ORI GIN AL PA PER

Temporal stability of niche use exposes sympatric Arcticcharr to alternative selection pressures

Rune Knudsen • Anna Siwertsson • Colin E. Adams • Monica Garduno-Paz •

Jason Newton • Per-Arne Amundsen

Received: 8 March 2010 / Accepted: 10 November 2010 / Published online: 24 November 2010� Springer Science+Business Media B.V. 2010

Abstract There is now strong evidence that foraging niche specialisation plays a critical

role in the very early stages of resource driven speciation. Here we test critical elements of

models defining this process using a known polymorphic population of Arctic charr from

subarctic Norway. We test the long-term stability of niche specialisation amongst foraging

predators and discuss the possibility that contrasting foraging specialists are exposed to

differing selection regimes. Inter-individual foraging niche stability was measured by

combining two time-integrated ecological tracers of the foraging niche (each individual’s

d13C and d15N stable isotope (SI) signatures and their food borne parasite fauna) with a

short-term measure of foraging niche use (stomach contents composition). Three dietary

subgroups of predators were identified, including zooplankton, gammarid and benthivore

specialists foragers. Zooplanktivorous specialists had muscle low in d 13C, a high abun-

dance of parasites transmitted from pelagic copepods, a smaller head, longer snout and a

more slender body-form than gammaridivorous specialist individuals which had muscle

more enriched in d 13C and high abundance of parasites transmitted from benthic

Gammarus. Benthivorous individuals were intermediate between the other two foraging

groups according to muscle SI-signals (d13C) and loadings of parasites transmitted from

Electronic supplementary material The online version of this article (doi:10.1007/s10682-010-9451-9)contains supplementary material, which is available to authorized users.

R. Knudsen (&) � A. Siwertsson � C. E. Adams � P.-A. AmundsenDepartment of Arctic and Marine Biology, University of Tromsø, 9037 Tromsø, Norwaye-mail: rune.knudsen@uit.no

C. E. AdamsScottish Centre for Ecology and the Natural Environment, University of Glasgow, Rowardennan,Glasgow G63 0AW, Scotland, UK

M. Garduno-PazFacultad de Ciencias, Universidad Autonoma del Estado de Mexico, Instituto Literario No. 100,Toluca, Estado de Mexico C.P. 50150, Mexico

J. NewtonNERC Life Sciences Mass Spectrometry Facility, SUERC, East Kilbride, GlasgowG75 0QF, Scotland, UK

123

Evol Ecol (2011) 25:589–604DOI 10.1007/s10682-010-9451-9

both copepods and Gammarus. The close relationship between subgroups identified by

stomach contents, time-integrated tracers of niche use (SI and parasites) and functional

trophic morphology (niche adaptations) demonstrate a long-term temporally stable niche

use of each individual predator. Differential habitat use and contrasting parasite commu-

nities and loadings, show differential exposure to different suites of selection pressures for

different foraging specialists. Results also show that individual specialisation in trophic

behaviour and thus exposure to different suites of selection pressures are stable over time,

and thus provide a platform for disruptive selection to operate within this sympatric

system.

Keywords Stable isotopes � Food web transmitted parasites � Functional morphology �Temporal niche stability � Salvelinus alpinus

Introduction

There is now compelling theoretical and empirical evidence that ecological processes can

play a significant role in the early stages of speciation (Schluter 1996, 2001). Models

describing this process typically invoke a behavioural specialisation on alternative

resources by individuals in a population; most frequently thought of in this context are

foraging resources (West-Eberhard 1989, 2005; Dieckmann and Doebeli 1999; Skulason

et al. 1999). For species that have the capacity for phenotypic plasticity, this may result in

the expression of alternative phenotypes through exposure of foraging specialists to

alternative environments (Adams et al. 2003; Snowberg and Bolnick 2008; Garduno-Paz

and Adams 2010). If this stage is reached, individuals expressing alternative phenotypes

and adopting different ecological specialisms may then be subject to disruptive selection

related directly to their use of different resources, or as a result of differential expression of

alternative morphological or behavioural phenotypes (West-Eberhard 1989, 2005; Skula-

son et al. 1999; Rueffler et al. 2007; Duckworth 2009). Thus both phenotypic plasticity and

genetic components play an important role in the process of divergence (Schluter and

Conte 2009; Pfennig et al. 2010) also in young species flocks (Losos 2010). Although the

process is far from fully understood, empirical evidence is now mounting, particularly for

fish inhabiting post-glacial lakes (but also for other animal groups, see e.g. Herrel et al.

2008; Funk 2010) that the above sequence of events has occurred, and is still occurring, in

a number of species (Bolnick et al. 2003).

Amongst freshwater fishes, lacustrine Arctic charr (Salvelinus alpinus (L.)), show clear

evidence of clear differential resource use (usually foraging resources) frequently associ-

ated with the expression of alternative phenotypes (resource polymorphisms sensuSkulason and Smith 1995) with a functional role (Adams and Huntingford 2002a;

Klemetsen et al. 2002, 2006; Knudsen et al. 2006). Alternative phenotypes are frequently

found in sympatry (Klemetsen 2010) and often take the form of a specialist zooplankton

feeders foraging in the limnetic zone and a specialist macro-invertebrate feeders, foraging

in the benthic zone of lakes (reviewed by Robinson and Parsons 2002; Hendry 2009).

However in some lakes, additional foraging specialisms have been identified in this species

(Snorrason et al. 1994; Adams et al. 1998; Knudsen et al. 2006). Similar parallel cases of

resource specialisation have also been recorded in other lacustrine fish species in post-

glacial systems (e.g. Kristjansson et al. 2002; Kahilainen and Østbye 2006). Arctic charr

have been shown to express, at least some, of the phenotypic variation described from

alternative phenotypes from the wild through ontogenetic plasticity (Adams and

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123

Huntingford 2002a, 2004). For some populations, expressed alternative phenotypes also

have an inherited genetic component (Sandlund et al. 1992; Adams and Huntingford

2002b, 2004; Klemetsen et al. 2002, 2006). Thus, it is likely that across the species,

populations are exhibiting multiple stages on the route to evolutionary divergence

(Skulason et al. 1999; Losos 2010).

There are two critical pre-requisites for evolutionary divergence resulting from resource

specialisation to operate. Firstly, the alternative resource specialisms adopted by individ-

uals need to be stable over time. For the ontogenetic effect of phenotypic plasticity to

modify the expressed phenotype, individuals should retain their niche specialisms over a

period close to the generation time. For disruptive selection to result in evolutionary

divergence, resource specialisms need to be expressed in a population over multiple

generations (see Woo et al. 2008; Knudsen et al. 2010). Secondly, for disruptive selection

to operate, organisms occupying separate ecological niches need to be exposed to different

selection pressures, disruptive selection could result in alternative expressed phenotypes of

resource specialists (Abrams 2006; Doebeli et al. 2007). Here we test if these two pre-

requisites for resource-specialism mediated evolution exist, in a model system where

Arctic charr display discrete alternative phenotypes which show foraging specialisms and

which are presumed to be in the process for incipient speciation (Knudsen et al. 2007).

The Arctic charr in Fjellfrøsvatn, Northern Norway show discrete trophic specialisation

related to macro-habitat segregation (Amundsen et al. 2008; Knudsen et al. 2007, 2010).

This trophic specialisation exposes individuals with different specialisms to different

burdens of specific trophic transmitted parasite species (Knudsen et al. 2004), which could

act as strong parasite-mediated selection forces on the host populations (Maan et al. 2008;

Matthews et al. 2010). Here we, use an individual based approach to test three specific

hypotheses in this model system. First, we hypothesise that resource-use specialisation

persists over the life-time of the individual [i.e. that they do not fluctuate over short periods

(such as seasonally)]. To test this we examine short-term (stomach contents) and long term

(stable isotopes analysis and parasite fauna) integrators of diet. Secondly, we hypothesise

that the expression of resource specialisation in this model system remains temporally

stable over periods consistent with evolutionary divergence. This we test by comparing

resource use data in this study with historical data. Thirdly, we hypothesise that resource

specialisation are accompanied by functional morphological adaptations. We discuss our

result in a context that resource specialists are subject to differential selection pressures

consistent with divergent selection.

Materials and methods

Fjellfrøsvatn (69�050N, 19�200E, 125 m a.s.l) is a dimictic, oligotrophic lake situated in

subarctic Norway. It has a surface area of 6.5 km2, a maximum depth of 88 m and well

developed pelagic and littoral habitats. The littoral zone (\10 m) constitutes slightly less

than one-third of the lake surface area. The only fish species present are Arctic charr and

brown trout (Salmo trutta L.).

Mature Arctic charr develop secondary sexual traits (Skarstein and Folstad 1996). To

limit this source of variation in our study of functional morphological traits, we restricted

the analyses to immature individuals only. Arctic charr were sampled from two habitats in

October 1997, the littoral (using benthic gill-nets, set in 0–10 m water depth) and pelagic

(using offshore, floating, gill-nets, set above[30 m water depth). All nets used were multi-

mesh gill-nets (10, 12.5, 15, 18.5, 22, 26, 35 and 45 mm, knot to knot). In total 78 charr of

Evol Ecol (2011) 25:589–604 591

123

mean fork length 21.5 cm (±1.9 SD, range: 18.9–25.1 cm) and mean age 5.2 years (±0.7

SD, range: 4–7 years) were collected and used as a basis for all analyses described below.

Furthermore, historical data are included to provide an insight into long-term stability of

specialised trophic behaviour (see online Supplementary Fig. 2). The temporal stability of

resource use (diet and parasite infection; see details below) of the present Arctic charr

population was explored using a long-term data set (from 1992 to 2009; except the years

1993–1995, 2008; see online Supplementary Table 1 for the number of fish at each

sampling period) that were collected as described.

Prey items in the stomachs of individual fish were identified and their relative contri-

butions to the total stomach fullness estimated according to the technique of Amundsen

et al. (1996). Food items were categorised into two main groups (and five subgroups; A–E)

as either limnetic prey: A) zooplankton, or several different benthic prey: B) molluscs, C)

insect larvae, D) Gammarus lacustris L., and E) semi-pelagic chydorids. Individual charr

were categorised as either zooplanktivores or benthivores based on the dominance ([50%

abundance) of limnetic or benthic prey respectively in the stomach contents. Additionally,

the benthivorous fish were divided into benthic generalists and Gammarus-specialists, the

latter group defined by a dominance of G. lacustris (C50% abundance) in stomachs. For

historical data, detailed stomach analyses were performed only in 1992 (n = 237), 1997

(n = 78) and 2006 (n = 37). In the other years only course-grained diet data (i.e. abun-

dance of zooplankton, G. lacustris, and benthic prey) are available (see Supplementary

Table 1 for details).

Dietary specialisation was quantified by comparing each individual’s diet niche with the

population diet niche, and the proportional similarity was calculated according to Bolnick

et al. (2002):

PSi ¼X

j

min pij; qj

� �

where pij is the proportion (% abundance) of prey type j (see the diet categories above) in

the diet of individual i, and qj is the proportion of prey type j in the whole population (%

abundance is estimated according to Amundsen et al. (1996)). Values of PSi varies between

0 (no overlap between individual and population) and 1 (total overlap). The lower the PSi-

value, the more specialised the individuals are compared to the overall population (see

Bolnick et al. 2003; Sargeant 2007). Differences in mean PSi-values between dietary

groups were tested using t-tests (SYSTAT, v. 13, 2009, SYSTAT Software, Inc.).

For stable isotope analysis, a sample of muscle tissue was taken from below the dorsal

fin from each individual fish (e.g. Pinnegar and Polunin 1999). Stable isotope ratios of a

consumer reflect the isotopic signatures of the prey consumed, during the time period that

the tissue was synthesised. Muscle tissue is commonly used to determine long-term diet, in

temperate fish, typically reflecting a summer-period of somatic growth (Perga and Ger-

deaux 2005). Zooplankton and four different groups of benthic prey (molluscs, G. lacus-tris, insect larvae and chydorids) were collected and analysed for stable isotopes. To ensure

sufficient material for analysis, each analysis was conducted on several whole animals

combined. Samples were oven dried at 60�C for 24 h and pulverised using a mortar and

pestle, before subsamples were weighed into tin capsules. Isotopic analyses of carbon and

nitrogen were carried out at the NERC Life Sciences Mass Spectrometry Facility, East

Kilbride, Scotland, by continuous flow isotope ratio mass spectrometry (CF-IRMS), using

a Costech ECS 4010 elemental analyser interfaced with a ThermoFisher Scientific Delta

XPPlus IRMS. Stable isotope ratios are expressed conventionally as parts per thousand

592 Evol Ecol (2011) 25:589–604

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(%) delta values (d13C and d15N), in relation to the international standards for carbon

(PeeDee Belemnite) and nitrogen (atmospheric nitrogen). Precision, obtained from repli-

cate analyses of internal gelatin and alanine standards, was \0.1% (carbon) and \0.2 %(nitrogen). To get information about the relative contribution of different food sources

based on stable isotope composition we used a recently developed Bayesian mixing model

(SIAR, Parnell et al. 2010), which allows for incorporation of uncertainties in both prey

isotopic estimates and trophic fractionation. We used trophic fractionation factor values

from the literature, mean (±SD) 0.4 (±1.3) % for d13C and 3.4 (±1.0) % for d15N (Post

2002). Correlations between posterior distributions of proportions of different prey showed

that the model had problems differentiating between zooplankton and chydorids (corre-

lation coefficient: -0.78) and between G. lacustris and molluscs (-0.59). Thus, com-

parisons between the three different diet groups of charr were performed for the three prey

categories: (1) zooplankton/chydorids, (2) insect larvae and (3) G. lacustris/molluscs. For

each prey category, probability values for differences between any two diet groups were

obtained by the proportion of the total number of simulations (30,000) where one group

had a higher contribution of the prey than the other. The Bayesian mixing model was fitted

using R software (R Development Core Team 2009) and the package SIAR (version 4.0, by

Andrew Parnell and Andrew Jackson).

The parasite community of each individual fish was described by a subset of endo-

parasite species. Adult trematodes (Crepidostomum spp.) and cestodes (Cyathocephalustruncatus (Pallas), Eubothrium salvelini (Schrank), Proteocephalus sp.) from the intes-

tines, and larval cestodes (Diphyllobothrium dendriticum (Nitzsch) and D. ditremum(Creplin)) were enumerated. The nematode Cystidicola farionis (Fisher) occurring in the

swim-bladder, was enumerated as L3-larvae (recruitment stage), preadults (L4-larvae) or

adults. All of these parasites are transmitted to the fish by species-specific intermediate

hosts such as pelagic copepods (E. salvelini, Proteocephalus sp., D. dendriticum, D. di-tremum), insect larvae (Crepidostomum spp.) and Gammarus lacustris (C. truncatus, C.farionis). The stability of parasite infection through time was explored by using long-term

data from 1992 to 2009 (see details above, n = 552) of the Gammarus-transmitted para-

sites (C. farionis and C. truncatus). A similar dataset for the copepod transmitted parasites

Diphyllobothrium spp. was only available for 1992 (n = 237), 1997 (n = 78) and 2006

(n = 37) when standardised sampling was performed (see Knudsen et al. 2004). The terms

prevalence and abundance of parasites are used according to Bush et al. (1997). Differ-

ences in parasite abundances between dietary groups was tested using either Mann–

Whitney U-tests or Kruskal–Wallis tests (SYSTAT, v. 13, 2009, SYSTAT Software, Inc.).

A Constrained Correspondence Analyses (CCA) followed by a Monte Carlo permuta-

tion test, was used to model relationships between stable isotope values, food borne par-

asites and resource use (stomach content) (Legendre and Legendre 1998). This analysis

was performed using the R software (R Development Core Team 2009) and the package

Vegan (version 1.8-8, by Jari Oksanen).

Landmark-based geometric morphometrics techniques were used to describe shape

variation of charr (Rohlf and Marcus 1993; Adams et al. 2004). The left side of each fish

was photographed under identical light conditions using Kodak 64 ASA EPR Ectachrome

film. The photographs were later digitised using the Kodak Photo CD-system. Shape-

analyses were performed separately for head and body, and in total 21 landmarks (see

online supplementary Fig. 1 for location of landmarks) were digitised using TPS Dig2

(Rohlf 2006). Landmark configurations were aligned, translated and rotated in a Gen-

eralized Procrustes Analysis (GPA) superimposition in order to remove position, orien-

tation and scale effects (Rohlf and Slice 1990). The GPA results in a data matrix with new

Evol Ecol (2011) 25:589–604 593

123

shape variables, Procrustes coordinates, which were used in statistical analyses. To test for

allometric effects of shape, shape variables were regressed on centroid size (body

P = 0.48; head P = 0.17). Centroid size in geometric morphometrics is defined as the

square root of the summed, square distance of all landmarks about their centroid (Zelditch

et al. 2004). There was no allometic effects over the size range of fish used in this study.

Shape differences between different diet groups were explored using Canonical Variates

Analyses (CVA) on Procrustes coordinates of body and head separately. Statistical

inference of group differences was made by pairwise permutation tests of the Mahalanobis

distances in shape between groups. MorphoJ 1.01a (Klingenberg 2008) was used for the

GPA and statistical analyses of shape.

Results

Dietary specialisation

Twenty nine (37%) of the total 78 Arctic charr, were categorised as zooplankton specialists

([50% prey abundance), 23 (30%) as gammarid specialists and 26 (33%) as general

benthic feeders. Only one gammarid specialist and one benthic feeder were caught in the

pelagic gillnets. Throughout the sampling period from 1992 to 2009 (n = 552), 98% of the

catch in the pelagic nets comprised zooplankton specialists. The zooplankton specialists

generally had lower PSi-value (i.e. higher degree of individual specialism) than both the

gammaridivore specialists (PSi-values: 0.41 ± 0.1 SD and 0.53 ± 0.07, respectively; t test:

t = -5.1, P = 0.000) (Fig. 1) and the benthivore group (PSi-value: 0.56 ± 0.18 SD; t test:

t = -3.6, P = 0.001), but there were no differences between the latter two dietary groups

(t test: t = -0.8, P = 0.448). The somewhat higher and more variable PSi-values of the

benthivore group indicating that these charr are the most generalist feeding group.

0

0.1

0.2

0.3

0.4

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00

PSi-values

Pro

port

ion

of in

divi

dual

s

Fig. 1 Individual diet specialisation quantified as the proportional similarity (PSi) of the diet utilisation ineach individual and the total population of Arctic charr. Low PSi-values indicate low overlap between theindividuals and overall population diet niche, i.e. high level of individual specialisation. Zooplanktivorespecialists (open bars), gammaridivore specialists (black bars) and benthivores (grey bars)

594 Evol Ecol (2011) 25:589–604

123

Stable isotope ratios of muscle tissue were significantly different between the three

dietary groups of Arctic charr for carbon (d13C; ANOVA tests, P \ 0.001) but not for

nitrogen (d15N; P [ 0.05; Fig. 2c). These observed differences in d13C-values corre-

sponded with isotope values of the different prey items, with G. lacustris and gammari-

divorous fish being most enriched in d13C and zooplankton and zooplanktivorous fish

having the lowest d13C-values (Fig. 2c). However, the isotopic data suggest that individual

charr eat a mixture of prey items as all foraging groups have intermediate d13C-values

compared to the range present in putative prey. The results of the isotope mixing model

(SIAR) showed similar general patterns in prey contributions as the stomach content data

(Fig. 2a, b). There were significant differences between zooplanktivore and gammaridivore

specialists, both in the contribution of zooplankton/chydorids (P \ 0.05; mean

(b)(a)

Pre

y ab

unda

nce

Pro

port

ion

0

25

50

75

100

0

.1

.2

.3

.4

.5

(c)

δ13C

2.0

3.0

4.0

zoo chy ins mol gamzoo chy ins mol gam

-33 -29 -25 -21 -17

δ15N

5.0

Z G

B

m

gi

cz

Foraging groups:Zooplanktivore(Z)Benthivore(B)Gammaridivore(G)

Fig. 2 Contribution of different prey resources to the three different diet groups of Arctic charr:zooplanktivores (open bars), gammaridivores (black bars) and benthivores (grey bars) based on a diet fromstomach contents (mean % abundance ±SE) and b results from the isotope mixing model (SIAR; meanproportion ±95% credibility intervals). In (c) the variation in d13C and d15N between dietary groups ofArctic charr (corrected for trophic fractionation) and different prey items (mean ±SE) are shown.Abbreviations fish: Z = zooplanktivores; B = benthivores; G = gammaridivores and prey: z, zoo = zoo-plankton; c, cyd = chydorids; i, ins = insect larvae; g, gam = G. lacustris; m, mol = molluscs

Evol Ecol (2011) 25:589–604 595

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contribution for zooplanktivores: 63%, gammaridivores: 40%) and G. lacustris/molluscs

(P \ 0.05; zooplanktivores: 20%, gammaridivores: 42%). In benthivorous fish, the con-

tribution of these prey groups was intermediate to the two specialist groups. On the

individual level, the stomach contents corresponded nicely with the isotopic data (d13C) as

there was a positive correlation between the d13C-values and the abundance of G. lacustrisin the stomachs but a negative correlation with the abundance of zooplankton (Spearman

rank: 0.61 and -0.50, respectively; P \ 0.01).

Parasite loadings

Parasite infections were also different between foraging subgroups (Table 1). Zooplank-

tivorous fish had highest infection rate of the copepod transmitted Diphyllobothrium spp.

but much lower abundances of the Gammarus transmitted, C. farionis and C. truncatus,compared with benthivore and gammaridivore fish (Mann–Whitney U-tests, P \ 0.001).

Gammaridivore specialists also had a significantly higher abundance of Gammarustransmitted parasites but a lower abundance of copepod transmitted parasites than the

benthivore diet group (M-W U-tests, P \ 0.001). These patterns among the three foraging

groups were consistent throughout the sampling period from 1992 to 2009 (Supplementary

Fig. 2 a, b). During this whole period, zooplanktivore foragers had a significantly higher

abundance of copepod transmitted parasites (Diphyllobothrium spp.) and lower abundance

of Gammarus transmitted parasites (C. farionis and C. truncatus combined) than both

gammaridivore specialists and the benthivore group (Kruskal–Wallis tests, P \ 0.001).

Similarly, the association between the present day diet and the former diet, indicated by

parasites present in individual fish was clear. The abundance of Gammarus transmitted

parasites (C. farionis and C. truncatus combined) was positively correlated with the

abundance of G. lacustris but negatively correlated with abundance of zooplankton in fish

stomach contents (Spearman rank: 0.72, and -0.57, respectively; P \ 0.01). On the other

hand, the abundance of the zooplankton transmitted Diphyllobothrium spp. was negatively

correlated with the abundance of G. lacustris but positively correlated with zooplankton

abundance in the diet (Spearman rank = -0.41 and 0.22, respectively; P \ 0.05).

Table 1 Prevalence (Prev; %) and abundance (�x) of parasite species in different foraging groups: zoo-planktivores (from pelagic and littoral habitats), benthivores and gammaridivores of Arctic charr

Parasite species Zooplanktivores (n = 29) Benthivores (n = 26) Gammaridivores (n = 23)

Prev �x a b Prev �x c Prev �x

Diphyllobothrium spp. (C) 100 36.6 * ** 100 29.9 ** 96 16.4

Proteocephalus sp. (C) 48 0.8 ns ns 77 0.3 ns 17 0.2

E. salvelini (C) 89 3.3 ns ns 100 4.8 ns 87 3.9

Crepidostomum spp. (I) 100 18.2 ** ** 92 31.5 ** 100 59.9

C. truncatus (G) 45 1.7 ** ** 92 14.2 ** 100 45.9

C. farionis (G) 76 10.9 ** ** 96 29.7 ** 96 58.7

Intermediate host for parasites are given: copepods (C), insects (I), Gammarus lacustris (G); n = numberof fish. Statistical differences (Mann–Whitney U tests) between different dietary groups of Arctic charr(zooplanktivores vs. benthivores (a), and vs. gammaridivores (b); benthivores vs. gammaridivores (c) aredenoted: ** P \ 0.001, * P \ 0.01, ns = not significant

596 Evol Ecol (2011) 25:589–604

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Relationships between dietary specialisations, stable isotopes and food transmitted

parasites

The CCA showed that the two different measures of time-integrated former niche use

(stable isotope ratios (d13C and d15N) and the abundance of different parasite species) were

significantly correlated (Monte Carlo test, P \ 0.005; Fig. 3). The first axis explains 32%

of the total variation. Low values of CCA I are indicative of low values of d13C (to the left,

Fig. 3) and associated with high infection rates of Diphyllobothrium spp. transmitted by

pelagic copepods, which was typical for the zooplanktivore (Z) specialist diet-group. High

values of CCA I, to the right side of Fig. 3, indicate that high values of d13C are associated

with high abundances of C. farionis and C. truncatus, both species transmitted to the fish

via G. lacustris as an intermediate host. This combination was found in gammaridivore (G)

specialists. The benthivore (B) predators had intermediate CCA I values indicating, lower

densities of both pelagic and benthic parasites and intermediate d13C-values. The second

axis explains only 0.4% of total variation, but describes a relationship between high values

of d15N, high abundance of the copepod transmitted parasites E. salvelini and Proteo-cephalus sp. and the benthivore feeding group. However, none of these associations with

the benthivorous diet group showed any statistical significance compared to other feeding

groups (see previous results).

Relationships between dietary specialisations and trophic morphology

Dietary groups classified from stomach contents (zooplanktivore, benthivore, gammarid-

ivore) were separated in both head and body morphology (Fig. 4a, b). All three dietary

groups were significantly different in body shape (Table 2). The first canonical axis (CV1)

in the CVA of body shape, separated zooplanktivore from gammaridivore/benthivore

foraging groups and accounted for 61% of the variation (Fig. 4a). The second axis CV2

(39% of variation) separated benthivore from gammaridivore dietary groups. Individuals

that belong to the zooplanktivore specialist group typically had a more slender body form

ProtProt

DiphDiph

EubEub

CrepCrep

CfCf ADAD

CtrunCtrunCfCf LL33

ZG

B

δδ1515 NN

δδ1313 CC

-1.0

-0.5

0.0

0.5

CCA I (31.6% / 98.6%)

CC

A II

(0.

44%

/ 1.

4%)

-10

1

-1.0 -0.5 0.0 0.5 1.0

-1 0 1Fig. 3 Relationship betweenstable isotope values (d13C, d15N,shown as arrows) and abundanceof different parasite speciesillustrated by a ConstrainedCorrespondence Analysis.Centroids for the different dietarygroups: zooplanktivores (Z),bentivores (B) andgammaridivores (G) are shownfor reference. Abbreviations forparasites:Diph = Diphyllobothrium spp.(c), Prot = Proteocephalus sp.(c), Eub = E. salvelini (c),Crep = Crepidostomum spp. (i),Ctrun = C. truncatus (g), CfL3 = C. farionis (larval stage, g),Cf AD = C. farionis (adult stage,g); transmission mode: copepods(c), insects (i), Gammarus (g)

Evol Ecol (2011) 25:589–604 597

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than the other dietary groups (Fig 4a). The head shape of gammaridivore fish was sig-

nificantly different from the other two groups, while the zooplanktivore and benthivore

dietary groups showed a non-significant trend towards a difference (Table 2). The CVA

plot showed some structuring of the data, where the CV1 axis (70% of variation) axis

separated all foraging groups, i.e. gammaridivore vs. benthivore vs. zooplanktivore

(Fig. 4b). The CV2 axis (30% of variation) separated benthivore vs. zooplanktivore/

gammaridivore foraging groups (Fig. 4b). Individuals in the gammaridivore diet group

(a)

-3

-2

-1

0

1

2

3

-3 -2 -1 0 1 2 3 4

Canonical variate 1C

anon

ical

var

iate

2

-3

-2

-1

0

1

2

3

4

5

-4 -2 0 2 4

Canonical variate 1

Can

onic

al v

aria

te2

(b)

Fig. 4 CVA of a body shape and b head shape in relation to different foraging groups: zooplanktivores(open symbols); benthivores (grey symbols); gammaridivores (filled symbols). Shapes corresponding toextreme values of CV1 and CV2 are exaggerated 2 times for easier interpretation. Arrow-lines (broken,solid) on axes correspond to lines (broken, solid) in body/head shape drawings

598 Evol Ecol (2011) 25:589–604

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typically had blunter snouts and more robust heads, while zooplanktivore individuals had

longer snouts and lower head depths compared to the two other feeding groups.

Discussion

In this study we tested 3 hypotheses directly related to resource use and its role in eco-

logically driven evolutionary divergence. Phenotypic plasticity is known to result in epi-

genetic modification of traits in charr when individuals are exposed to differing resources

over periods which are broadly similar to an individual’s lifetime. Here we show that

individual Arctic charr demonstrating foraging specialisms, identified by measures that

integrate over very short temporal periods (hours) showed clear evidence of persistent

foraging specialisation over much longer temporal periods (months and years). In Fjell-

frøsvatn, Arctic charr individuals exhibiting dietary specialism, defined by their stomach

contents, providing a temporally restricted measure of diet over the preceding hours only,

also showed significant differences in two trophic tracers with a much longer integration

period. Muscle stable isotopes (SI) signatures integrate over months (Perga and Gerdeaux

2005) and parasite fauna integrates over months and years (Knudsen et al. 2004). Thus,

individuals identified as zooplanktivores based on their stomach contents, had depleted

d13C values in muscle tissue, a high number of copepod transmitted parasites (CTP) and a

low abundance of Gammarus-transmitted parasites (GTP). In contrast, individuals with

stomach contents dominated by macrobenthos, exhibited enriched d13C values, a high

number of GTP and a low abundance of CTP. Individuals categorised as gammarid spe-

cialists on the basis of their stomach contents, showed the most enriched muscle d13C and

had the highest abundance of Gammarus-transmitted parasites. Despite that foraging

behaviour is often regarded as a highly labile trait (e.g. West-Eberhardt 2005; Duckworth

2009), we conclude that individual foraging specialisms identified from stomach contents,

do persist in Fjellfrøsvatn charr over periods that are sufficient to induce ontogenetic

plastic responses in individuals (e.g. Day and McPhail 1996; Adams et al. 2003; Alexander

and Adams 2004; Garduno-Paz et al. 2010) that is, broadly equivalent to the life-time of an

individual. This finding is consistent with models where alternative resource specialism

drives phenotypic plasticity in the earliest stages of divergence (Skulason et al. 1999).

Secondly we show, at the level of the population, stability in foraging specialisms that

persist over a period of almost two decades. The expression of alternative foraging spe-

cialisms that persists over multiple generations is a key requirement for ecologically driven

evolution through resource specialisation (Dieckmann and Doebeli 1999; Schluter 2000).

Thirdly we show that individuals demonstrating foraging specialisations also showed

morphological adaptations with a functional significance related to foraging. Thus the

gammaridivore specialists had blunter snout and more robust head and body compared

with zooplanktivore specialists. Benthivorous individuals were intermediate between the

Table 2 Pairwise comparisons (P-values from permutation tests of Mahalanobis distances in shape) ofdifferences in trophic morphology (body and head shape) between the three dietary groups of Arctic charr

Body shape Head shape

Zooplanktivores vs. gammaridivores \0.001 \0.001

Zooplanktivores vs. benthivores \0.001 0.066

Benthivores vs. gammaridivores \0.001 0.003

Evol Ecol (2011) 25:589–604 599

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two other foraging groups according to both head shape and body shape. Similar patterns

of functional body shape differences between zooplanktivore and benthivore specialists

have been widely reported amongst a number of postglacial fishes in general, and spe-

cifically in Arctic charr elsewhere (Robinson and Parsons 2002; Knudsen et al. 2007;

Hendry 2009).

Despite high levels of flexibility in foraging behaviour (e.g. West-Eberhard 2005;

Duckworth 2009), variation in behavioural traits relating to foraging and prey selection in

Arctic charr has been shown in some populations, to have at least some genetic component

to its expression (Adams and Huntingford 2002a,b; Klemetsen et al. 2002, 2006; Albertson

et al. 2005; Roger and Bernatchez 2007). Similarly, expression of alternative head mor-

phologies associated with foraging specialisms, has recently been linked to ecologically

based, disruptive selection in fish and seems to be genetically controlled by a very few loci

(Albertson et al. 2005; Tripathi et al. 2009) but has also been shown to be highly plastic

(Adams et al. 2003; Alexander and Adams 2004; Adams and Huntingford 2004). The

ultimate mechanisms regulating the expression of resource specialisation and morpho-

logical traits are not known for the model system investigated here. However it is most

likely that expression of the variation in foraging behaviour and morphology shown here is

the result of an interplay between genes and the environment (Schluter 2000; Adams and

Huntingford 2004; Abrams 2006; Rueffler et al. 2007).

One important outcome of this study in the context of evolutionary divergence, is that

the foraging specialists described here are subject to differential selection pressures.

Individuals adopting differing foraging specialisms in this study occupied very different

and contrasting habitat types. Thus the zooplankton specialists were predominantly col-

lected from, and clearly foraged in, the pelagic zone of the lake. In contrast the macro-

benthos and Gammarus foraging specialists were mainly collected from, and foraged in,

the littoral zone of the lake. These two environments differ substantially across a wide

range of physical and biotic characteristics (e.g. light penetration, habitat heterogeneity,

predation vulnerability, wave energy, etc.) strongly suggesting that specialists occupying

these alternative habitats would be subject to different suite of selection pressures. In

addition, the predator characteristics (e.g. the behavioural search image and capture

techniques) required of successful specialists foraging on the three different prey resources

in this study are very different (Day and McPhail 1996; Fraser et al. 2008).

Parasites have been shown to result in selection pressures with evolutionary conse-

quences for the host population (e.g. Maan et al. 2008; Blanchet et al. 2009; MacColl

2009). The foraging specialists in this study exhibited very different parasite fauna and

infection rates. This is also likely to have a significant impact on the selection pressures to

which each foraging specialist was exposed. Several of the endoparasites observed in the

current study are transmitted by foraging on specific intermediate hosts. In this study

zooplankton specialists had a higher abundance of Diphyllobothrium spp., and a lower

abundance of Crepidostomum spp., C. truncatus and C. farionis parasites than both

macrobenthos and gammarus specialists. The macrobenthos specialists had a higher par-

asite abundance of Diphyllobothrium spp., and a lower abundance of Crepidostomum spp.,

C. truncatus and C. farionis parasites than gammarus specialists. C. farionis, C. truncatus,

and Diphyllobothrium spp. have been shown to place direct fitness costs on their fish host

(see e.g. Curtis 1984; Knudsen et al. 2004, 2008). In sticklebacks limnetic and benthic

morph pairs show differential immune responses (Matthews et al. 2010) probably in order

to be able to specifically deal with the contrasting parasite faunas often found among

trophically diverged species pairs (Knudsen et al. 2003; MacColl 2009). Parasites often

have a dose dependent effect on their host, where the most parasitised individuals run the

600 Evol Ecol (2011) 25:589–604

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highest risk of being affected. Thus, dietary specialists run a high risk of suffering from

parasite infections as they accumulate high densities of a restricted group of parasites

(Amundsen et al. 2003; Knudsen et al. 2004), but may also benefit from a reduced

investment in a costly immune defence (Moret and Schmid-Hempel 2000). By concen-

trating the immune responses against a few parasite species, specialist foragers may be able

to respond effectively to manage infection levels. An ability to withstand the effects of a

specific, restricted group of parasite larvae may then allow a specialist predator to exploit a

potentially infected prey group without accumulating the high costs associated with

defense against a more diverse range of parasite types (Lafferty et al. 2000). Thus it is

highly likely that parasites are causing parasite-mediated differential selection pressures

amongst the three specialist charr foragers in Fjellfrøsvatn and that these and other iden-

tified differential selection pressures have remained consistent for at least 2 decades (see

also Knudsen 2010).

In the early process of ecologically driven speciation, theoretical models typically

include resource specialisation by individuals in a population adapted specifically to dif-

ferential resource usage in alternative environments (Dieckmann and Doebeli 1999; Sch-

luter 2000; Rueffler et al. 2007). The importance of dietary specialisation in sustaining

isolated morphs was recently highlighted in a collapsing divergent morph pair of stick-

lebacks (Behm et al. 2010). In the present study, where multiple co-occurring behavioural

specialists, adapted to a variety of distinct ecological niches, may have arisen as a result of

disruptive selection initiating the speciation process. Populations may appear at different

stages within the evolutionary diversification process, but not all diverging populations will

result in speciation (Hendry 2009; Nosil et al. 2009). If ecologically significant traits are

directly linked to mate choice (magic traits; sensu Gavrilets 2004), positive assortative

mating could be an important mechanism in promoting the likelihood of reproductive

isolation. Adaptive ecological traits related to visual cues and signals like trophic behav-

iour, body size and colouration (e.g. dietary-based carotenoid red ornamentation) seem

important in mate recognition and assortative mating of both reproductively isolated

postglacial fishes and in unimodal populations that are adapted to different dietary niches

(Snowberg and Bolnick 2008; Head et al. 2009; Stelkens and Seehausen 2009). Thus, the

behaviour and/or morphological traits related to alternative niche adaptation observed in

this study could eventually be involved in reducing gene flow among distinct phenotypes.

The observed incidence of multiple co-occurring specialist ecotypes adapted to a variety

of distinct ecological niches, has support in recent model studies, highlighting that adaptive

radiation may result in stable multiple morphs (e.g. Abrams 2006; Bolnick 2006; Doebeli

et al. 2007). In Fjellfrøsvatn, a deep-water morph has been found to be reproductively

isolated and, genetically and ecologically distinct from the ecotypes examined here

(Klemetsen et al. 1997; Westgaard et al. 2004; Amundsen et al. 2008) with specific

behavioural and morphological adaptations allowing the utilisation of a narrow profundal

soft bottom dietary niche (Klemetsen et al. 2002, 2006; Knudsen et al. 2006). Other studies

have also highlighted that locally adapted benthic morphs may live in sympatry (e.g.

Snorrason et al. 1994; Kahilainen and Østbye 2006). These results suggest that local

adaptations (Ravigne et al. 2009; Losos 2010) to alternative niches can occur beyond the

traditional view of divergence into littoral-pelagic morph-pairs which has been commonly

observed among postglacial fishes (reviewed by Robinson and Parsons 2002).

We conclude that these results suggest that differential selection pressures from several,

separate ecological processes are involved in shaping the stable foraging niches of several

different co-occurring ecotypes. The present lake ecosystem with stable resource spe-

cialists represents a promising candidate for complementary studies addressing how

Evol Ecol (2011) 25:589–604 601

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plasticity and heritability of traits are involved in the early process of speciation and which

definitive mechanisms are involved in the reproductive isolation process. Our findings are

thus particularly important in respect to understanding the mechanisms of incipient spe-

ciation, which appear to be related to a subtle interplay between ecological and evolu-

tionary processes at the individual and population level.

Acknowledgments We thank Laina Dalsbø, Jan Evjen and Pal Sørensen for field and laboratory assis-tance, Raul Primicerio for statistical advice and Jennifer Dodd for grammar checking of the manuscript. Wealso thank two anonymous referees for their constructive criticisms.

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