Morphological variability and developmentalinstability in subpopulations of the Eurasianbadger (Meles meles) in DenmarkCino Pertoldi1,2*, Lars A. Bach2, Aksel Bo Madsen1, Ettore Randi3 and Volker Loeschcke2

1Department of Landscape Ecology, National Environmental Research Institute, Kalø

Grenavej 14, DK-8410 Rønde, Denmark, 2Department of Ecology and Genetics, University

of Aarhus, Aarhus C, Denmark and 3Istituto Nazionale per la Fauna Selvatica, Ozzano

Emilia (Bo), Italy

Abstract

Aim Local populations from different geographical regions may differ in the selectionregimes to which they are exposed. Differences in environmental factors and populationdensity may affect the relative importance of different selective forces (e.g. natural vs.sexual selection). We suggest a direction of investigation concerned with the develop-mental instability of morphological traits. The goal is to disclose putative small-scalegeographical differences in the evolutionary forces, which may be hard to detect.

Location Craniometrical investigations were carried out on ninety-eight skulls andteeth of the Eurasian badger (Meles meles) collected during the period 1995–97 fromthree different populations in Denmark. One of these thrives at low population density,whereas the two others are characterized by high local density.

Methods The skulls were investigated for developmental instability (DI) using fluctu-ating asymmetry (FA) as its estimator. FA was measured on canines, molars, premolarteeth and other skull and mandible traits. For the statistical analyses, we applied non-parametric permutation tests.

Results Evidence was found suggesting differentiation among populations in meandegree of FA, and the FA values measured on canines were higher in the high-densitypopulations. FA of the canines was significantly higher in males than females, in contrastto FA of the other traits. Evidence of a negative relationship between canine size andtheir FA was found, whereas no significant correlations were found between the molarand premolar teeth measures and their FA.

Main conclusions Our results suggest that canines could be under directional selectionstemming from intrasexual competition, which may be stronger in high-density zones.The other teeth investigated seem to be under a stabilizing regime hence their FA ismainly affected by environmental stresses. The negative relationship between canine sizeand FA found in males suggests the capacity of badgers to respond in an evolutionaryway to environmental changes, despite the low genetic variability previously found at themolecular level.

Keywords

Fluctuating asymmetry, developmental instability, badger, sexual selection, populationdensity.

*Correspondence: Cino Pertoldi, Department of Landscape Ecology, National Environmental Research Institute, Kalø Grenavej 14, DK-8410 Rønde, Denmark.

E-mail: cpb@dmu.dk

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INTRODUCTION

Estimates based on the Danish badger game bag records,ranging from 1941 to 1977, show that the badger populationsize has decreased more than 50% since the 1950s(Strandgaard & Asferg, 1980).

The geographical distribution of the Danish game bagrecord presumably reflects differences in population densityin the different regions, at least to a degree where we canqualitatively consider populations as maintaining high orlow densities. Previous studies (Forchhammer et al., 1998;Stenseth et al., 1998; Cattadori et al., 1999; Forchhammer& Asferg, 2000) have found hunting statistics to be a goodapproximation of actual population abundances.

Three distinct zones emerged from genetic investigations ofvariation in allozymes (Pertoldi et al., 2000), minisatellites(Pertoldi et al., 2002) and microsatellites (Bijlsma et al.,2000; C. Pertoldi, A.B. Madsen, M.M. Baagoe, R. Bijlsma, L.van de Zande, M.M. Hansen, E. Randi & V. Loeschcke, pers.comm.) conducted on badger populations collected between1995 and 1997. Hence such substructure remains the basis ofthis study. These genetic investigations revealed a relativelylow level of genetic variability in the Danish badgers andevidence of genetic differentiation among the populations inthe three zones. A significantly lower genetic variability wasfound in zone 3 as compared with zones 1 and 2 (Pertoldiet al., 2000, 2002; Pertoldi et al., in prep.), which might bedue to the lower population density in this zone.

Two of the three zones considered (zones 1 and 2) aredensely inhabited by badgers, which is believed to be a resultof the high amount of suitable habitat. A mean annual bag of(mean � SE) 0.036 � 0.002 badgers km)2 for zone 1,0.037 � 0.002 badgers km)2 for zone 2 and 0.012 �0.0018 badgers km)2 for zone 3 were calculated averagingthe Danish game bag in the period 1973–93. The relativelylow SE of the mean annual bag of the badgers seems toreflect the lack of temporal fluctuation, which in turn indi-cates relatively stable population dynamics (data from theDanish badger bag records are available from the first authoron request). After 1993 no additional bag records areavailable as the badgers were protected from hunting;however, we assumed that the badger population density hasnot changed considerably in the last 10 years as no relevanthabitat changes (e.g. the ratio between rural area and forest)in the three study zones have occurred since (data fromDanmark Statistik: http://www.dst.dk).

Fluctuations from year to year have also been suggested tobe <15% for badgers and do not appear to coincide in dif-ferent parts of the country (Strandgaard & Asferg, 1980).The reasons for the relatively low badger density in zone 3 isbelieved to be related not only to habitat, intense agriculturalpractice and low forest coverage, but also to the fox controlcampaigns following the outbreak of rabies conducted atdifferent periods in this zone (Strandgaard & Asferg, 1980).

The Eurasian badger is unique among the Mustelidae inthat adults form large, territorial and highly stable socialgroups (Kruuk, 1978), and show an unusually high degree ofnatal philopatry (Kruuk & Parish, 1982; Cheeseman et al.,

1987). Hence, mating structure and social organization ofbadgers are believed to strongly limit the gene flow betweengeographical regions. Furthermore, there is evidence that thefrequency of male dispersal declines at high populationdensities (Woodroffe et al., 1993). The limited gene flowamong geographical regions is also reflected by the geneticdifferentiation found when conducting genetic investigationsby means of different molecular markers (Pertoldi et al.,2000, 2002; Pertoldi et al., in prep.), which suggests anextremely limited contact among the zones considered in thisinvestigation.

During ontogeny individuals are exposed to more or lessstressful conditions from the surrounding environment. Theability to withstand such stressful conditions may be esti-mated by measures of developmental instability (DI) (Palmer& Strobeck, 1986; Parsons, 1992). The most commonlyused estimate of DI is fluctuating asymmetry (FA), which canbe defined as random deviations from perfect symmetry in abilaterally symmetrical trait (Palmer & Strobeck, 1986). Yet,empirical studies supporting its general utility for monitoringendangered species or populations are largely lacking orcontradictory (e.g. Fowler & Whitlock, 1994). The theore-tical argument that stressed individuals should have high DIand, hence, higher FA is supported by positive relationshipsbetween FA and environmental stressors such as pollutionand genetic stressors such as inbreeding, although somestudies have indicated that these relationships might not beubiquitous (Møller & Swaddle, 1997 and references there-in). In relation to conservation efforts, organisms withenhanced levels of FA may provide sensitive monitors ofsuch stresses before detrimental impacts occur in the popu-lation (�the early warning system�, Clarke, 1995).

Several authors have focused on traits, which are pre-sumed to be under sexual selection, as for example thecanine teeth. Intrasexual competition has been used as anexplanation of sexual dimorphism in canine size in somespecies of cervids (Ralls et al., 1975) and in primates (Har-vey et al., 1988). If larger canines signal good general healthconditions, canine size will be negatively correlated with FA(see reviews in Møller & Pomiankowski, 1993; Møller &Swaddle, 1997). Therefore, measures of canines were alsoincluded in our investigation, which are the only dimorphicteeth in badgers (Wiig, 1986; Lups & Roper, 1988) andhence suspected to be influenced by sexual selection. Sexu-ally selected traits usually appear to show high genetic var-iation, and it is thought that they reflect a history of strongdirectional selection, which has led to the loss of develop-mental canalization and to the evolution of strong condition-dependent expression (Bjorksten et al., 2000).

Tooth DI is particularly suitable for studying the conse-quences of severe stress (Manning & Chamberlain, 1993;Badyaev, 1998 and references therein). In fact, in black bearsdevelopment of bunodont teeth occurs over a short period oftime and is strongly influenced by diet, prenatal exposure tostressors, disease, and consanguinity (Manville, 1992).

The aim of this study is to examine the variation andspatial pattern of FA in the three badger populationsand hence assess the possible role of FA as a measure of

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environmental stress. Despite the potential use of measuresof DI in connection to conservation (Leary & Allendorf,1989; Clarke, 1995), few studies have examined the pat-terns of FA of endangered species in relation to environ-mental or genetic stress. The detection of significantdifferences in the levels of FA among populations wouldsuggest the presence of ecological forces producing suchpatterns. Therefore, we expect to observe concordancebetween FA and the specific conditions of the local popu-lations. Several traits were chosen for investigation as pre-vious studies have shown that traits under different selectiveregimes show different responses to environmental stressors(Møller & Pomiankowski, 1993).

We also expect that FA in canine teeth increases in areas ofhigh density because of higher male–male competition,whereas we do not expect to observe this patterns in femalebadgers for which the intrasexual competition is relatively va-gue as compared with males (Kruuk, 1978; Roper et al., 1986).

Furthermore, we expect higher FA in both sexes in thelow-density area where the badgers are exposed to lessfavourable habitat conditions.

MATERIALS AND METHODS

Skull collection

We utilized for our investigation ninety-eight skulls of bad-gers collected between 1995 and 1997, stored at the DanishNational Environmental Research Institute Kalø (DMU).For every specimen we know the exact finding place with anapproximation of 10 km2. Most of the collected skulls stemfrom traffic killed badgers, and some are damaged. Nomeasurements were attempted on broken or worn parts of

the skull and the teeth; therefore, we have missing values (theworn teeth represented <5% of the material analysed). Weconsidered skulls collected from three distinct geographicalzones (Fig. 1). Because of the genetic differentiation foundamong the badgers collected in these three geographicalzones (see Introduction), we considered the badgers living inthe three zones as members of three distinct populationswith very limited contact: population 1 (n ¼ 28 skulls),population 2 (n ¼ 42) and population 3 (n ¼ 28). In orderto exclude the possibility of performing the analysis based ona non-uniform sampling within populations, we subdividedeach of the zones of collection in eight squares of20 · 20 km (none of the squares were devoid of badgers). Asubsequent G-test of the number of samples within eachsquares indicates a uniform sampling within zones, as theintra-zone variances in all the three zones were significantlylower than the mean (Bishop, 1983).

Measurement error, test for normality of FA and for

directional asymmetry

The badgers were sexed and we measured eight tooth traitsand five skull traits on both sides of the skull with a dialcalliper to the accuracy of 0.01 mm. The tooth measure-ments were made only on fully erupted teeth (see Table 1 forabbreviations of the traits). For the investigation of FA weconsidered three indices: FA1, FA4 and FA5 (Palmer &Strobeck, 1986). FA1 is the absolute value of asymmetry(absolute FA), FA4 is the variance of (r ) l) and FA5 is theP

(r ) l)2. As the absolute values of FA are half-normallydistributed and because our collection is of limited size wedecided to use nonparametric statistics and resample statis-tics for our investigation (Davison & Hinkley, 1997). Aresample programme (made available by the authors onrequest) was designed in order to compare the data samples.The resample tests were conducted as Monte Carlo stylepermutation tests with replacement (10,000 iterations).Because of the large number of tests, which we have per-formed in our investigation, a sequential Bonferroni test(Rice, 1989) was applied to all the nonparametric tests toavoid significant results arising as a consequence of a largenumber of related tests. Following Miller’s (1981) sugges-tions, we made a separate probability statement for the testsof the two sexes and of the three populations.

We measured each trait five times and the median of thefive measures was chosen. A two-way ANOVA was conductedon thirty individuals whose trait medians were measuredtwice, to test for the significance of FA relative to measure-ment error and for detecting the presence of directionalasymmetry (DA) (following Palmer, 1994). The interaction�mean square� (MS) containing information about FA wastested against error MS (reflecting measurement error)showing that FA was significantly larger than measurementerrors in all cases (6.28 £ interaction MS £ 81.32, 0.00 <error MS £ 0.07, 95 £ d.f. ¼ 29, P < 0.001).

The (r ) l) distributions were tested for departure fromnormality with a Lilliefors test and for presence of DA with aone-sample t-test (Sokal & Rohlf, 1981). No significantFigure 1 Map of Denmark with the three zones of collection.

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Developmental instability in Eurasian badgers 951

deviation of the mean of the trait (l ) r) distributionswere found (one-sample t-test males: 0.063 < P < 0.88,22 < n < 43, females: 0.051 < P < 0.863, 29 < n < 47),

and no significant departure from normality (Lillieforstest, males: 0.058 < P < 0.23, 22 < n < 43, females:0.061 < P < 0.12, 29 < n < 47). In conducting the

Table 1 Resampling test of differences between variance in the two sexes of (r ) l): FA4, index of width, length and height of the traits

measured on the badgers� skull from populations (Pop.) 1, 2 and 3 (for description of the measured traits see Lups & Roper, 1988; Pertoldi et al.,1997; Pertoldi et al., 2000)

Pop. trait Abbreviations

Males

(n), var(r ) l)Females

(n), var(r ) l)P (FA4) (resample

statistics)

Upper canines

1 uCw (upper canine width) (15), 0.27 (10), 0.002 (m > f)***2 uCw (15), 0.402 (25), 0.012 (m > f)***

3 uCw (13), 0.062 (12), 0.002 (m > f)***

1 uCl (upper canine length) (15), 0.789 (10), 0.101 (m > f)***2 UCL (15), 0.069 (25), 0.054 (m > f)*

3 UCL (13), 0.032 (11), 0.004 (m > f)***

Mandibular canines

1 mCw (mandible canine width) (13), 0.006 (9), 1.07 · 10)4 (m > f)***2 mCw (13), 0.095 (22), 3.23 · 10)5 (m > f)***

3 mCw (10), 0.025 (10), 0.001 (m > f)***

1 mCl (mandible canine length) (13), 0.27 (9), 4.44 · 10)5 (m > f)***

2 mCl (13), 0.043 (22), 1.82 · 10)5 (m > f)***3 mCl (10), 0.03 (9), 0.1 (f > m)*

Upper molars

1 uM1l (upper molar 1 length) (13), 7.7 · 10)5 (8), 5 · 10)5 (m > f), n.s.2 uM1l (12), 1.97 · 10)4 (18), 0.001 (f > m)***

3 uM1l (5), 0.16 (10), 4.1 · 10)4 (m > f)***

Mandibular molars

1 mM1l (mandible molar 1 length) (10), 4 · 10)5 (8), 2.99 · 10)4 (f > m)***2 mM1l (11), 3.64 · 10)5 (19), 1.29 · 10)4 (f > m)**

3 mM1l (5), 0.002 (10), 1.57 · 10)4 (m > f)***

1 mM2l (mandible molar 2 length) (7), 0.001 (8), 2.2 · 10)4 (m > f)**

2 mM2l (9), 2.36 · 10)4 (12), 0.001 (f > m)*3 mM2l (5), 0.002 (10), 3.29 · 10)4 (m > f)**

Mandibular premolars

1 mP4l (mandible premolar 4 length) (13), 1.94 · 10)4 (8), 2.27 · 10)4 (f > m), n.s.

2 mP4l (11), 2 · 10)5 (18), 9.28 · 10)5 (f > m)**3 mP4l (5), 0.002 (10), 2.5 · 10)4 (m > f)**

Skull traits

1 A (distance between opistokranionand zygomatic process of frontal bone)

(8), 0.059 (7), 0.172 (f > m), n.s.

2 A (12), 0.05 (18), 0.008 (m > f)***

3 A (6), 0.282 (8), 1.12 · 10)4 (m > f)***

1 MOh (Max. inner orbital height) (12), 0.037 (6), 0.004 (m > f)*2 MOh (12), 0.005 (16), 0.053 (f > m)***

3 MOh (4), 0.052 (8), 0.023 (m > f), n.s.

Mandibular traits

1 mTl (mandible total length:infradentale – processus condylis)

(11), 0.206 (6), 0.402 (f > m), n.s.

2 mTl (10), 0.142 (14), 0.074 (m > f), n.s.

3 mTl (5), 0.435 (9), 0.084 (m > f), n.s.1 mUh (mandible upper height) (14), 0.001 (7), 0.002 (f > m), n.s.

2 mUh (12), 0.001 (19), 0.001 (m ¼ f), n.s.

3 mUh (6), 0.411 (11), 0.001 (m > f)***

1 mAl (mandible angular length:infradentale processus – angularis)

(10), 0.269 (7), 0.416 (m > f), n.s.

2 mAl (7), 0.101 (14), 0.003 (m > f)***

3 mAl (5), 0.633 (8), 0.322 (m > f), n.s.

*P < 0.05, **P < 0.01, ***P < 0.001; n.s., non-significant. u, upper; m, mandibular; w, width; l, length; h, height.

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Lilliefors test and the one-sample t-test, we pooled the threepopulations together (in order to obtain a sufficiently largesample size), but we considered the two sexes separately. Wedid not check for differences in FA of the skull traits betweenfully-grown (adults) and immature (subadults) skulls,because of the limited sample size for this comparison.However, the proportion of adults and subadults wasequally distributed over the three populations and the twosexes (results not shown).

Differences of FA between sexes

All the tests for significance made on skull traits and teethtraits for differences in FA between sexes, were made com-paring FA4 indexes with a resample statistics, wherehomogeneity of variances between two samples was tested.For that purpose we tested the individuals from the threepopulations separately.

FA differences among populations

A Kruskal–Wallis one-way nonparametric analysis of vari-ance (FA of traits-population) was made, in order to detectdifferences of the degree of FA (FA1 index) among popula-tions. A resample test (testing for pairwise differencesbetween means) were performed in order to check forsignificant differences between FA1 and FA5 values.

Dependency of FA on the size of teeth traits

A Spearman rank test (Zar, 1984) and a resample test (testingsignificance of the correlation) were utilized to assess if FA1of the teeth is correlated with the teeth trait length.

RESULTS

Differences of FA between sexes

There was evidence for a significantly higher FA of malecanines as compared with FA of female canines in all threepopulations (Table 1).

Significant differences of FA (FA4 index) values betweensexes of the skulls traits and molars and premolars werefound (resample statistic), in all three populations, but theresults did not show a consistent pattern (Table 1).

FA differences among populations

There was some evidence for significant differences of can-ine’s FA1 among the three populations ( Kruskal–Wallis test)(Table 2). The resample tests for pairwise comparisons ofFA1 and FA5 were significant only for males, and revealed ahigher FA of the canines of badgers collected in population 1and 2 (high-density area) as compared with population 3(low-density area), and of population 1 as compared with 2(see Table 2).

For the remaining teeth and skull traits, strong evidence ofsignificant differences of FA among the three populations

was also found for males (Kruskal–Wallis test), whereas,weaker evidence was found for females (see Table 2). Theresample tests (testing for pairwise differences of FA1 andFA5) were significant for several traits for males, andrevealed a higher FA (FA1 and FA5) of the skull traits ofbadgers collected in population 3 (low-density area) ascompared with traits� FA of the skulls of badgers collected inpopulation 1 and 2 (high-density area) (see Table 2). Forfemales, we found only two significant differentiation of FAamong populations (trait mAL) (see Table 2).

Dependency of FA on the size of the traits

Some significant negative correlations were found betweenmales� canines length and width and their degree of FA in allthe three populations, whereas, for females some significantpositive and negative correlations were found in populations1 and 2 (Table 3). The two tests performed: Pearson–Spearman test and resample tests (testing for significance ofcorrelation) were not always all significant or were notalways significant at the same level (Table 3).

No significant correlations were found between molar andpremolar lengths and their FA, performing Spearman ranktest and resample statistic (testing for significance of corre-lation) (Table 3).

DISCUSSION

FA differences between sexes

The strong evidence of higher FA in male canines comparedwith female canines could suggest that sexual selection af-fects the sexes differently in these populations. Such findingsare in accordance with previous studies suggesting that malecanines of several mammals are affected by sexual selection(Dayan & Simberloff, 1994). Furthermore, from Table 1, itis clear that FA in canines is considerably higher than the FAof the other teeth. This finding is in accordance with Møller& Pomiankowski (1993), who suggest that sexually selectedtraits are directionally selected and therefore show higherlevels of FA than traits exposed to a stabilizing selectionregime.

The suspect that males� canines are under sexual selectionare further supported by evidence of a negative relationshipbetween FA and canine length and width in male badgers(see Table 3). The skull traits show an FA comparable withthe canines, but their FA values may be associated with aweaker stabilizing selection regime. The biomechanical costof asymmetry of a skull trait with a low functionality is lowcompared with, for example, the cost of an asymmetricalmolar, which shows a relatively low FA. As mentionedearlier, the most conspicuously dimorphic teeth in badgersare typically the canines (Wiig, 1986; Lups & Roper, 1988),which are significantly larger in males. No sex differencesexist in the amount or type of food consumed by adultbadgers as determined from stomach content (Lups &Roper, 1988). It seems unlikely that the canines are fre-quently used in capturing or despatching prey as badgers

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Developmental instability in Eurasian badgers 953

Table 2 Kruskal–Wallis test for comparing the absolute mean value of FA (FA1) of the length and width of upper and mandibolar canine (UCW, UCL, MCW and MCL) of male and

female badgers in populations (Pop.) 1, 2 and 3. {R(FA1)} and {R(FA5)} are the resampling test for pairwise comparisons

Traits Abbreviations Sex

Pop. 1

(n), mean � SD

Pop. 2

(n), mean � SD

Pop. 3

(n), mean � SD

K–W test

(H-value) P {R(FA1)} {R(FA5)}

Upper canines

uCw m (15), 0.68 � 0.469 (15), 0.581 � 0.632 (13), 0.069 � 0.25 4.959 *** {Pop. 1 > Pop. 2}*,

{Pop. 1 > Pop. 3}***,

{Pop. 2 > Pop. 3}**

{Pop. 1 > Pop. 2}*,

{Pop. 1 > Pop. 3}**,

{Pop. 2 > Pop. 3}**uCl m (15), 0.589 � 0.657 (15), 0.156 � 0.247 (13), 0.089 � 0.154 3.625 n.s. {Pop. 1 > Pop. 2}* {Pop. 1 > Pop. 2}*

Mandibular

canines

mCw m (13), 0.065 � 0.075 (13), 0.145 � 0.289 (10), 0.093 � 0.159 0.737 n.s.mCl m (13), 0.345 � 0.376 (13), 0.104 � 0.185 (10), 0.11 � 0.173 6.964 *� {Pop. 1 > Pop. 3}** {Pop. 1 > Pop. 3}***

Upper molars

uM1l m (13), 0.01 � 0.001 (12), 0.008 � 0.014 (5), 0.296 � 0.4 14.962 *** {Pop. 3 > Pop. 1}*** {Pop. 3 > Pop. 1}***Mandibular

molars

mM1l m (10), 0.018 � 0.006 (11), 0.002 � 0.006 (5), 0.1 � 0.045 16.669 *** {Pop. 3 > Pop. 1}*** {Pop. 3 > Pop. 1}**

mM2l m (7), 0.016 � 0.017 (9), 0.008 � 0.013 (5), 0.068 � 0.027 10.287 ** {Pop. 3 > Pop. 1}*** {Pop. 3 > Pop. 1}***,{Pop. 3 > Pop. 2}*

Mandibular

premolars

mP4l m (13), 0.012 � 0.07 (11), 0.02 � 0.004 (5), 0.092 � 0.04 19.346 *** {Pop. 3 > Pop. 1}* {Pop. 3 > Pop. 1}*,{Pop. 3 > Pop. 2}*

Skull traits

A m (8), 0.15 � 0.238 (12), 0.109 � 0.208 (6), 0.462 � 0.23 7.815 *� {Pop. 3 > Pop. 1}*** {Pop. 3 > Pop. 1}***,,{Pop. 3 > Pop. 2}**

MOh m (12), 0.131 � 0.14 (12), 0.085 � 0.052 (4), 0.358 � 0.228 3.4 n.s. {Pop. 3 > Pop. 1}** {Pop. 3 > Pop. 1}*,

{Pop. 3 > Pop. 2}*

Mandibulartraits

mTl m (11), 0.211 � 0.41 (10), 0.222 � 0.296 (5), 0.598 � 0.29 7.32 *�mUh m (14), 0.026 � 0.02 (12), 0.023 � 0.021 (6), 0.48 � 0.369 11.376 **� {Pop. 3 > Pop. 1}*** {Pop. 3 > Pop. 1}**

mAl m (10), 0.289 � 0.424 (7), 0.202 � 0.255 (5), 0.608 � 0.427 4.063 n.s.Upper

canines

uCw f (10), 0.025 � 0.049 (25), 0.036 � 0.109 (12), 0.031 � 0.036 2.458 n.s.

uCl f (10), 0.109 � 0.313 (25), 0.093 � 0.223 (11), 0.046 � 0.061 1.437 n.s.Mandibular

canines

mCw f (9), 0.038 � 0.01 (22), 0.043 � 0.006 (10), 0.039 � 0.014 3.172 n.s.mCl f (9), 0.002 � 0.007 (22), 0.01 � 0.004 (9), 0.122 � 0.308 8.411 *�

Upper molars

uM1l f (8), 0.01 � 0,01 (18), 0.019 � 0.031 (10), 0.015 � 0.013 1.66 n.s.

Mandibular molarsmM1l f (8), 0.016 � 0.012 (18), 0.007 � 0.01 (10), 0.015 � 0.01 6.162 *�mM2l f (8), 0.01 � 0.013 (12), 0.015 � 0.02 (10), 0.018 � 0.018 0.922 n.s.

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feed mainly on invertebrates and on small items of plantmaterial (Kruuk, 1978).

Therefore, an alternative explanation is that the canineshave evolved at least partly in conjunction with aggressivebehaviour. Badgers are strongly territorial and fierce fightsbetween the members of adjacent social groups occur atterritory boundaries (Kruuk, 1978; Roper et al., 1986). Theterritorial behaviour is less pronounced in low-density areas(Cresswell & Harris, 1988). Territorial fights between malebadgers are common. Badgers are known to bite each other,for example, around the neck, and wounding can be severeand occasionally fatal (Gallagher & Nelson, 1979). Femalebadgers are also very aggressive with other specimens of thesame sex when defending their litters (Kruuk, 1989;Woodroffe et al., 1993; Woodroffe & Macdonald, 1995).However, data on bite wounding show that fighting amongmales is more common as compared with females (Lups &Roper, 1988), suggesting that male competition is higher.

The lack of a clear pattern of relationship between femalesFA and their canine size (see Table 3) could be explained bya weaker intensity of intrasexual competition betweenfemales as compared with males.

The lack of any significant correlations between FA andmolar and premolar length (see Table 3) could point to thepossibility that a second-order polynomial regression will fitthe relationship, creating the typical U-shaped relationshipfound in traits under stabilizing selection (Møller & Pomi-ankowski, 1993). Unfortunately, we could not test thisrelationship because of the too small sample size (seeTable 3).

FA differences among populations

The evidence for a higher FA in canines in populations 1 and 2compared with population 3 (low-density area) can be inter-preted as the outcome of a stronger intrasexual competitionoccurring in populations 1 and 2, as a result of more intenseterritorial behaviour in the densely inhabited zones. The dif-ferences of FA found between populations 1 and 2 couldreflect differences in the degree of intrasexual competitionalthough the Danish game bag record indicates that the twopopulations have approximately the same population density.However, this result may be a reflection of different currentpopulation densities not reflected in the historical bag record.Skull traits, molars and premolars showed the opposite pat-tern compared with canines. A generally higher FA was foundin population 3 as compared with population 1. This resultwas not unexpected, as high levels of FA are typically associ-ated with disturbed environments (Parsons, 1992). We do notthink that increased FA in the most disturbed population mayhave resulted from inbreeding depression (as a consequence ofsmall population sizes). Given the natural inbreeding levelfound in badgers, strong negative effects of consanguinity andnegative effects of low genetic variability seem less likely. Infact, inbreeding is usually deleterious in species that normallyoutbreed, whereas, when inbreeding is part of the naturalsocial system of a species, inbreeding depression is far lesssevere, and the genetic load is usually low (Soule, 1987 andT

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Pop.

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;h,

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� 2003 Blackwell Publishing Ltd, Journal of Biogeography, 30, 949–958

Developmental instability in Eurasian badgers 955

references therein). Alternatively, the high levels of FAobserved in population 3 seems more likely to stem fromvarious sources of environmental stress. Models of badgersocial organization suggest that if carrying capacity is reachedor reduced, different level of competition, following a gradientbetween scramble and contest competition in which domin-ance will be an advantage (Revilla & Palomares 2001). Accessto reproduction is asymmetric among group members,determining social status (Woodroffe & Macdonald, 1995).Under optimal conditions, competition for trophic resourcesis thought to be negligible within groups (Woodroffe et al.,1993). If the territory is equally accessible to all individuals,there should be no asymmetry in access to the key trophicresource and, hence, all individuals would be expected tosuffer equally from food stress (scramble competition). Incontrast, if badgers are unequal competitors, with a domin-ance hierarchy in access not only to reproduction but also tofood when it is limiting, dominant animals should preferen-tially use the key habitat that contains the resource (contestcompetition) (Revilla & Palomares 2001). Therefore, thedifferent population density can create different degrees ofstress acting on the different populations and in fact suchstressors can act with different intensities on the individualscomposing the populations depending on the density.

CONCLUSIONS

We found indications suggesting that DI of canines and ofthe remaining skull and teeth traits are associated with themode of selection, as they showed different patterns ofrelationship between FA and the trait sizes. Our resultssupport the hypothesis that population-level FA estimation

can be a very sensitive indicator of environmental stress.This hypothesis is relatively accepted and supported byseveral investigations (see Møller & Swaddle, 1997 andreferences therein). It is also generally accepted that direc-tional selection may select against strict developmentalcontrol (Møller & Pomiankowski, 1993) thereby makingsexually selected traits more responsive to resource avail-ability and allocation during growth compared with non-sexually selected traits (Watson & Thornhill, 1994).Whereas traits subject to long-term stabilizing selectionwould demonstrate the opposite effect, restricting the effectof environmental and genetic factors on the expression of thegenotype (Pomiankowski & Møller, 1995). Our findings arehowever, in contrast with several investigations in which anincreased FA of sexually selected traits have been found to bepositively associated with increasing environmental stressand a consequent reduction of the population size (seeBadyaev, 1998 and references therein). In fact, we foundhigher FA in sexually selected traits in the badger popula-tions living at higher density (where the environmental stressis presumed to be lower as compared with the populationliving at lower density).

The observed pattern could be due to the fact that sexuallyselected traits are more responsive to variation in environ-mental conditions, as compared with traits, which are understabilizing regimes. However, this property can be obscuredif the selective pressure on canines overwhelms the effect ofenvironmental disturbance. The environmental disturbanceseems also to overwhelm other stressors. For example, socialstresses that are acting more intensely in the densely inhab-ited zones, as compared with the low-density zone. Socialstress is a form of stress not linked to fighting, but is rather

Table 3 Spearman rank correlation analysis for the correlation between FA1 and the length of the trait of male and female badgers collected in

populations (Pop.) 1, 2 and 3 (n) is the sample size, [b] is the correlation coeffecient, the asterisks after the [b] indicate the level of significancy ofthe Spearman test, the asterisks in R indicate the level of significancy of the resampling test (testing significance of the correlation)

Traits Abbreviations Sex

Pop. 1

(n), [b], {R}

Pop. 2

(n), [b], {R}

Pop. 3

(n), [b], {R}

Upper canines uCw m [n.s.] {n.s.} [n.s.] {n.s.} [n.s.] {n.s.}

Upper canines uCl m (15), ()0.713]*** {***} [n.s.] {n.s.} (13), [)1.223]*� {***}Mandibular canines mCw m [n.s.] {n.s.} (13), [)2.409]*** {***} [n.s.] {n.s.}

Mandibular canines mCl m (13), [)2.852]*** {***} [n.s.] {n.s.} (10), [)2.543]*� {***}

Upper molars uM1l m (10), [)0.088] n.s. {n.s.} [n.s.] {n.s.} [n.s.] {n.s.}Mandibular molars mM1l m [n.s.] {n.s.} [n.s.] {n.s.} [n.s.] {n.s.}

Mandibular molars mM2l m [n.s.] {n.s.} [n.s.] {n.s.} [n.s.] {n.s.}

Mandibular premolars mP4l m [n.s.] {n.s.} [n.s.] {n.s.} [n.s.] {n.s.}

Upper canines uCw f (10), (0.218] n.s. {**} (25), [0.776]*** {***} [n.s.] {n.s.}Upper canines uCl f (10), [)3.277]*** {***} (25), [)2.17]*** {***} [n.s.] {n.s.}

Mandibular canines mCw f [n.s.] {n.s.} (22), [)0.055] n.s. {n.s.} [n.s.] {n.s.}

Mandibular canines mCl f [n.s.] {n.s.} [n.s.] {n.s.} [n.s.] {n.s.}

Upper molars uM1l f [n.s.] {n.s.} [n.s.] {n.s.} [n.s.] {n.s.}Mandibular molars mM1l f [n.s.] {n.s.} [n.s.] {n.s.} [n.s.] {n.s.}

Mandibular molars mM2l f [n.s.] {n.s.} [n.s.] {n.s.} [n.s.] {n.s.}

Mandibular premolars mP4l f [n.s.] {n.s.} [n.s.] {n.s.} [n.s.] {n.s.}

*P < 0.05; **P < 0.01; ***P < 0.001; n.s., non significant.

�Result no longer significant after sequential Bonferroni correction (K ¼ 8).

(u, upper; m, mandibular; w, width; l, length; h, height)

� 2003 Blackwell Publishing Ltd, Journal of Biogeography, 30, 949–958

956 C. Pertoldi et al.

mediated by hormones or olfactory cues that could restrictforaging areas for low ranking individuals or producereproductive suppression both in males and females(Cheeseman et al., 1987; Woodroffe et al., 1993). Hence,we suspect that that different population densities producedifferent degrees of directional forces, which are translatedinto different degrees of FA, if we consider the FA measuredon canines. FA will in such case be a reliable indicator of thedegree of selection rather than an indicator of environmentaldisturbance. Strong conclusions, however, cannot be drawn,because of the limited sample sizes of our material. How-ever, we cannot exclude the possibility that different levels ofgenetic variation in the different populations could havecontributed to the observed FA patterns (Vøllestad et al.,1999).

In this study, we have seen that the results of the statisticaltests were not necessarily concordant. The reasons mainlystem from the different assumptions on which the tests arebased and from their sensitivity to the violations of theseassumptions. The different statistical power of the FA indi-ces (which means different discriminatory capacity), havesurely contributed to the discrepancy between the resultsobtained testing the different indices (see Palmer & Stro-beck, 1986 for a discussion of the different FA indices dis-criminatory capacity). Therefore, the use of several FAindices is strongly recommended, especially when dealingwith small sample sizes (which is the case in our investiga-tion). In particular the FA5 index has shown in our inves-tigation to have a higher statistical power as compared withother indices. This finding is in accordance with the con-clusions of Palmer & Strobeck (1986) that FA5 is a superiorindex when dealing with small sample sizes. However, themain problem is the limited sample size increasing the pos-sibility of committing a type II error. The possibility of usingstatistical packages, which allow bootstrapping (as the re-sample statistic) can at least limit the problems associatedwith small sample sizes.

The use of the Bonferroni correction can lead us to com-mit error type I. Especially, when measuring several traitsand having small numbers of individuals (which limit thepossibility of obtaining high levels of significance in thetests). Therefore, we suggest the use of the resample statis-tics, which utilize 100% of the information available and isless sensitive towards small sample sizes and deviations fromnormality.

ACKNOWLEDGMENTS

We are grateful to the Danish Natural Science ResearchCouncil for financial support to Cino Pertoldi (grant no.21-01-0526). Furthermore, two anonymous reviewers areacknowledged for invaluable comments on the manuscript.

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BIOSKETCHES

Cino Pertoldi is a Population Geneticist with interest inconservation issues and environmental stress. He iscurrently working as a researcher at the Danish Envi-ronmental Research Institute. He is also affiliated withUniversity of Aarhus and the National Museum ofNatural History, Madrid.

Lars A. Bach is an evolutionary biologist working at theComputer Science Department, University of Aarhuswith interest in adaptive population modelling andspatial population structure.

Aksel Bo Madsen is an expert on Carnivores at theDanish National Environmental Research Institute withspecial interest in the conservation of mustelidae species.

Ettore Randi is the head of the Istituto Nazionale per laFauna Selvatica, Bologna, with special interest in the useof molecular markers.

Volker Loeschcke is Professor of the Department ofEcology and Genetics, University of Aarhus. He has ageneral interest in evolutionary genetics, with specialemphasis on stress adaptation. He currently leads theCentre for Environmental Stress Research (ACES).

� 2003 Blackwell Publishing Ltd, Journal of Biogeography, 30, 949–958

958 C. Pertoldi et al.