrustes distance between diet treatmentsDifference in partial Procrustes distance between diet treatments
LB0.00300.0058
PL0.0056*0.0024
VB0.00180.0065
VS0.0075*0.0011

Ontogenetic trajectories of morphological development were widely influenced by diet (Table 6). Allometric effects were minimal, with only the VB ecomorph showing no significant difference in trajectory when allometric variation was included. This suggests that ontogenetic trajectory is a highly plastic trait in charr ecomorphs.

Table 6.   Diet-induced allometric repatterning measured as angles between ontogenetic vectors in each Icelandic Arctic charr ecomorph (LB = large benthivore, PL = planktivore, VB = brown, VS = silver). Plasticity was induced by rearing charr on either a benthic or limnetic diet treatment. Asterisks indicate significant differences in ontogenetic vector angles at α = 0.05.
 LBPLVBVS
Angle52.0°*23.6°21.5°*41.4°*
Angle (allometry removed)43.7*19.7*29.8*36.3*

Visualization of morphological changes between diets and ontogenetic stages

The discriminant function analyses used to model shape indicated that diet had a significant effect on morphology (all < 0.01). We found only very subtle differences in the visualization of morphological variation between the data set including allometric variation compared to when it was removed and therefore only display the former. In general, limnetic diets induced a more slender, fusiform body shape in fish relative to those reared on benthic diets (Figs 2 and 3). Fish reared on limnetic diets also had larger eyes than those reared on a benthic diet except for the relatively larger eyes of the LB ecomorph reared on a benthic diet and sampled at 90 days. Finally, limnetic diets resulted in heads characterized by a narrower and more elongated snout.

image

Figure 2.  Diet-induced variation in the body form of Arctic charr ecomorphs (LB = large benthivore, SB = small benthivore, PL = planktivore) from Thingvallavatn. Deformation of the gridlines represents a 3× magnification of the observed difference in body form between each treatment group within an ecomorph at a specified treatment stage.

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image

Figure 3.  Diet-induced variation in the body form of Arctic charr ecomorphs (VB = brown, VS = silver) from Vatnshlidarvatn. Deformation of the gridlines represents a 3× magnification of the observed difference in body form between each treatment group within an ecomorph at a specified treatment stage.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Here, we add to our understanding of the ontogenetic processes underlying adaptive divergence and provide support for resource polymorphism theory by demonstrating evidence for less plasticity in more divergent populations. Additionally, we demonstrate that plasticity plays a strong role in determining ontogenetic repatterning, whereas heterochrony is more strongly influenced by heritable differences between ecomorphs. Previous study on the fish in this experiment demonstrated that ecomorphs hypothesized to be more derived (LB and VB ecomorphs) showed a greater degree of canalization (Parsons et al., 2010). Additional evidence here, which may explain this canalization, suggests that both LB and VB ecomorphs have somehow decoupled allometric responses to diet from overall rates of shape change. In other words, the rate of shape change in these ecomorphs is largely independent of size and diet. Finally, shape responses to diet correspond to predictions about fish body shape in relation to benthic and limnetic habitats (Webb, 1984; Parsons & Robinson, 2007). To date, the expression of shape over ontogeny in adaptively diverging populations has been studied on few occasions (e.g. Skúlason et al., 1989; Holtmeier, 2001). However, prior studies have not involved comparisons between ecomorphs reared under different diet treatments and from populations that vary in their magnitude of divergence.

As predicted, the total contribution of ecomorph effects (interactions included) had a larger role over ontogeny in the more divergent Thingvallavatn population compared with the subtly divergent ecomorphs of Vatnshlidarvatn. Overall, plasticity plays a relatively small role in determining shape in juvenile charr, but had a larger effect in Vatnshlidarvatn with an increasing influence over time. Taken together, this shows (i) that genetic variation related to morphological development is present among charr ecomorphs at early stages and (ii) that the availability of this variation changes over development, in charr.

The lower levels of plasticity in Thingvallavatn charr suggests that development may be more canalized in Thingvallavatn, supporting a longstanding hypothesis for this system (Skúlason et al., 1999; West-Eberhard, 2003, p. 548). An immediate implication is that environmental canalization may be prevalent in many other divergent populations of charr. More broadly, these findings provide an empirical basis for suggestions that genetic assimilation, the loss of plasticity relative to ancestors, is involved in the evolution of adaptive divergence in general (Wimberger, 1994; Skúlason & Smith, 1995). However, we caution against this interpretation until more is known about the levels of plasticity in anadromous charr, the putative ancestor to these populations. Nonetheless, molecular data suggest that charr invaded Iceland during a single recolonization event (Brunner et al., 2001; Wilson et al., 2004), implying that genetic variation was similar among the founders of these rapidly diverging populations. Further study assessing the contribution of environmental and genetic variation in additional charr populations, which differ in their degree of morphological divergence, could help provide further support for canalization.

One critique that may be levelled against our suggestion that plasticity played a significant role in charr divergence is that it contributes a relatively small amount of the total phenotypic variation. Therefore, this may lead to the assumption that plasticity cannot account for the adaptive differences observed between ecomorphs. Two counter arguments, which we discuss below, are that (i) small changes in phenotypes can account for large differences in relative fitness and that (ii) the evolution of environmental canalization may occur very rapidly producing low levels of observable plasticity.

Empirical evidence suggests that small differences in phenotype can account for large differences in relative fitness. For example, a study on polymorphic African finches (Pyrenestes;Smith, 1993) found that a difference of only 1 mm in the width of the lower mandible accounted for more than a 50% change in fitness. Additionally, in pumpkinseed sunfish (Lepomis gibbosus), a species that displays a very subtle benthic/limnetic polymorphism in body form, substantial trade-offs in ecological performance have been detected between the two ecomorphs (Robinson et al., 1996). Plastic morphological responses in fish, however slight, have also been shown to significantly affect foraging performance in sticklebacks, sunfish and Arctic charr (Day & McPhail, 1996; Andersson, 2003; Parsons & Robinson, 2007).

Our data suggest that reduced levels of plasticity accompany greater magnitudes of divergence. We contend that the small amounts of observable plasticity present in our charr are due to the rapid evolution of canalization, perhaps mainly through the fixation of the developmental rates of morphological change in each ecomorph. However, the time frame for the evolution of charr ecomorphs (∼10 000 ybp) may exceed the period where most of the observable evolutionary process of canalization has occurred. Prior study has shown that environmental canalization can occur very rapidly under laboratory conditions (see Waddington, 1952, 1953; Pigliucci & Murren, 2003). Conversely, ecological divergence and genetic differentiation can also occur in just a few generations (see Hendry et al., 2000 for an example of rapid divergence in a related salmonid). We concede that more investigation into the possible process of canalization in Icelandic charr is needed. Future investigation could determine the relative levels of plasticity in anadromous charr reared on the benthic and limnetic treatments used here to determine an ancestral level of plasticity.

Evolvability and signatures of selection over ontogeny

Variance due to the contribution of ecomorph and diet effects changed over charr ontogeny. This was especially notable in the degree of variation attributable to ecomorph effects, which nearly doubled over time (Table 2). This pattern could reflect at least two interrelated processes. First, selection pressures may become stronger over ontogeny as performance trade-offs on prey increase (Wimberger, 1994). This should in turn produce an accumulation of genetically based differences between ecomorphs over ontogeny. Second, there could be more relaxed selection early in ontogeny when embryos subsist off of their yolk sac and do not rely on specialized functional morphology for exogenous feeding until increases in gape allow for diet switches (Sandlund et al., 1988; Parsons et al., 2010).

Are the observed ontogenetic patterns in charr directly relatable to selection pressures and adaptation? Charr ecomorphs have relatively specialized diets within both lakes, suggesting a role for selection (Malmquist et al., 1992; Snorrason et al., 1992; Jónsson & Skúlason, 2000). Within Thingvallavatn, there are specific ontogenetic stages where niche shifts occur in the LB and piscivorous ecomorphs (Snorrason et al., 1992). This suggests that ontogeny presents key stages for selection on charr morphology. A foraging experiment on the fish in the current study has shown that our observed changes in shape relate to the rate and efficiency of prey consumption (Parsons, 2008). In turn, these putatively adaptive properties of shape change should also affect growth rate. Growth rates of juveniles likely influence size-dependant niche shifts that occur later in life. Indeed, Björklund et al. (2003) tracked charr growth over 2.5 years and found that smaller individuals in the earliest life stages also tended to be smallest later. This suggests that shape changes that occur early in ontogeny could have important adaptive implications later in life (Holtmeier, 2001).

Following the idea that the early life stages we have studied have important influences later, we must consider the conditions that may influence selection prior to these stages. Spawning site fidelity and differences in the time of spawning have been associated with ecological divergence in charr, and these factors could generate environmental differences during early charr life history (Skúlason et al., 1989; Adams et al., 2006). Adaptations to early environmental differences may therefore exist and explain the presence of diet by ecomorph interactions. These interactions may provide an early developmental bias that acts as an ontogenetic ‘leader’ in development, which helps to curb the increasingly larger trade-offs involved with switching habitats.

Charr heterochrony and ontogenetic repatterning

The detection of heritable differences in the developmental rate of morphology among ecomorphs implicates a role for heterochrony in their divergence. However, there is some degree of plasticity in the rate of development as comparisons between ecomorphs showed that diet, in combination with allometry, could sometimes enhance differences in developmental rate. This suggests that differences in developmental rate between ecomorphs could be enhanced under natural conditions where they inhabit different habitats. This may be particularly important during ontogenetic niche shifts when gape limitations for some ecomorphs are overcome, and followed by bursts of rapid growth (Snorrason et al., 1992).

All ecomorphs showed strong evidence of heritable differences in ontogenetic trajectory, regardless of diet treatment. This implies that charr ecomorphs, in addition to having heritable differences in developmental rate, also progress along different ontogenetic trajectories to produce divergent phenotypes. However, unlike developmental rate, there is broad support for plasticity in ontogenetic trajectory. This suggests that there is the potential for further developmental canalization in charr ecomorphs. In this context, it is noteworthy that the PL ecomorph shows relatively little plasticity in ontogenetic trajectory. The PL ecomorph arguably faces both the simplest and most predictable fluctuations in prey availability, which includes yearly blooms of chironomid pupae in the spring and Daphnia and Cyclops in mid-summer in the pelagic zone of Thingvallavatn (Malmquist et al., 1992; Snorrason et al., 1992). In the presence of predictable ecological fluctuations, it may be most advantageous to maintain plasticity but reduce its costs by limiting responses to environmental cues along a fixed trajectory. Such a strategy could be important in other taxa experiencing predictable fluctuations in environments and may pertain directly to the evolution of genetic accommodation, whereby plastic response can become fine-tuned (McLeod, 1984; Shapiro, 1984; Smith, 1991; Pfennig, 1992; West-Eberhard, 2003).

Evidence supporting the presence of both heterochrony and ontogenetic repatterning was found among charr ecomorphs. This supports the hypotheses of Zelditch & Fink (1996) that morphology should typically evolve by changing both spatial location and temporal aspects of ontogeny. Based on our data, we suggest that heterochrony alone is not an adequate explanation for the divergence of phenotypes in populations undergoing rapid ecological divergence. It appears that evolutionary divergence occurs both for rates and for trajectories of shape ontogeny among charr ecomorphs (Wray & McClay, 1989; Zelditch & Fink, 1996; Zelditch et al., 2000).

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Our findings highlight what can be discovered through the application of an ontogenetic approach to the studies of plasticity and adaptive divergence. Also, our study represents an early empirical test for determining the developmental processes underlying canalization in a resource polymorphism (Wimberger, 1994; Skúlason & Smith, 1995). Our detection of genetic variation in ontogenetic plasticity among ecomorphs provides requisite evidence for its evolution. Also, changes in the influence of ecomorph and diet factors over ontogeny suggest that selection has varying opportunities over development. However, more information on levels of selection over ontogeny is needed to understand the adaptive potential of this variation.

Our findings also suggest that studies on heterochrony can benefit from the consideration of potentially important environmental influences that determine differences in the rate and timing of developmental events. To understand the evolution of development, biologists are encouraged to move beyond studies that involve single environments and stages. Furthermore, we suggest researchers to broaden their investigations to include potentially adaptive changes in the trajectory of development (Zelditch & Fink, 1996). Phenotypes are the direct targets of selection and are the result of interactions between the genotype and its given environment during development (Mayr, 1997; Young & Badyaev, 2007). In general, selection has been the focus of research on the evolution of phenotypic divergence in resource polymorphisms. Our findings suggest that ontogeny, especially in relation to environmental influences, could be another promising area of focus. This could compliment existing research programmes on selection by providing an explanation for the origins of phenotypic variation – what selection needs to operate.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

We are grateful to K. Cottenie, R. Danzmann, R. McLaughlin, R. Young, W.J. Cooper, R.C. Albertson, D. Pfennig, B. Frédérich and an anonymous reviewer who all provided valuable input. B. Kristjánsson, R. Sturlaugsdóttir, E. Svavarsson, T. Tunney and I. Arnarson were especially helpful over the course of the rearing experiment. Funding for this study was provided by Ontario Graduate Scholarships to K.J.P. and by NSERC Discovery grants to M.M.F.

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  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References
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