This article was published in the above mentioned Springer issue.The material, including all portions thereof, is protected by copyright;all rights are held exclusively by Springer Science + Business Media.

The material is for personal use only;commercial use is not permitted.

Unauthorized reproduction, transfer and/or usemay be a violation of criminal as well as civil law.

ISSN 0340-5443, Volume 64, Number 4

ORIGINAL PAPER

Are behavioral syndromes invariant? Spatiotemporalvariation in shy/bold behavior in squid

David L. Sinn & Natalie A. Moltschaniwskyj &Erik Wapstra & Sasha R. X. Dall

Received: 15 May 2008 /Revised: 17 November 2009 /Accepted: 17 November 2009 /Published online: 10 December 2009# Springer-Verlag 2009

Abstract Behavioral syndromes are correlated suites ofbehavior, analogous to human personality traits. Most workto date has been taken from limited “snapshots” in spaceand time, with the implicit assumption that a behavioralsyndrome is an invariant property, fixed by evolutionaryconstraints or adaptations. However, directional selectionon two mechanistically independent traits (selective co-variance) could also result in correlated behaviors. Previ-ously, we have shown that shy/bold behavior in Southerndumpling squid (Euprymna tasmanica) across predatorencounter and feeding risk contexts is genetically andphenotypically uncoupled, and hence potentially free tovary independently. Here, we collected data on shy/boldbehaviors from two independent wild populations of squidin two different years to test whether behavioral correla-tions across these same two functional contexts vary

through time and space. We detected significant influencesof population, sex, and body size on the expression ofboldness in squid within each functional context, and thiswas coupled with significant differences in relative popu-lation density and adult sex ratio. Despite these changes inbehavior and demographic parameters, we found thatcorrelations between boldness scores across the twofunctional contexts were largely absent in both wildpopulations of squid in both years. Our work suggests thatsome animal groups may be largely characterized bycontext-specific behavioral expression. A theoretical frame-work which conceptualizes behavioral syndromes resultingfrom context-specific behavioral rules may be needed tofully understand why behaviors are sometimes correlated,and why sometimes they are not.

Keywords Behavioral syndromes . Shy/bold . Selectivecovariance . State-dependent optimization

Animals within the same age, size, and sex class oftendisplay consistent inter-individual behavioral differencesin a number of functional contexts related to fitness, suchas foraging or mating behavior (Wilson 1998; Gosling2001). For example, individuals often differ in theirwillingness to take risks: some individuals are consistentlybolder than other conspecifics (Wilson et al. 1994). Anindividual's shy/bold behavioral phenotype often corre-lates with many important life history traits such asreproduction (Both et al. 2005; Duckworth 2006; Sinn etal. 2006) and survival (Réale and Festa-Bianchet 2003;Dingemanse and Réale 2005). Here, we use the terminol-ogy “behavioral phenotype” to refer to an individual'sconsistent behavioral trait expression in any one functionalcontext through time, while we use the term “behavioral

Communicated by T. Bakker

D. L. Sinn :N. A. MoltschaniwskyjSchool of Aquaculture, University of Tasmania,Launceston, Tasmania

D. L. SinnSchool of Plant Science, University of Tasmania,Hobart, Tasmania

D. L. Sinn (*) : E. WapstraSchool of Zoology, University of Tasmania,Private Bag 5,Hobart TAS 7001, Australiae-mail: david.sinn@utas.edu.au

S. R. X. DallCentre for Ecology & Conservation, School of Biosciences,University of Exeter, Cornwall Campus,Penryn, UK

Behav Ecol Sociobiol (2010) 64:693–702DOI 10.1007/s00265-009-0887-2

Author's personal copy

syndrome” to refer to cases where two or more behavioralphenotypes (shy/bold or otherwise) are correlated at thepopulation level across functional contexts (Bell 2007).Despite the clear benefits of maintaining a high degree ofbehavioral plasticity, recent work suggests that behavioralphenotypes and syndromes (i.e., reduced behavioralplasticity) are widespread (Gosling 2001) and mayinfluence and reflect important life history trade-offs, suchas growth versus mortality or future versus currentreproductive success (Sih et al. 2004b; Stamps 2007; Wolfet al. 2007). For example, some populations of sticklebackfish are characterized by a behavioral syndrome betweenboldness and aggression: individual fish that are moreaggressive towards conspecifics are also bolder towardspredators. While high levels of competitive aggressivenessmay be favorable in acquiring mates, inappropriate boldnessmay also have survival costs (Huntingford 1976; Bell 2005;Bell and Sih 2007; Dingemanse et al. 2007).

Empirical work on behavioral syndromes has thus farfocused on documenting patterns of behavioral correlationsacross contexts and explaining the strategic consequencesof these behavioral trait correlations, usually within arestricted time frame such as one cohort or generation(e.g., Huntingford 1982; Bell 2005; Duckworth 2006;Alvarez and Bell 2007; Dingemanse et al. 2007; Brydgeset al. 2008). There is often an implicit assumption that a“snapshot” is representative of longer-term evolutionarypatterns and that the behavioral correlation is invariant in agiven population through time. However, recent worksuggests that behavioral syndromes within a populationand cohort can be environmentally induced (e.g., throughaddition of predators: Bell and Sih 2007), and thuscorrelated behaviors across contexts, when observed, maynot be fixed properties of populations. Furthermore, manystudies have also found that behavioral phenotypes areoften expressed in a context-specific manner. In otherwords, many populations are characterized by a lack of asyndrome (e.g., Coleman and Wilson 1998; Sinn andMoltschaniwskyj 2005; van Oers et al. 2005). For example,in some populations of pumpkinseed sunfish, individualsthat consistently behave boldly towards predator encountersdo so independently of their consistent “bold” responsetowards novel food sources (Coleman and Wilson 1998).Understanding when behaviors are correlated across differ-ent functional contexts and when they are not should giveinsight into the processes by which integrated suites ofbehaviors guide and constrain adaptive behavioral evolu-tion (Bell 2007; Bell and Sih 2007; Réale et al. 2007).

In general, when correlated behaviors have beenobserved, two possible hypotheses have been proposedto explain the correlation per se. The first of these, theconstraint hypothesis, posits that correlations result fromlimitations on the way that animals are organized, where

the constraint is usually genetic, hormonal, or develop-mental (Sih et al. 2004a; Bell 2005; but see Cheverud1984 for a discussion of how developmental and geneticconstraints may arise through natural selection andtherefore represent adaptations). The second hypothesis,the adaptive hypothesis, posits that correlated behaviorsoccur because the linkage itself is adaptive. In this case,selection occurs for particular syndromes (Bell 2005; Wolfet al. 2007). Several non-selective mechanisms could alsoexplain fixed correlated suites of traits, such as drift ormutation covariance (Armbruster and Schwaegerle 1996).However, one additional selective explanation for variablebehavioral syndromes is that correlated behaviors repre-sent an emergent property of two mechanistically andfunctionally independent traits. In other words, selectioncould favor mechanisms which allow for the independentexpression of behaviors in any two given contexts (e.g.,Coleman and Wilson 1998; Sinn and Moltschaniwskyj2005), but due to fluctuating environmental and socialconditions, correlated behaviors arise and disappear as anemergent property of the behavioral system. Oppositedirectional selection in two contexts would result innegative covariance between two behavioral traits, whileselection in the same direction in both contexts wouldresult in positive covariance. While rule-based behaviorwithin single functional contexts is likely to be common(McNamara et al. 1999; Hutchinson and Gigerenzer2005), the possibility that population-level variation inbehavioral syndromes may result from context-specificbehavioral phenotypes has yet to be examined.

Previously, we documented context-specific shy/boldbehavioral phenotypes in Southern dumpling squid(Euprymna tasmanica, Pfeffer, 1884), which are consistentover long periods (25%) of their lifespan (Sinn andMoltschaniwskyj 2005; Sinn et al. 2008). In behavioraltests simulating a predator encounter (threat test) andforaging (feeding test), bolder squid are ambivalent towardsor even attack a threat stimulus, while shyer squid tend toflee by jetting and inking after fewer “attacks” with a novelstimulus. Boldness in feeding contexts is characterized byshorter latencies to feed, more feeding attempts over longerdistances, and faster feeding rates in the presence of anobserver, whom squid perceive as a threat. Shy/boldbehaviors in different functional contexts may be mecha-nistically independent in squid. Phenotypically, shy/boldbehaviors are expressed in a context-specific manner acrossthe entire lifespan, and genetic correlations betweenboldness in threat and feeding contexts, at least in somepopulations, are not significantly different from zero (Sinnet al. 2006, 2008). Here, we use data on shy/bold behaviorscollected from two wild populations of dumpling squid intwo different years to test whether behavioral correlationsacross contexts vary through time and space. Due to

694 Behav Ecol Sociobiol (2010) 64:693–702 Author's personal copy

features of their life history, including a short 5–8-monthlifespan (Norman and Reid 2000), non-overlapping gen-erations (Boyle and Boletzky 1996), and “slow” dispersalthrough crawling (e.g., Kimbell et al. 2002), this samplingdesign allowed us to test correlated behaviors in distinctcohorts and populations of squid.

While our main question of interest was to test thestability of the observed patterns of (un)correlated behav-iors across contexts in squid, little empirical data has beenpresented on biological and social influences on behavioralcorrelations in wild populations of animals. Given that wehave previously documented widespread context-specificexpression of boldness in squid (Sinn and Moltschaniwskyj2005; Sinn et al. 2006, 2008), we also examined howseveral demographic factors influenced the expression ofbehavioral phenotypes within each functional context. Wemeasured two demographic variables (relative populationdensity and adult sex ratio) and two squid biological “state”variables (body size and condition) since theoreticalanalyses suggest that interactions between biological statesand resource acquisition can generate adaptive “personalitydifferences” (Dall et al. 2004).

Methods

Study sites

Squid were collected from two geographically disjunctpopulations (531 km by sea) in Tasmania, Australia (Kelso:41°6′S, 146°47′E; Margate: 43°1′S×147°16′E) in January,February, and March of 2003 and 2004 (Table 1). Kelso,located at the mouth of a large river (the Tamar), ischaracterized by large semi-diurnal tidal influences. Squidat Kelso were found mainly in a small area of sand flat(100×500 m) on the side of the river basin. Margate is arelatively large, calm shallow inlet of a larger bay andexperiences diurnal tides with less overall water flowcompared with Kelso. Squid at Margate were in a largeroverall area (500×500 m) which was mainly covered in seagrass (approximately 65%).

Subject animals

E. tasmanica are small (adult size 4–7 cm) non-gregariousbenthic squid found in shallow inshore waters of southeast

Table 1 Demographic, state, and behavioral data for dumpling squid (Euprymna tasmanica) over 2 years at two geographically disjunctpopulations in Tasmania, Australia

Site and year N Average density(squid/h)

Adultsex ratio

Body size in mm(mean±SD)

Body condition(mean±SD)

Threat boldnessscores (mean±SD)

Feeding boldnessscores (mean±SD)

Kelso 2003 total 62 16.03 0.66 18.0±4.4 −0.03±0.67 0.26±2.89 0.99±2.69

Adult males 27 21.7±3.5 0.03±0.92 0.46±2.50 1.49±2.51

Adult females 14 18.7±2.7 −0.28±0.36 1.68±4.02 1.00±2.64

Subadult males 6 13.6±1.5 0.26±0.62 −1.24±0.98 1.11±3.19

Subadult females 8 13.8±1.0 0.36±0.33 −0.19±2.68 −1.64±3.31Kelso 2004 total 59 20.42 0.42 20.2±5.0 −0.70±0.45 0.05±2.76 0.08±2.70

Adult males 18 22.7±3.3 0.66±0.71 −0.29±2.85 −0.03±2.73Adult females 25 22.3±3.7 −0.60±0.92 0.39±3.21 −0.46±2.93Subadult males 0

Subadult females 4 14.0±0.8 0.15±0.32 2.33±1.28 0.90±1.53

Margate 2003 total 33 7.86 0.61 17.0±5.1 0.83±1.24 0.30±4.15 −0.33±3.01Adult males 8 22.0±1.7 1.00±1.31 −0.71±2.20 −0.72±3.31Adult females 5 22.8±5.4 1.92±2.41 4.13±7.44 1.07±3.17

Subadult males 3 14.5±0.5 0.52±0.37 −1.81±0.36 −2.38±3.68Subadult females 9 13.9±1.1 0.38±0.40 0.43±3.42 −0.75±3.06Margate 2004 total 41 6.66 0.41 19.8±7.6 0.03±1.14 −0.71±3.23 −1.18±2.96Adult males 9 23.9±5.1 −0.56±0.83 −0.11±4.81 −0.44±3.24Adult females 13 25.7±7.4 0.27±1.70 0.65±3.35 −1.78±3.02Subadult males 0

Subadult females 2 14.5±0.7 0.53±0.23 −1.93±1.49 −0.76±5.30

Several individuals that were classified as “adult” according to body size were not sexed: Kelso 2003 one individual, Kelso 2004 two individuals,Margate 2003 one individual, and Margate 2004 two individuals. Several subadult individuals could not be reliably sexed based on externalmorphological characteristics: Kelso 2003 six individuals, Kelso 2004 ten individuals, Margate 2003 seven individuals, Margate 2004 15individuals. Data from these individuals is included in the totals for threat and feeding boldness scores but not for the break-down by sex for thatsite/year

Behav Ecol Sociobiol (2010) 64:693–702 695 Author's personal copy

Australia (Norman and Reid 2000). Dumpling squid arenocturnal and spend daylight hours buried in sand substrateemerging near dusk to forage and find mates. Ourobservations over the past 6 years suggest that, while nota schooling species, dumpling squid are found in socialaggregations in the wild, usually associated with sandybottom substrates near sea grass beds. Most adult squidfeed on a wide variety of vertebrate (fishes) and inverte-brate (copepods, crustacean shrimp, and marine worms)prey, while larger fishes are their main predators (Packard1972). As in many animals, feeding in sepiolid squidinvolves reduced predator vigilance and predation risk(Messenger 1977). While little is known of the reproductivebehavior of Euprymna, both sexes are promiscuous, andmultiple reproductive events for both males and females,immediately followed by death, occur over a relativelyshort time period (approximately 3 weeks). Like otherclosely related cephalopods (e.g., Hanlon 1996; Naud et al.2004), this short reproductive period for adults is likely toresult in intense male–male competition for females, femaleharassment and coercion by males, and cryptic femalesperm choice.

Squid were collected at night at both sites by two diversusing SCUBA. All squid were either counted (e.g., juvenilerecruits) or collected, and we were unable to detect anycontrollable bias in our collection methods. For example,once sighted, fewer than ten individuals escaped captureover the 2 years of collection. Information regardingrelative population density, adult sex ratio, squid body size,and condition were collected at each site during each year.For all dives, two divers followed approximate straight-linedive transects for 30 min before returning along a parallelpath. Relative densities of the two populations werecalculated by summing the total number of squid collectedand seen on dives for all months in a given year (one dive/month for 3 months each year). The total number of squidcollected and seen was then divided by the total dive timesummed across both divers in hours, resulting in a relativemeasure of squid per hour for each site for each year. Eachcaptured adult squid was sexed, and all captured squid weremeasured for mantle length (ML) and wet weight (WW).We calculated a condition index for each individual basedon studentized residuals of ML on WW. Slopes ofregression lines for ML and WW were not significantlydifferent between males and females (two-tailed t144=0.38,p=0.70), therefore, a single sex-independent regression linewas used to calculate condition indices. We furtherclassified subject squid as either subadult (<15.6 mm ML)or reproductively mature (>15.6 mm ML) based onprevious studies (Sinn et al. 2008). We calculated the sexratio of mature adults for each site during each year bydividing the number of reproductively mature adult malesby the total number of adults.

Squid were transported within 2–12 h to a closed-seawatersystem at the University of Tasmania, Launceston, Australia,where they were held for behavioral testing. Squid werehoused and tested separately in 34 cm long×29 cm wide×13 cm deep opaque plastic tubs at ambient conditions (18°C,33–34 ppt salinity). Immediately upon housing, squid werefed to excess with mysid shrimp and allowed to acclimatizefor 48 h prior to behavioral tests, which were carried out in thesame tubs that squid were housed in.

Behavioral tests

Behavioral test procedures were identical to those describedin Sinn and Moltschaniwskyj (2005). Briefly, 6 to 12 hbefore testing, all test containers were covered with opaqueplastic lids (to avoid disturbing adjacent subjects), andtesting began 1–2 h after the start of the dark phase. Allsquid were subjected to two behavioral tests given by asingle experimenter (DLS) on two test days (48 and 96 hpost-capture), with both tests designed to simulate aseparate functional context.

Threat test The first test represented a simulated predatorencounter and consisted of the experimenter touching thesquid with a threat stimulus on one of its arms, up to tentimes or until the squid moved away. The threat stimuluswas a 50-ml plastic eyedropper, colored to enhance visualacuity. Four behaviors were measured in threat tests: thenumber of touches required before the squid moved away,the first behavioral response towards the stimulus (acategorical variable, ranging from 1 to 9 with increasingnumbers indicating more bold or aggressive responses: 1=jet, 2=fin swim, 3=amble, 4=ink, 5=bury, 6=no reaction,7=color change, 8=arm flower posture, and 9=grab), thefrequency of jetting, and the number of times a squidgrabbed the threat stimulus. See Sinn and Moltschaniwskyj(2005) for detailed descriptions of these behaviors.

Feeding test Feeding tests were conducted 60–120 minafter threat tests and consisted of a live food presentation(mysid shrimp) in the presence of the experimenter, whomsquid treat as a potential threat. Three behaviors wererecorded in feeding tests: latency to first feeding attempt,number of feeding attempts, and handling time for fooditems (time spent feeding (s)/number of captured mysids).After initial feeding during acclimatization, all squid weresubsequently fed only during feed tests, and the test orderof individual squid was randomized on each test day.

Behaviors in both tests were recorded for 5 min afterintroduction of the stimulus using an audio cassetterecorder and hand-held timer. An a priori 5-s rule was usedfor all frequency counts, in which behaviors were scored asa multiple frequencies only if there was at least a 5-s break

696 Behav Ecol Sociobiol (2010) 64:693–702 Author's personal copy

between occurrences. Squid were tested within 1 week onlybecause shy/bold behavior in both test contexts is highlyrepeatable across longer (i.e., 2–4 weeks) time periods (ρ>0.50, Sinn and Moltschaniwskyj 2005; Sinn et al. 2008). Toreduce within-individual within-test variability in behavior(Epstein 1983; Fleeson 2004), observable behaviors withineach test context were summed across the two test days,representative of an individual's overall “average” behaviorin the two tests.

Shy/bold scores

In a previous study using a large sample of wild-caughtsquid from Kelso, we developed aggregate scales represent-ing shy/bold behavior in threat and feed tests based onprincipal components analyses (PCA) of the same behav-iors measured here (Sinn and Moltschaniwskyj 2005).Similarly, the behaviors assessed within each test in thecurrent subjects (four in threat, three in feeding) were alsohighly inter-correlated within each test and loaded stronglyon a common component in PCA analyses in eachpopulation (Table 2). Thus, in order to reduce the numberof variables used in subsequent analyses and to facilitateuse of a reliable single score representative of boldness, wecomputed separate scale scores for each squid for each testseparately (Tabachnick and Fidell 1996), resulting in twounique scores per squid. Specifically, for the threat test, wecomputed scale scores by summing the normalized varia-bles measured in this test (number of touches, first behaviorafter touch, frequency grabs, and frequency jet (reversekeyed)). For the feeding test, boldness scale scores werecomputed by summing the normalized variables measuredin this test (number of feeding strikes, latency to firstfeeding strike (reverse keyed), and handling time (reverse

keyed)). Normalization for each variable was according tothe grand mean for that variable for both populations.

In both test contexts, higher scores represent “bolder”overall responses. For example, high scores in threat testsindicated squid which did not flee from the threat stimulusbut instead were ambivalent to the threat, or even attackedit by grabbing and signaling aggressively towards it. Highscores in feed tests indicate squid which took more feedingstrikes, had shorter feeding latencies, and had shorterhandling times in the presence of an observer. Seven squidfrom Kelso did not receive feeding tests in 2003 due tologistical reasons, and sample sizes for subsequent analysesvaried, due either to exclusion of subadult animals ormissing data for some individuals for some variables. Theresulting N for each analysis is given below.

Demographic differences between sites

Variation in reproductively mature adult sex ratios were testedusing loglinear analyses (N=119, Quinn and Keough 2002).The significance of all interactions between sex, site, andyear was tested by using log-likelihood ratio tests comparingthe full model with its appropriate restricted version (i.e.,dropping the two- or three-way interaction and comparingthe fits of the models). Differences in relative density of thetwo squid populations was tested using a two-way analysisof variance (ANOVA) with site and year as fixed factors andeach collection month within a year as a replicate (N=6).Differences in log ML and body condition between sites andyears were both analyzed with two separate three-wayANOVAs (sex, site, and year as fixed factors; N=151).

Variation in mean-level shy/bold behaviors

We assessed the role of population, year of collection, squidgender, and their interactions in explaining variation inthreat boldness and feeding boldness scores with a three-way multivariate analysis of covariance (MANCOVA:Tabachnick and Fidell 1996). Categorical predictors andtheir interactions were entered as fixed factors. Squid MLand condition were used as covariates in the MANCOVA;there were no significant univariate interactions betweenboth covariates and the three factors of interest for either ofthe shy/bold scores (all two-, three-, and four-way inter-actions in ANCOVA models, p>0.05). Canonical discrim-inant analysis and univariate F tests were used to examineany significant main effects or interactions, while univariateF tests were used to examine any significant covariateeffects (Quinn and Keough 2002). Equality of the variance–covariance matrices for each group and multivariate outlierswere examined by Box's M test and examination ofMahalanobis distances prior to interpreting the MANCOVA.Variance–covariance matrices were homogenous (Box's M=

Table 2 Loadings of discrete behaviors on site-specific principalcomponents from dumpling squid (Euprymna tasmanica) observed inthreat and feeding tests (N=121 in threat tests for Kelso, N=114 infeeding tests for Kelso, and N=74 for both tests for Margate)

Behavior Kelso Margate

Threat test

Number of touches 0.87 0.91

First behavior after touch 0.91 0.92

Grab 0.80 0.88

Jet −0.64 −0.45Cumulative variance explained (%) 66.2 66.2

Feed test

Number of feeding strikes 0.98 0.98

Latency to first feed −0.96 −0.98Handling time (time(s)/mysid) −0.94 −0.96Cumulative variance explained (%) 91.9 94.6

Behav Ecol Sociobiol (2010) 64:693–702 697 Author's personal copy

28.91, P=0.16), and boxplots of Mahalanobis distancesindicated no multivariate outliers. Squid with missing data(many subadults could not be reliably sexed) were notincluded in the MANCOVA model (N=145).

Variation in correlations between behaviors across contexts

Population-, year-, and life stage-specific correlations wereexamined using Spearman-rank correlations between bold-ness scores in the two behavioral tests for each site, year,and life stage combination (Kelso 2003 subadults, N=16;Kelso 2003 adults, N=39; Margate 2003 subadults, N=19;Margate 2003 adults, N=14; Kelso 2004 subadults, N=14;Kelso 2004 adults, N=45; Margate 2004 subadults, N=17,Margate 2004 adults, N=24). If boldness was context-specific within a population during a given year, thenacross test correlations would be low; conversely, highcorrelations between scores across test contexts wouldindicate boldness that was phenotypically coupled. Non-parametric correlations were used because shy/bold scoresin feeding tests were not normally distributed. We adjustedfor multiple comparisons for shy/bold ranks across contextsusing the false discovery rate procedure (Benjamini andHochberg 1995) and note significant results according tothis procedure. We used SAS 9.1 for demographic analysesand SPSS 14.0 for behavioral analyses.

Results

Demographic differences between sites

There was no significant interaction between site and yearin explaining patterns in sex ratios (χ2

1 ¼ 0:03, p=0.86). Atboth sites, there was a significant switch from a male-biasedadult population in 2003 to a female-biased one in 2004(χ2

1 ¼ 6:28, p=0.01; Table 1). Variation in relative densityof squid was not explained by the interaction between siteand year (F1, 8=0.73, p=0.42), or year as a main effect(F1, 8=0.17, p=0.69). Instead, the population of squid atKelso was roughly twice as dense as that at Margate duringboth years of study (F1, 8=11.29, p=0.001; Table 1). Therewere no significant two- or three-way interactions betweensex, site, and year in explaining variation in mean body sizein squid. However, squid in 2004 were significantly largerthan the 2003 cohort (F1, 143=30.4, p<0.001), and maleswere significantly larger than females in both years and atboth sites (F1, 143=7.11, p=0.01). Similarly, there were nosignificant two- or three-way interactions between sex, site,and year in mean condition of squid. However, squid in2003 were in better condition than the 2004 cohort (F1, 143=16.05, p<0.001), with squid from Margate in better overallcondition than Kelso squid (F1, 143=20.98, p<0.001).

Thus, the overall demographic pattern was of a high(Kelso) and low (Margate) density squid population whichwere both male-biased in 2003 and female-biased in 2004.Males were larger than females, but both sexes were smallerduring the male-biased year and larger in the female-biasedone. Margate squid were in better overall condition thanKelso squid, but all squid were in better condition in themale-biased year than in the female-biased one.

Variation in mean-level shy/bold behaviors

Amongst individuals from both populations of squid, weobserved considerable variation in boldness scores (Table 1).The MANCOVA including the two covariates (bodycondition and size) and three fixed factors indicatedsignificant multivariate effects of body condition, body size,population, and sex on boldness scores, while year ofcollection and all higher-order main effect interactions werenonsignificant (Table 3). The pooled within-groups correla-tion between site and standardized discriminant functionsindicated that feeding boldness scores were the mostimportant in discriminating between squid from Kelso andMargate (feeding boldness score coefficient=0.98; threatboldness score coefficient=0.07). Kelso squid on averagehad higher feeding boldness scores over both years thanMargate squid (unstandardized discriminant functions eval-uated at group means: Kelso=0.15, Margate=−0.29). Thesediscriminant functions were supported by univariate F tests,which indicated that only feeding boldness scores explainedsignificant differences between the sites (threat boldness:F1, 135=2.20, p=0.14; feeding boldness: F1, 135=5.24, p=0.02). Similarly, an assessment of the pooled within-groupscorrelation between sex and standardized discriminantfunctions (threat boldness score coefficient=0.82; feedingboldness score coefficient=−0.63) along with discriminantfunctions at group means (females=0.18; males=−0.21)indicated that females were bolder in threat tests but shyer

Table 3 Multivariate analysis of covariance with threat boldness andfeeding boldness scores as the dependent variables (N=145)

Source Pillai's trace df F P

Intercept 0.055 2, 134 3.91 0.022

Body size (ML) 0.068 2, 134 4.92 0.009

Body condition 0.059 2, 134 4.23 0.017

Site 0.059 2, 134 4.17 0.018

Year 0.007 2, 134 0.44 0.644

Sex 0.045 2, 134 3.12 0.047

Site×year 0.004 2, 134 0.29 0.749

Site×sex 0.003 2, 134 0.19 0.827

Year×sex 0.028 2, 134 1.96 0.145

Site×year×sex 0.034 2, 134 2.36 0.099

698 Behav Ecol Sociobiol (2010) 64:693–702 Author's personal copy

in feeding tests while males showed the opposite pattern.However, univariate F tests indicated that only mean-levelthreat boldness scores were significantly different betweenthe sexes (threat boldness: F1, 135=5.72, p=0.02; feedingboldness: F1, 135=1.08, p=0.30). Univariate F tests indicatedthat body condition and body size explained significantvariation in threat boldness scores (condition: F1, 135=7.15,p=0.008; size: F1, 135=9.34, p=0.003), but not feedingboldness scores (condition: F1, 135=0.68, p=0.41; size:F1, 135=1.26, p=0.26). Larger squid (slope coefficient=0.23) and those that were in better condition (slopecoefficient=0.97) were bolder in threat tests.

Thus, the overall mean-level behavioral pattern wassquid from Kelso having higher mean feeding boldnessscores than Margate squid, while females regardless of sitewere bolder than males in threat tests. Within each sex,larger squid and those in better condition regardless of site,year, or sex were bolder in threat tests than squid that weresmaller or in worse condition.

Variation in correlations between shy/bold behaviors

Between predator encounter and foraging test contexts, thecorrelations between boldness scores for subadults werer16=−0.30 (p=0.26) and r14=0.11 (p=0.71) at Kelso in2003 and 2004, respectively, and r19=−0.28 (p=0.24) andr17=−0.16 (p=0.54) at Margate in 2003 and 2004,respectively. The correlations for reproductively matureadults were r39=−0.35 (p=0.03) and r45=0.09 (p=0.54) atKelso in 2003 and 2004, respectively, and r14=0.05 (p=0.87) and r24=−0.25 (p=0.24) at Margate in 2003 and2004, respectively. While it was clear that most correlationsbetween behaviors at both sites were not significantlydifferent from zero, the correlation for Kelso adults in 2003was an exception to this (Fig. 1). However, after correctingfor multiple comparisons, the correlation amongst adults atKelso in 2003 was also not statistically significant.

Discussion

Often, when researchers (including ourselves) have identi-fied behavioral correlations (or lack thereof) across contextsin their study system, the implicit conclusion is that thesuite of correlated behaviors is an invariant property of thepopulation or species, shaped by natural selection (e.g.,Johnson and Sih 2005; Sinn and Moltschaniwskyj 2005;Stapley and Keogh 2005; Duckworth 2006). However,most studies of behavioral correlations across contexts havebeen based on single cohorts from any given studypopulation. This may be problematic if longer-termselection regimes are inferred from these studies, and thestability through time for correlations per se is unknown. If

syndromes can result from selective covariance on mecha-nistically independent, context-specific trait expression, thenevolutionary interpretations based on limited “snapshots” intime may limit any conclusions about the functionalconsequences of behavioral syndromes.

We found that several squid demographic parametersthat could be considered proxies for competition for foodand mates varied between years and populations (relativedensity and adult sex ratio; West-Eberhard 1979). Relativedensities of squid in one population were twice that of theother, and both populations underwent a significant shiftfrom a male- to a female-biased adult sex ratio. While thereis currently no field data on the factors that alter thestrength of social competition in dumpling squid, it is likelythat due to their short reproductive life history and spatialclumping in the wild, males compete over females (e.g.,Rankin and Kokko 2007) and coerce females duringcopulation (e.g., Hanlon 1996; Maxwell and Hanlon2000; D. Sinn personal observation). It is also likely thatsquid populations with male-biased sex ratios have, onaverage, higher levels of competition for females (e.g.,

5

10

15

20

25

30

35

40

0 10 20 30 40 50

5

10

15

20

25

30

35

40

45

50 A

0 20 5030 4010

Threat boldness scores

Fee

din

g b

old

nes

s sc

ore

s

50

45B

Fig. 1 Scatterplots of rank boldness scores across two contexts fromKelso population squid (Euprymna tasmanica) in two different years.Solid lines represent least-squares regression line. a 2003. b 2004

Behav Ecol Sociobiol (2010) 64:693–702 699 Author's personal copy

Mougeot et al. 2003) and that competition for food andmates increases with increasing squid densities (Boal et al.1999; Maxwell and Hanlon 2000). Concurrently with thesedemographic changes, shy/bold scores within each func-tional context tested here also varied according to popula-tion of origin, sex, and size. Squid at the highest density site(Kelso) were consistently more feeding bold than squidfrom the lower density site (Margate), females were bolderthan males in predator encounter tests, and larger squidwithin both sexes were bolder in predator encounters. Thus,if being larger or female tends to equate to being boldertowards threats in squid and increased competition forresources results in increased incentive to be bolder infeeding situations, then context-specific directional selec-tion in these two functional contexts could result incorrelations at the population level if larger squid orfemales experienced less competition for food, whilesmaller squid or males experienced more. Taken together,our results on changes in behaviors within contexts and indemographic patterns suggest that interactions betweensquid size/sex and the level of contest competition anindividual was subject to may have occurred during ourstudy. Clearly, food availability and predation risk shouldalso influence the strength of selection induced bycompetitive regimes and the potential interactions betweeninternal state variables (e.g., sex and size; Heithaus et al.2007), and further theory and empirical examples areneeded to test these ideas.

Despite the observed variation in social conditions andchanges in behavioral responses by squid within both testcontexts, correlations between boldness scores across thetwo functional contexts were largely uncoupled in bothwild populations of dumpling squid across both years.Previously, we have shown that boldness in dumpling squid(at least in these two contexts) may be phenotypically andgenetically uncoupled, and hence potentially free to varyindependently (Sinn and Moltschaniwskyj 2005; Sinn et al.2006, 2008). In addition to conceptualizing behavioralsyndromes as either fixed evolutionary constraints oradaptations, some animal systems may be largely charac-terized as having behavioral phenotypes that are expressedin a context-specific manner. This is in contrast to manycurrent studies on behavioral syndromes, where theemphasis is placed on different behaviors in differentfunctional contexts (i.e., conspecific aggression and bold-ness towards predators), and where these different behav-iors are sometimes correlated and are sometimes not (Bell2007; Bell and Sih 2007; Dingemanse et al. 2007). Ourresults suggest that behavioral flexibility (i.e., context-specific behavioral phenotypes) may exist at the level of thesame trait in different contexts, and therefore, if behavioralcorrelations across contexts occurred in systems such assquid, they would most likely emerge as a result of the

expression of independent behavioral “rules” (potentiallyadaptive) that determine behavioral responses. In this case,instead of the idea of correlational selection occurring at thelevel of adaptive trait combinations (Lande and Arnold1983; Endler 1986), a behavioral syndrome would resultfrom selection on context-specific behavioral phenotypes. Itis worth noting that if we had, by chance alone, sampledadult squid from a single cohort in a single population(Kelso in 2003), we would have detected a small butsignificant negative relationship between boldness in thetwo test contexts and may have inferred that this behavioralcorrelation represented a fixed property of the population.In addition to emphasizing the need for longer-termobservations in wild populations, this result also illustrateshow selective covariance on context-specific traits mightresult in emergent behavioral syndromes.

To date, there has been a relative paucity of studies onthe role of social conditions in generating across-contextbehavioral correlations (but see: Sih and Watters 2005;Magnhagen 2007). This is despite the fact that behavior isoften influenced by social competition (McNamara et al.1999), and theoretical work suggests that behavioralsyndromes can also be generated by such rule-basedbehavior (Rands et al. 2003; Dall et al. 2004). Our studyis one of the first to analyze patterns of individual behavioracross a range of different behavioral and demographiccircumstances in the field, which has allowed us to identifywidespread context-specific behavioral expression of bold-ness, and to generate hypotheses which may explain whybehaviors are sometimes correlated, and sometimes not.Clearly, further work is required to confirm our interpreta-tion, including both developing the theory of state-dependent games in animal populations as well as empiricalmanipulations of behavioral phenotypes in different com-petitive environments.

Acknowledgements This work was conducted in partial fulfillmentof the requirements for D.L. Sinn's PhD degree at the University ofTasmania. Funding was provided by a Holsworth Wildlife ResearchFund grant (M0013237) and Australian International PostgraduateResearch scholarship to DLS. Tom Fox-Smith, Jonothan Newman,and Colin Johnson provided essential logistical support in the field.Kees van Oers, Piet Drent, and Luc Alain-Giraldeau provided helpfuldiscussions; Alison Bell, Jo McEvoy, and two anonymous reviewersgave constructive criticism on earlier drafts that greatly improved thefinal version. Experimental methods for all squid conformed toAustralian law and the standards set by the University of TasmaniaAnimal Ethics Committee (2003).

References

Alvarez D, Bell AM (2007) Sticklebacks from streams are more boldthan sticklebacks from ponds. Behav Processes 76:215–217

Armbruster WS, Schwaegerle KE (1996) Causes of covariation ofphenotypic traits among populations. J Evol Biol 9:261–276

700 Behav Ecol Sociobiol (2010) 64:693–702 Author's personal copy

Bell AM (2005) Behavioural differences between individuals and twopopulations of stickleback (Gasterosteus aculeatus). J Evol Biol18:464–473

Bell AM (2007) Future directions in behavioural syndromes research.Proc R Soc Biol Sci Ser B 274:755–761

Bell AM, Sih A (2007) Exposure to predation generates personality inthreespined sticklebacks (Gasterosteus aculeatus). Ecol Lett10:828–834

Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate:a practical and powerful approach to multiple testing. J Roy StatSoc B 57:289–300

Boal JG, Hylton RA, Gonzalez SA, Hanlon RT (1999) Effects ofcrowding on the social behavior of cuttlefish (Sepia officinalis).Contemp Top Lab Anim Sci 38:49–55

Both C, Dingemanse NJ, Drent PJ, Tinbergen JM (2005) Pairs ofextreme avian personalities have highest reproductive success. JAnim Ecol 74:667–674

Boyle PR, Boletzky Sv (1996) Cephalopod populations: definitionand dynamics. Philos T Roy Soc B 351:985–1002

Brydges NM, Colegrave N, Heathcote RJP, Braithwaite VA (2008)Habitat stability and predation pressure affect temperamentbehaviours in populations of three-spined sticklebacks. J AnimEcol 77:229–235

Cheverud JM (1984) Quantitative genetics and developmentalconstraints on evolution by natural selection. J Theor Biol110:155–172

Coleman K, Wilson DS (1998) Shyness and boldness in pumpkinseedsunfish: individual differences are context-specific. Anim Behav56:927–936

Dall SRX, Houston AI, McNamara JM (2004) The behaviouralecology of personality: consistent individual differences from anadaptive perspective. Ecol Lett 7:734–739

Dingemanse NJ, Réale D (2005) Natural selection and animalpersonality. Behaviour 142:1159–1184

Dingemanse NJ, Wright J, Kazem AJN, Thomas DK, Hickling R,Dawnay N (2007) Behavioural syndromes differ predictablybetween 12 populations of three-spined stickleback. J Anim Ecol76:1128–1138

Duckworth RA (2006) Behavioral correlations across breedingcontexts provide a mechanism for a cost of aggression. BehavEcol 17:1011–1019

Endler JA (1986) Natural selection in the wild. Princeton UniversityPress, Princeton

Epstein S (1983) Aggregation and beyond: some basic issues on theprediction of behavior. J Personal 51:360–392

Fleeson W (2004) Moving personality beyond the person-situationdebate. Curr Dir Psychol Sci 13:83–87

Gosling SD (2001) From mice to men: what can we learn aboutpersonality from animal research? Psychol Bull 127:45–86

Hanlon RT (1996) Evolutionary games that squids play: fighting,courting, sneaking, and mating behaviors used for sexualselection in Loligo pealei. Biol Bull 191:309–310

Heithaus MR, Frid A, Wirsing AJ, Dill LM, Fourqurean JW,Burkholder D, Thomson J, Bejder L (2007) State-dependentrisk-taking by green sea turtles mediates top-down effects of tigershark intimidation in a marine ecosystem. J Anim Ecol 76:837–844

Huntingford FA (1976) The relationship between anti-predatorbehaviour and aggression among conspecifics in the three-spined stickleback. Anim Behav 24:245–260

Huntingford FA (1982) Do inter- and intraspecific aggression vary inrelation to predation pressure in sticklebacks? Anim Behav30:909–916

Hutchinson JMC, Gigerenzer G (2005) Simple heuristics and rules ofthumb: where psychologists and behavioural biologists mightmeet. Behav Processes 69:97–124

Johnson JC, Sih A (2005) Precopulatory sexual cannibalism in fishingspiders (Dolomedes triton): a role for behavioural syndromes.Behav Ecol Sociobiol 58:390–396

Kimbell JR, McFall-Ngai MJ, Roderick GK (2002) Two geneticallydistinct populations of bobtail squid, Euprymna scolopes, existon the island of O'ahu. Pac Sci 56:347–355

Lande R, Arnold SJ (1983) The measurement of selection oncorrelated characters. Evolution 37:1210–1226

Magnhagen C (2007) Social influence on the correlation betweenbehaviours in young-of-the-year perch. Behav Ecol Sociobiol61:525–531

Maxwell MR, Hanlon RT (2000) Female reproductive output in thesquid Loligo pealeii: multiple egg clutches and implications for aspawning strategy. Mar Ecol Prog Ser 199:159–170

McNamara JM, Gasson CE, Houston AI (1999) Incorporating rulesfor responding into evolutionary games. Nature 401:368–371

Messenger JB (1977) Prey-capture and learning in the cuttlefish,Sepia. Symp Zool Soc Lond 38:347–376

Mougeot F, Redpath SM, Leckie F, Hudson PJ (2003) The effect ofaggressiveness on the population dynamics of a territorial bird.Nature 421:737–739

Naud MJ, Hanlon RT, Hall KC, Shaw PW, Havenhand JN (2004)Behavioral and genetic assessment of reproductive success in aspawning aggregation of the Australian giant cuttlefish, Sepiaapama. Anim Behav 67:1043–1050

Norman M, Reid A (2000) A guide to squid, cuttlefish, and octopusesof Australasia. The Gould League and CSIRO Publishing,Collingwood

Packard A (1972) Cephalopods and fish: the limits of convergence.Biol Rev 47:241–307

Quinn GP, Keough MJ (2002) Experimental design and data analysisfor biologists. Cambridge University Press, Cambridge

Rands SA, Cowlishaw G, Pettifor RA, Rowcliffe JM, Johnstone RA(2003) Spontaneous emergence of leaders and followers inforaging pairs. Nature 423:432–434

Rankin DJ, Kokko H (2007) Do males matter? The role of males inpopulation dynamics. Oikos 116:335–348

Réale D, Festa-Bianchet M (2003) Predator-induced naturalselection on temperament in bighorn ewes. Anim Behav65:463–470

Réale D, Reader SM, Sol D, Mcdougall PT, Dingemanse NJ (2007)Integrating animal temperament within ecology and evolution.Biol Rev 82:1–28

Sih A, Watters JV (2005) The mix matters: behavioural types andgroup dynamics in water striders. Behaviour 142:1417–1431

Sih A, Bell A, Johnson JC (2004a) Behavioral syndromes: anecological and evolutionary overview. Trends Ecol Evol19:373–378

Sih A, Bell AM, Johnson JC, Ziemba RE (2004b) Behavioralsyndromes: an integrative overview. Q Rev Biol 79:242–277

Sinn DL, Moltschaniwskyj NA (2005) Personality traits in dumplingsquid (Euprymna tasmanica): context-specific traits and theircorrelation with biological characteristics. J Comp Psychol119:99–110

Sinn DL, Apiolaza LA, Moltschaniwskyj NA (2006) Heritability andfitness-related consequences of squid personality traits. J EvolBiol 19:1437–1447

Sinn DL, Gosling SD, Moltschaniwskyj NA (2008) Development ofshy/bold behaviour in squid: context-specific phenotypes associatedwith developmental plasticity. Anim Behav 75:433–442

Stamps JA (2007) Growth-mortality tradeoffs and ‘personality traits’in animals. Ecol Lett 10:355–363

Stapley J, Keogh JS (2005) Behavioral syndromes influence matingsystems: floater pairs of a lizard have heavier offspring. BehavEcol 16:514–520

Behav Ecol Sociobiol (2010) 64:693–702 701 Author's personal copy

Tabachnick BG, Fidell LS (1996) Using multivariate statistics, 3rdedn. HarperCollins, New York

van Oers K, Klunder M, Drent PJ (2005) Context dependence ofpersonalities: risk-taking behavior in a social and a nonsocialsituation. Behav Ecol 16:716–723

West-Eberhard MJ (1979) Sexual selection, social competition, andevolution. Proc Am Philos Soc 123:222–234

Wilson DS (1998) Adaptive individual differences within singlepopulations. Philos T Roy Soc B 353:199–205

Wilson DS, Clark AB, Coleman K, Dearstyne T (1994) Shyness andboldness in humans and other animals. Trends Ecol Evol 9:442–446

Wolf M, Sander van Doorn S, Leimar O, Weissing FJ (2007) Life-history trade-offs favour the evolution of animal personalities.Nature 447:581–585

702 Behav Ecol Sociobiol (2010) 64:693–702 Author's personal copy