Directional and fluctuating asymmetry in the black-winged Calopteryx maculata (Beauvois) (: Calopterygidae).

Jason Pither and Philip D. Taylor

J. Pither (correspondence). Department of Biology, Queen’s University, Kingston, ON K7L 3N6. email: [email protected] P.D. Taylor. Department of Biology, Acadia University, Wolfville, NS B0P 1X0.

Abstract Directional asymmetry (DA) has received considerably less attention than fluctuating asymmetry (FA) in the literature. Evidence for DA, however, is building am ong taxa. We examined asymmetries in two wing traits within both sexes of the damselfly Calopteryx maculata (Beauvois) (Odonata: Calopterygidae), sampled from three sites in southeastern Ontario. After accounting for measurement error, we show that proximal segments within right fore and hind wings are consistently longer than left in all but one sample group. Full wing lengths, however, exhibited fluctuating rather than directional asymmetry. Mean asymmetry values for both traits (segment and length) occurred in the direction of right-wingedness significantly more often than expected by chance. Patterns of asymmetry were generally consistent among the sexes and sites, although males tended to exhibit more pronounced DA. W e suggest that the wings of C. maculata may undergo compensatory development such that full lengths are more bilaterally symmetric than their component parts.

Introduction flies, Klingenberg et al. 1998; wood butterfly; Windig and Nylin 1999; bumblebees; Klingenberg pers. comm.). Of the possible forms of subtle asymmetry in The degree to which methodological problems (Palmer bilateral characters, fluctuating asymmetry (FA) has and Strobeck 1997) have served to bias the literature received the most attention (e.g. Palmer 1994; Leung and away from reporting DA is unknown, but possibly Forbes 1996; Møller and Swaddle 1997). FA is defined significant (Rowe et al. 1997). T his bias may have been as subtle, random deviations from perfect symmetry (Van enhanced by the fact that DA is commonly deemed Valen 1962). FA arises when the stabilising processes uninterpretable in relation to stress or fitness relations inherent to organism development (i.e. developmental (the most common goal of FA papers; Graham et al. homeostasis) are unable to buffer against disruptive 1998). Indeed, whether in fact DA can be used in any factors during development (Palmer 1996). Increased way to infer developmental stability is still debated (e.g. levels of FA - beyond those which are expected solely Palmer 1994; M oller and T hornhill 199 7; Palmer and from the interplay of developmental "noise" and Strobeck 1997; Graham et al. 1998). stabilising processes - have been correlated with extreme Consider, for example, the case of FA in conditions of environment (e.g. high levels of pollutants; (Odonata: Zygoptera). Prior to 1997, several Graham et al. 1993), and restricted gene flow (e.g. papers purported to have found ideal FA in wing length, inbreeding or genetic bottlenecks; Leary et al. 1985; and reported that FA was correlated with measures of Wayne et al. 1986). Its potential as a tool for monitoring individual fitness and / or quality (Harvey and W alsh stress levels in natural populations, and as an indicator of 1993; Cordoba-Aguilar 1995; Bonn et al. 1996). individual quality or fitness (e.g. Leung and Forbes 1996; However, none of these papers properly evaluated the Moller 1997), has thus prompted this attention (Leary and contributions of both measurement error and other forms Allendorf 1989; Clarke 1993; Graham et al. 1993; Palmer of asymmetry (i.e. D A) to their quantification of FA. 1996). Subtle directional asymmetry (DA), the case Interestingly, subsequent experiments that used when one side’s character value is consistently greater appropriate methodology found inconsistent relationships than the other, has received considerably less attention between FA and measures of individual fitness and (e.g. Palmer 1994; Kraak 1997; Møller and Swaddle quality (Leung and Forb es 1997; Forbes et al. 1997), and 1997; Rowe et al. 1997). W e are aware of only a between FA and environmental stress (Hardersen and handful of studies that document the existence of Wratten 1998; Hardersen et al. 1999). Furthermore, significant DA within (crickets, Simmons and using these more appropriate methods, Leung and Forbes Ritchie 1996; honeybees, Smith et al. 1997; houseflies, (1997) and Hardersen et al. (1999) found some evidence Goulson et al. 1999; fruit flies, house flies, and tsetse for DA in the wings of their subject species. Thus, whether or not differences in damselfly wing lengths 0.05 mm x 0.05 mm. All measurements are presented in conform to a distribution consistent with ideal FA millimetres. (normal or leptokurtic distribution with mean zero; The scanned images of the wings were enlarged Palmer and Strobeck 1992) is debatable. Indeed, given on the computer screen to approximately 11 times their the recent findings of DA in several insect taxa (above), normal size and were measured using an on-screen it could be that shortcomings in methodology were pointer that is part of a freeware image analysis software masking a phenomenon that is widespread in the insect program (Image Tools; University of Texas). On all world (Klingengberg et al. 1998). individuals we measured a segment of the wing that runs

As part of a previous study relating wing parallel to the R1 vein of the fore and hindwings - morphology to landscape structure (Pither 1997), we henceforth referred to as the “segment” (Fig. 1). On a found preliminary evidence suggesting the presence of subset of the individuals we measured a trait that better DA in the wings of the damselfly Calopteryx maculata represents the full length of the wing - henceforth referred (Beauvois) (Odonata: Calopterygidae) within several to as the “length” - (Fig. 1). Pigmentation of the distal populations sampled in Nova Scotia. Here we present the portions of male wings was too dark to measure wing results of a follow-up study, conducted in eastern Ontario, length on all individuals (some wings were also slightly that set out to more explicitly exam ine patterns of damaged distally). Thus, for 14 males and 14 females (to asymmetry in the wings of males and females of this maintain balanced sample sizes) from each site (total 42 species. We determine whether fluctuating or directional individuals per sex), we performed this second asymmetry is present within multiple traits, and compare measurement on the forewings and hindwings. All patterns of asymmetry among the sexes and three sample measurements were taken twice (using the same image, sites. see below) by the same person, each time in a haphazard order. Materials and Methods (c) Measurement error (a) Sample collections Two potential sources of measurement error Twenty five male and 25 female C. maculata exist: human measurement error and error due to the were collected from each of three streams in the vicinity scanning process. The former source of error was of the Queen’s University Biological Station (QUBS) in evaluated for both traits using mixed-model ANOVAs, eastern Ontario, Canada: Forsyth Creek (henceforth following the methods of Palmer and Strobeck (1986). called Forsyth); (391000 m E, 4933300 m N), Peterson To test for both potential sources of error simultaneously Creek (Peterson); (394200 m E, 4933000 m N), and (thus providing an additional test of the human Black Creek (B lack); (390200 m E , 4954500 m N). Only measurement error), we scanned a total of 18 individual reproductively mature individuals were collected (ad ult wings (9 left and 9 right of a pair) twice, at perpendicular and immature damselflies are easily distinguished visually orientations to each other, and repeated segment by eye), and all sam pling was conducted over a short measurements (in a haphazard order) for each scanned period of time within 14 days of the first emergence image twice. We then ran a mixed-model ANOVA with recorded at each of the three sites. Maturation period for “scan” and “individual” each as random effects this species is typically 7-11 days (Waage 1972). (individual nested within scan), and repeated Collection dates (all in 1998) were 28 May (Peterson), 31 measurements as the residual error. This enabled a May (Black), and between 29 May and 1 June (Forsyth). partitioning of variance components due to the scanning A standard insect flight net was used, and individuals effect (random effect 1, 1 df), the difference in segment were placed within mesh cages upon capture. Within two size among individuals (random effect 2, 16 df), and hours of capture all individuals were placed in vials and within-individual measurement errors (residual error, 18 stored in a freezer at -10°C until measured. All df) (cf. Yezerinac et al. 1992). This entire exercise was individuals received identical treatment throughout. conducted for each sex. We also scanned five individual (b) Measurements female wings from the front and back (i.e. flip sides), and The forewings and hindwings of each individual were conducted a similar procedure. We additionally tested clipped at their base, and fastened between two acetate for a difference in calculated left minus right values of the sheets (forming an envelope) using clear adhesive tape. segments (average of two left measurements minus The first millimetre of the proximal end of each wing was average of two right measurements) for the two “flip taped to one of the sheets. Each envelope of acetate side” scans. sheets (containing 15 - 18 individuals’ wings) was (d) Testing for normality scanned once at a resolution of 600 dpi using an Hewlett Asymmetry for each trait was taken as the Packard 4c Scanjet flatbed scanner with a background average of left minus the average of right repeated light source (i.e. a transparency adapter). Scanning at this measurements. The resulting asymmetry data for each resolution produces images whose pixel dimensions are site and sex were tested for normality using a Fig. 1. Landmarks used for m easuring wing segments (sho rt white line) and lengths (long white line), shown here on a left forewing from a female C. maculata. Labels follow nomenclature in Walker (1953): ar - arculus; R 1 - radial vein 1; M1 - median vein branch 1; sn - subnodus. The segm ents were measured from the intersection of M1 and the subnodal vein to the arculus, and the lengths were measured from the distal terminus of the M1 vein to the arculus.

combination of skewness (G1) and kurtosis (G2) statistics et al. 1998). Variance in unsigned asymmetry was (Zar, 1999) and graphical techniques. compared among the sexes and sites using F-tests on the absolute values of left minus right values (i.e. Levene’s (e) Patterns of asymmetry among sites and sexes test; Palmer and Strobeck 1992). For each trait we For each trait (forewing and hindwing segment evaluated the effect of site and sex on mean signed and length asymmetry - total of 4 traits) and group (males asymmetry using two-way ANOVAs. To determine and females within each of 3 sites) we conducted mixed- whether there were allometric effects on magnitudes of model ANOVAs and t-tests to evaluate the significance asymmetry, we calculated Pearson product-moment of different forms of asymmetries, and departures from a correlation coefficients between trait sizes and mean of zero asymmetry respectively. Beyond asymmetries. establishing if a particular group’s mean asymmetry value was different from zero, we were interested in whether Results the direction of asymmetry was consistent among the (a) Measurement error different sample groups. To do this we used a binomial Mixed-model ANOVAs conducted o n the full test, wherein the null hypothesis is that positive and (for segments) and sub-sample (for lengths) data sets negative values of mean group asymmetries are equally verified measurement error did not contribute likely, and should thus occur at a ratio of 1:1. W e significantly to any forms of asymmetry (Table 1). The calculated the magnitude of trait asymmetry as a tests for the scanning effects (multiple scans and flip-side percentage of mean trait size (a scale-independent scans) and human measurement error resulted in variance measure that permits comparisons with other studies) by components of less than 0.1% for each. The asymmetry dividing the groups’ mean absolute value of left minus values calculated from the flip-side scans were not right by the groups’ mean character length (Klingenberg significantly different (paired t-test, P > 0.2). Thus, we Table 1. Results of a mixed model ANOVA conducted o n the wing segm ents (n = 75 for each sex) and wing lengths (n = 42 for each sex) of male and female C. maculata. Significance of the individual ind icates that the differences between individuals are larger than the measurement error; significance of side indicates the presence of DA; significance of individual × side interaction indicates that the FA is larger than the measurement error. Effect trait size refers to the correlation coefficient between trait size (0.5 × left +right) and unsigned asymmetry (absolute value of left minus right) (cf. Windig and Nylin 1999).

ANOVA

individual side ind. × side effect trait size

F p F p F p r p

Segment:

fore male 419.06 *** 27.06 *** 12.71 *** 0.08 ns

female 605.48 *** 8.20 ** 14.19 *** 0.03 ns

hind male 559.94 *** 10.23 ** 16.33 *** 0.19 ns

female 499.06 *** 5.31 * 18.36 *** 0.26 *

Length:

fore male 239061 *** 4.67 * 15.15 *** 0.02 ns

female 3644.2 *** 11.74 ** 35.44 *** !0.21 ns

hind male 3041.5 *** 1.44 ns 28.11 *** !0.24 ns

female 3339.6 *** 0.09 ns 53.61 *** 0.32 *

* P < 0.05, ** P < 0.01, *** P < 0.001

are confident that our data were not affected by the forewing segments exhibited significant DA at Peterson various potential sources of error. Creek only. Hindwing segment D A was significant in (b) Testing for normality both sexes at Peterson Creek, but was not significant in Skewness and kurtosis measures revealed that samples from Black or Forsyth creeks. Importantly, asymmetry distributions were symmetrical, and no eleven of the twelve groups’ mean asymmetry values evidence of platykurtosis was found. Some groups were in the same direction: right segments were longer exhibited slight leptokurtosis in a single trait, but in each than left (P = 0.003; binomial test). The magnitudes of case this was due to mild outliers (these were treated segment asymmetry as a percentage of mean trait size accordingly within the analyses). ranged from 0.69 % (forewing segments of females from (c) Types, magnitudes, and consistency of Peterson Creek) to 1.42 % (forewing segments of males asymmetry from Black Creek) (Table 2). The mixed-model ANOVAs revealed significant Forewing lengths were also directionally DA in the fore and hindwing segments of both males and asymmetric when considering the entire subsamples of females when individuals from each site were pooled males and females (n = 42 per sex) (Table 1). Mixed- together (n = 75 per sex) (Table 1). When segment model ANOVAs conducted on a site-by-site basis asymmetry was assessed on a group-by-group basis revealed significant length FA within both males and (mixed model ANOV As not shown; t-tests are females, over and above measurement error, within b oth summarised in Table 2), male forewing segments fore and hindwings (total of 12 ANOVAs; all individual exhibited significant DA at all sites,while female × side MS / residual MS: F13,28 > 2.09, P < 0.05). One of Table 1 continued

Table 2. Mean segment size of left and right forewings and hindwings (standard error), mean of left segment minus right segment (standard error), t-statistic (tested against zero), and unsigned directional asymmetry as a percentage of within- group mean segment size for damselflies sampled from three sites.

sample left (mm) right (mm) difference (mm) t % difference

Male forewing segment:

Forsyth 12.946 (0.099) 13.037 (0.107) - 0.092 (0.036) - 2.51* 1.17

Peterson 13.336 (0.070) 13.455 (0.083) - 0.119 (0.025) - 4.72*** 0.97

Black 12.891 (0.108) 13.000 (0.105) - 0.109 (0.044) † n/a** 1.42

Female forewing segmen t:

Forsyth 14.085 (0.112) 14.096 (0.110) - 0.012 (0.029) -0.41 0.79

Peterson 14.196 (0.098) 14.270 (0.100) - 0.074 (0.019) - 4.01*** 0.69

Black 14.065 (0.104) 14.138 (0.116) - 0.073 (0.043) † n/a 1.27

Male hind wing segmen t:

Forsyth 11.414 (0.096) 11.452 (0.083) - 0.038 (0.031) -1.21 1.04

Peterson 11.781 (0.065) 11.862 (0.077) - 0.081 (0.023) - 3.57** 0.99

Black 11.369 (0.086) 11.402 (0.086) - 0.033 (0.022) -1.52 0.8

Female hind wing segm ent:

Forsyth 12.396 (0.086) 12.384 (0.099) 0.011 (0.032) 0.35 1.1

Peterson 12.517 (0.075) 12.592 (0.085) - 0.076 (0.025) - 3.07** 0.89

Black 12.387 (0.085) 12.426 (0.089) - 0.040 (0.029) -1.38 0.93

†Wilcoxin rank sum test used in lieu of t-test due to leptokurtic distributions. * P < 0.025, ** P < 0.005, *** P < 0.001 (two-tailed tests). these groups (females from Black Creek) exhibited The variance of unsigned segment asymmetry in significant DA within forewing lengths (Table 3). Within the forewings of females differed significantly among the all other groups, mean asymmetry values were not sample sites (F2,72 = 4.23, P = 0.018; Levene’s test), but significantly different from zero, and were normally otherwise did not differ significantly among sites for any distributed, thus conforming to FA. The magnitudes of other group. The variance of unsigned segment length asymmetry as a percentage of mean trait size were asymmetry in the forewings of males was significantly smaller than observed in the segment asymmetries larger than in females (F74,74 = 1.58, P = 0.05; F-test), but (above), and ranged from 0.24 % (forewing lengths of otherwise did not differ significantly among sexes for any males from Black Creek) to 0.61 % (hindwing lengths of other trait. The variance of unsigned length asymmetry males from Forsyth Creek) (Table 3). Although the for both fore and hindwings was not significantly length asymmetries were extremely small, nine of the different between the sexes. twelve group means were asymmetric in the same Two-way ANOVAs (site and sex as factors) direction: right wings were longer than left (P = 0.053; indicated that signed forewing and hind wing segment binomial test). and length asymmetry did not vary significantly among (iv) Comparisons of asymmetry among groups sites, nor between the sexes (P > 0.05). Table 1 continued

Table 3. Mean length of left and right forewings and hindwings (standard error), mean of left length minus right length (standard error), t-statistic (tested against zero), and unsigned directional asymmetry as a percentage of within-group mean length for damselflies sampled from three sites.

sample left (mm) right (mm) difference (mm) t % difference

Male forewing length:

Forsyth 32.258 (0.317) 32.301 (0.318) !0.044 (0.052) !0.85 0.44

Peterson 33.379 (0.188) 33.380 (0.185) !0.001 (0.051) !0.02 0.48

Black 32.401 (0.200) 32.409 (0.185) !0.008 (0.028) !0.29 0.24

Female forewing length:

Forsyth 35.577 (0.280) 35.669 (0.252) !0.092 (0.043) !2.11 0.42

Peterson 36.086 (0.316) 36.149 (0.305) !0.062 (0.057) !1.10 0.48

Black 36.100 (0.208) 36.258 (0.211) !0.158 (0.057) !2.79* 0.57

Male hindwing length:

Forsyth 31.023 (0.320) 31.024 (0.292) !0.001 (0.069) !0.02 0.61

Peterson 32.139 (0.171) 32.089 (0.182) 0.050 (0.041) 1.23 0.40

Black 31.023 (0.175) 30.968 (0.178) 0.055 (0.037) 1.49 0.36

Female hindwing length:

Forsyth 34.257 (0.266) 34.262 (.0257) !0.005 (0.066) !0.07 0.49

Peterson 34.680 (0.267) 34.689 (0.288) !0.009 (0.055) !0.15 0.42

Black 34.718 (0.204) 34.682 (0.200) 0.035 (0.060) 0.60 0.54 * P < 0.05 (two-tailed test)

Table 4. Pearson correlation coefficients among traits for all male and female individuals (n = 75 per sex). Variables are forewing segment signed asymmetry (FWSD ), hindwing segment signed asymmetry (HWSD), forewing segment size (FWS ), and hindwing segment size (HW S).

Trait FWSD HWSD FWS

Males HWSD 0.038

FWS !0.142 !0.029

HWS !0.073 !0.048 0.946 c

Females HWSD 0.275

FWS !0.133 !0.401 c

HWS !0.189 !0.350 b 0.936 c a P < 0.1, b P < 0.05, c P < 0.01, d P < 0.001 (sequential Bonferroni-adjusted probabilities) Table 5. Pearson correlation coefficients among traits for subset male and female individuals (n = 42 per sex). Variables are forewing length signed asymmetry (FWLD ), forewing segment signed asymmetry (FWSD), hindwing length signed asymmetry (HWLD), hindwing segment signed asymmetry (HWSD), forewing length size (FWL), forewing segment size (FWS), hindwing length size (HW L), and hindwing segment size (HW S). The two values in bold correspond to the significant correlations for females listed in Table 4 (for full data set).

Sex Trait FWLD FWSD HWLD HWSD FWL FWS HWL

Males FWSD 0.176

HWLD 0.043 !0.214

HWSD !0.073 !0.183 0.072

FWL 0.126 0.085 0.217 !0.072

FWS 0.187 !0.115 0.085 !0.275 0.756 d

HWL 0.155 0.093 0.178 !0.058 0.983 d 0.730 d

HWS 0.251 !0.053 0.163 !0.261 0.773 d 0.945 d 0.759 d

Females FWSD 0.425

HWLD !0.164 !0.275

HWSD !0.329 !0.067 0.451*

FWL 0.210 !0.048 !0.059 !0.176

FWS 0.347 !0.011 !0.046 !0.236 0.591 c

HWL 0.220 !0.121 !0.043 !0.218 0.922 d 0.588 c

HWS 0.400 !0.057 0.032 !0.301 0.642 d 0.913 d 0.675 d a P < 0.1, b P < 0.05, c P < 0.01, d P < 0.001 (sequential Bonferroni-adjusted probabilities) * a single outlier renders this value significant at p = 0.05.

(v) Correlations among trait size and asymmetry correlation was not significant at Bonferroni-adjusted Not surprisingly, correlations among trait sizes alpha levels). (segments and lengths of fore and hindwings) were Finding relatively consistent DA within the wing significant in both males and females (Tables 4 and 5). segments but not within the wing lengths prompted us to In addition, signed segment asymmetry within the test the idea that the wings experience “compensatory hindwings of females was significantly correlated with variations” (Schultz 1926), whereby the development of fore and hingwing segment size (Table 4): females with the distal portions of the wings compensates for the longer average fore and hind wing segments tended to differences in left and right segm ent lengths (at least have increasingly longer right segments than left (Fig. 2). along the M1 vein; Fig. 1) . If this is the case, then t-tests These correlations, however, had little explanatory power comparing the absolute values of size-standardised length (< 17 % variation in segment asymmetry explained by and segment asymmetries (i.e. (L - R) / mean size) should either forewing or hindwing segment size). Correlations indicate that length asymmetries are significantly smaller between length and segment asymmetries were non- than segment asymmetries, and F-tests should reveal significant (Table 5). Only within female forewings was larger variances in unsigned segment asymmetries than in length and segment signed asym metry significantly length asymmetries. T his was indeed the case:size- correlated (Fig. 3) (note that within T able 5, this standardised length asymmetry within the fore and Fig. 2. Scatterplots of hindwing signed asymmetry within females in relation to forewing (a) and hindwing (b) segment size. Scaling of y-axes is identical in both plots. Lines represent locally weighted regressions with smoothing parameter = 2/3. Both relationships are significantly negative (see Table 4).

hindwings of males and females was significantly smaller within insects, and asym metries in external traits - as in magnitude than segment asymmetry (all 4 paired t- observed here - may result from carry-over effects during tests, P 41 d.f. < 0.005), and so too were their variances (all development. 4 F-tests, P 41, 41 d .f. < 0.05). Although we found significant FA when wing lengths were considered on a site-by-site basis for each Discussion sex, it is interesting to note that again, the mean L-R We have provided evidence that, while the full asymmetry values for each group tended to be in the lengths of damselfly wings exhibit FA (in all but one direction of right lengths longer than left (if only group), a proximal length-wise sub-section of the wings marginally) (Table 3). The probability of nine out of (the “segment”) exhibited significant DA, and did so in a twelve groups having a negative mean value of common direction among sample groups and traits: right asymmetry purely by chance is 5.3 %. Thus, although the segments were consistently longer than left segments. results of the t-tests on wing lengths were not significant, Additional data collected on individuals sampled within there appears to be some tendency for right wings to be Nova Scotia (Pither 1997) are consistent with these marginally longer than left. Our finding of FA within findings: 13 out of 14 groups exhibited mean right wing lengths is consistent with other (methodologically segment lengths that were longer than left (P = 0.0009; sound) studies concerning damselflies (Leung and Forbes binomial test) (we do not have length data for the Nova 1997; Forbes et al. 1997; Hardersen and Wratten 1998; Scotia individuals). In the present study we also found Hardersen et al. 1999; Carchini et al. 2000). None of that correlations between segment asymmetry and length these other studies, however, analysed wing traits asymmetry within individuals were non-significant in all comparable to our “segments”, so it remains to be seen but one group (Fig. 3). These results, as well as those of whether DA within the proximal sections of damselfly the supplementary analyses conducted a-posteriori (last wings is a general phenomenon. set of Results section above) support the possibility that Previous findings of DA within insects are the distal portions of the wings of C. macula ta develop in considerably heterogeneous. Simmons and Ritchie a way that compensates for asymmetries in proximal wing (1996) found DA in the harps of field crickets: on components. We are unsure of the details of wing average, right harps were larger than left. This result, development in damselflies, but, as others have pointed however, involved individuals poo led from three separate out (Klingenberg et al. 1998), there exist systematic left- populations (sample sizes 60, 39 and 26), and it was right asymmetries in the development of internal organs Fig. 3. Scatterplots of forewing length asymmetry versus forewing segment asymmetry for 42 males (left panel) and 42 females (right panel). D iagonal lines represent 1:1 concordance. For m ales, r = 0.176, P > 0.05; for females, r = 0.425, P = 0.005. Hindwing correlations were not significant.

unclear as to whether DA was significant at each or any to more often have larger left wings than right, but of the sites when considered separately. Such a significance levels varied among samples (Klingenberg et consideration can be important. For example, when we al. 1998). Windig and Nylin (1999) found that fore and pooled our length data across sample sites, forewing hindwing areas and forewing widths were directionally lengths of both sexes showed significant DA, each asymmetric within male, but not female, speckled wood consistent in direction (Table 1). When evaluated on a butterflies: on average right wings had larger trait sizes site-by-site basis, length DA was non-significant (Table than left. Our findings are consistent with Windig and 3). Nylin (1999) and Smith et al. (1997) in that trait DA was Smith et al. (1997) used procrustes methods more pronounced, on average, within males than within (which analyse multiple morphometric measures females (both concerning DA, and variance in simultaneously) to demonstrate the presence of DA in the asymmetry). wings of honeybees (Apis mellifera). DA was more Based on these few methodologically rigorous prominent in the wings of males than in females, and their studies involving insects, it seems that, when present, DA measure of asymmetry involved inequalities in wing vein is inconsistent in direction and significance among placement - somewhat similar to our “segments”. Using species, and also within species among populations. Our measures of mean centroid size, male tsetse flies findings do, however, suggest a common direction for at (Glossina palpalis gambiensis) were shown to have larger least segment asymmetry within C. maculata, and perhaps right wings than left (Klingenberg and McIntyre 1998). also length asym metry. Otronen (1998) found the same pattern in the small One feature that is shared by the aforementioned claspers of the fly Dryom za anilis. The pattern is less studies is the use of high-precision measurement methods. clear for the housefly: using proscrustes methods, The reported magnitudes of D A are typically extremely Klingengberg et al. (1998) found right wings to be larger small, and thus require such methods to be detected. For than left (mean centroid size), while Goulson et al. (1999) example, we found that average differences in right and used a length index similar to our segments and found left left wing segments and lengths were typically less than a wings to be significantly longer in this trait within both tenth of a millimetre in magnitude (Tables 2 and 3). This reared and wild individuals (and both sexes) originating range is comparable to findings elsewhere (Otronen 1998; from a single site. Drosophila melanogaster (a species Klingenberg et al. 1998). As others have pointed out that has been the subject of many FA studies) was shown (Rowe et al. 1997; Goulson et al. 1999), such a stringent requirement for precise measurement techniques has References likely led to a misleadingly low detection rate (and consequently, report rate) of forms of asymmetry other Bennet, D.M., and Hoffmann, A.A. 1998. Effects of size than FA. Shortcomings in measurement techniques and fluctuating asymmetry on field fitness of the would more likely be manifest as FA rather than DA parasitoid Trichogramma carverae (Rowe et al. 1997). We carefully verified that neither (Hymenoptera: Trichogrammatidae). J. Anim. systematic nor random errors influenced our Ecol. 67: 580-591. measurements. We are thus confident that the patterns of Bonn, A., Gasse, M ., Rolff, J., and Martens, A. 1996. segment and length asymmetry revealed here (and in data Increased fluctuating asymmetry in the from Nova Scotia) are real, not artifacts. damselfly puella is correlated with To our knowledge, only Leung and Forbes ectoparasitic water mites: implications for (1997) and Hardersen et al. (1999) have reported fluctuating asymmetry theory. Oecologia, 108: evidence for DA in damselfly wing lengths. The former 596-598. study found DA in the hindwings of teneral fem ale Carchini, G., Chiarotti, F., Di Domenico, M., and Enallagma ebrium (Hagen). The trait measured was a Paganotti, G. 2000. Fluctuating asymmetry, size segment from the nod us to the pterostigma (i.e. the and mating success in males of Ischnura elegans portion of the wing distal to our segments). Because this (Vander Linden) (Odonata: Coenigrionidae). was the only sample out of four assessed that exhibited Anim. Behav. 59: 177-182. DA, they attributed their finding to chance, and retained Clarke, G.M. 1993. P atterns of developmental stability of all of their results for subsequent FA-quality assessments. Chyrsopa perla L. (Neuroptera: Chryso pidae) in Interestingly, for a similar detection rate (5 of 16 tests), response to environmental pollution. Env. Hardersen et al. (1999) chose to discard their Entomol. 22: 1362-1366. directionally asymmetric measures of wing lengths on Córdoba-Aguilar, A. 1995. Fluctuating asymmetry in similar grounds. We suspect this latter procedure to be paired and unpaired damselfly males Ischnura common in FA research, and suggest that this too could denticollis (Burmeister) (Odonata: contribute to an under-representation of DA within the ). J. Ethol. 13: 129-132. asymmetry literature. Other studies examining Forbes, M., Leung, B., and Schalk, G. 1997. Fluctuating asymmetry in damselfly wings (Harvey and Walsh 1993; asymmetry in Coenagrion resolutum (Hagen) in Cordoba-Aguilar 1 995; Bonn et al. 1996) would benefit relation to age and male pairing success from re-evaluation using more precise measuring methods (Zygoptera: Coenagrionidae). Odonatologica, and complete analyses. 26: 9-16. This study demo nstrates that while the full Graham, J.H., Freeman, D.C., and Emlen, J.M. 1993. lengths of C. maculata wings exhibit fluctuating Developmental stability: a sensitive indicator of asymmetry, a proximal segment measured along a populations under stress. In Environmental prominent length-wise vein shows consistent directional toxicology and risk assessment, ASTM STP asymmetry. Supplemental analyses provided evidence 1179. Edited by W.G. Landis, J.S. Hughes, and consistent with the idea that the wings may undergo M.A. Lewis. Philadelphia. pp. 136-158. compensatory development, such that total wing length Graham, J.H., Emlen, J.M., Freeman, D.C., Leamy, L.J., asymmetry may be less variable than sup-part asymmetry. and Kieser, J.A. 1998. Directional asymmetry We are unaware of any other studies demonstrating such and the measurement of developmental consistency among sample groups in subtle DA of any instability. Biol. J. Linn. Soc. 64: 1-16. insect wing traits. Goulson, D., Bristow, L., Elderfield, E., Brinklow, K., Perry-Jones, B., and Chapman, J.W. 1999. Size, symmetry, and sexual selection in the housefly, Musca domestica. Evolution, 53: 527-534. Acknowledgements Hardersen, S., and Wratten, S.D. 1998. The effects of We thank the numerous landowners who kindly allowed carbaryl exposure of the penultimate larval access to sampling sites. Logistical support was provided instar of Xanthocnemis zealandica (Odonata: by the “Sandbox” at Acadia University (Nova Scotia Zygoptera) on emergence and fluctuating data), the Queen’s University Biological Station, and asymmetry. Ecotoxicology, 7: 1-8. Mark Forbes. Discussions with Mark Forbes and Bruce Hardersen, S., Wratten, S.D., and Frampton, C.M. 1999. Smith were very constructive. Bob Montgomerie, C.P. Does carbaryl increase fluctuating asymmetry in Klingenberg, and one anonymous reviewer provided damselflies under field conditions? A mesocosm extremely helpful comments on earlier drafts of the experiment with Xanthocnemis zealandica manuscript. (Odonata: Zygoptera). J. Appl. Ecol. 36: 534- 543. Bioscience, 46: 518-532. Harvey, I.F., and Walsh K.J. 1993. Fluctuating Palmer, A.R., and Strobeck C. 1992. Fluctuating asymmetry and lifetime mating success are asymmetry as a measure of developmental correlated in males of the damselfly Coenagrion stability: Implications of non-normal puella (Odonata: Coenagrionidae). Ecol. Entom. distributions and power of statistical tests. Acta 18: 198-202. Zool. Fennica, 191: 57-72. Leary, R.F., Allendorf, F.W., and Knudsen, K.L. 1985. Palmer, A.R., and Strobeck C. 1997. Fluctuating Inheritance of meristic variation and the asymmetry and developmental stability: evolution of develop mental stability in rainbow heritability of observable variation vs. trout. Evolution, 39: 308-314. heritability of inferred cause. J. Evol. Biol. 10: Leary, R.F., and Allendorf, F.W. 1989. Fluctuating 39-49. asymmetry as an indicator of stress in Pither, J. 1997. Responses in the habitat occupancy, conservation biology. TREE , 4: 214-217. movement behavior, and wing morphology of Leung, B., and Forbes, M. 1996. Fluctuating asymmetry two species of calopterygid damselflies to in relation to stress and fitness: Effects of trait landscape structure. M.Sc. Thesis. Acadia type as revealed by meta-analyis. Ecoscience, 3: University, Wolfville, NS. 400-413. Rowe, L., Repasky, R.R., and Palmer, R. 1997. Size- Leung, B., and Forbes, M. 1997. Fluctuating asymmetry dependent asymmetry: fluctuating asymmetry in relation to indices of quality and fitness in the versu antisymmetry and its relevance to damselfly, Enallagma ebrium (Hagen). condition-dependent signaling. Evolution, 51: Oecologia, 110: 472-477. 1401-1408. Klingenberg, C.P., and McIntyre, G.S. 1998. Geometric Schultz, A.H. 1926. Fetail growth of man and other morphometrics of develop mental instability: primates. Quart. Rev. Biol. 1: 465-521. analyzing patterns of fluctuating asymmetry Simmons, L.W., and Ritchie, M.G. 1996. Symmetry in with procrustes methods. Evolution, 52: 1363- the songs of crickets. Proc. R. Soc. Lond. B, 1375. 263: 305-311. Klingenberg, C.P., McIntyre, G.S., and Zaklan, S.D. Smith, D.R., Crespi, B.J., and Bookstein, F.L. 1997. 1998. Left-right asymmetry of fly wings and the Fluctuating asymmetry in the honey bee, Apis evolution of body axes. Proc. R. Soc. Lond. B, mellifera: effects of ploidy and hybridization. J. 265: 1255-1259. Evol. Bio l. 10: 551-574. Kraak, S.B.M. 1997. Fluctuating around directional Taylor, P.D., and Merriam H.G. 1995. W ing asymmetry? TREE 12: 230. morphology of a forest damselfly is related to Møller, A.P. 1997 . Developmental stability and fitness: landscape structure. Oikos, 73: 43-48. a review. Am . Nat. 149: 916-932. University of Texas Health Sciences Centre. Image T ool. Møller A.P., and Swaddle, J.P. 1997. Developmental 1997. Freeware, available through the internet stability and evolution. Oxford University Press, at: http//macorb.uthscsa.edu/dig/itdesc.html Oxford. Van Valen, L. 1962. A study of fluctuating asymmetry. Møller A.P., and T hornhill, R. 19 97. A meta-analysis of Evolution, 16: 125-142. the heritability of developmental stability. J. Waage, J.K. 1972. Longevity and mob ility of adult Evol. Bio l. 10: 1-16. Calopteryx macula ta (Beauvois, 1805) Monedero, J.L., Chavarrías, D., and López-Fanjul, C. (Zygoptera: Calopterygidae). Odonatologica, 1: 1997. The lack of mutational variance for 155-162. fluctuating and directional asymmetry in Walker, E.M. 1953. The Odonata of Canada and Alaska. Drosop hila melanogaster. Proc. R. Soc. Lond. Vol. 1 Part 2. U niversity of Toronto Press, B, 264: 233-237. Toronto. Otronen, M. 1998. Male asymmetry and postcopulatory Wayne, R.K., Mode, W.S., and O’Brien, S.J. 1986. sexual selection in the fly Dryom za anilis. Morphological variability and asymmetry in the Behav. E col. Sociobiol. 42: 185-191. cheetah (Acinonyx jubatus), a genetically Palmer, A.R. 1994. Fluctuating asymmetry analysis: a uniform species. Evolution, 40: 78-85. primer. In Developmental instability: its origins Windig, J.J., and Nylin, S. 1999. Adaptive wing and evolutionary implications. Edited by T.A. asymmetry in males of the speckled wood Markow. Kluwer, T he Netherlands. pp. 335- butterfly (Pararge aegeria)? Proc. R. Soc. 364. Lond. B. 266: 1413-1418. Palmer, A.R. 1996. Waltzing with asymmetry. Yezerinac, S.M., Lougheed, S.C., and Handford, P. 1992. Measurement error and morphometric studies: statistical power and observer experience. Syst. Biol. 41: 471-482. Zar, J.H. 1999. Biostatistical analysis, 4th edition. Prentice Hall Inc. New Jersey. pp. 67-76.