SEXUAL DIMORPHISM OF NUCELLA LAPILLUS (GASTROPODA: MURICIDAE) IN NORTH WALES, UK

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1 J. Moll. Stud. (2000), 66, The Malacological Society of London 2000 SEXUAL DIMORPHISM OF NUCELLA LAPILLUS (GASTROPODA: MURICIDAE) IN NORTH WALES, UK MIN HO SON and ROGER N. HUGHES* Korea Inter-University Institute of Ocean Science, Pukyong National University, Pusan , Korea *School of Biological Sciences, University of Wales, Bangor, Gwynedd, LL57 2UW, UK (Received 22 November 1999; accepted 10 April 2000) ABSTRACT Multivariate statistical methods were employed to examine sexual dimorphism in size and shape of Nucella lapillus collected from 16 sheltered sites along coasts of Anglesey and the Lleyn Peninsula, North Wales, UK. Females were significantly larger than males in overall size; among 12 relative measures of shell shape, two ratios (shell width/shell length and aperture length/shell length) were significantly different between males and females, but these differences usually decreased with increasing age (shell length). The observed hypoallometric dimorphism could be a result of selection on increased female fecundity, which may be positively correlated with shell size in N. lapillus as in other gastropod species. INTRODUCTION Sexual dimorphism is widespread among animals (Shine, 1989) and has attracted the attention of biologists for centuries (Slatkin, 1984; Shuster, 1990). In the majority of gonochoristic animal species, including marine gastropods (Hughes, 1986; Reid, 1986), males and females differ in body size, but the direction and extent of this difference varies widely both within and between taxa (Arak, 1988). Sexual dimorphism, especially in body size, is generally considered to have some adaptive significance as a consequence of differential selection for body size in the two sexes (Fairbairn, 1990), perhaps associated with intrinsic differences between the reproductive roles of males and females (Hedrick & Temeles, 1989). Several workers have reported dimorphism in size and shape between male and female marine snails, despite certain claims that sexual dimorphism in the shell is rare (Fretter, 1984) or generally not pronounced in gastropods (Webber, 1977). Sexual dimorphism has been shown, for example, in Strombus pugilis (Colton, 1905) and Buccinum undatum (Hallers-Tjabbes, 1979). The common dogwhelk, Nucella lapillus (Linnaeus, 1758), a gonochoristic intertidal gastropod, has long been recognised as a highly polymorphic member of the littoral community of northern European and New England coasts (Colton, 1922). Most studies have focused extensively on interpopulation variation in shell morphology in relation to physical and/or biological habitat gradients (e.g. Crothers, 1974; Seed, 1978; Hughes & Elner, 1979; Currey & Hughes, 1982), but information on aspects of sexual dimorphism in N. lapillus is sparse and contradictory. Pelseneer (1926) briefly noted that females are broader than similar sized males and Moore (1938) found small, but significant, size difference in mean shell height ( shell length in the present study) between males and females in a Plymouth population. Most authors, however, have been unable to determine the sex of living animals on the basis of shell form or body colour, although the largest individuals usually proved to be females (Crothers, 1985). The question is there significant sexual dimorphism in size and shape of N. lapillus?, therefore, remains unanswered. To resolve this, we sampled N. lapillus from 16 populations and applied multivariate statistical methods to analyse shell characteristics. MATERIALS AND METHODS Sampling and Measuring Nucella lapillus (n 3083) were collected from 16 sheltered sites between November 1997 and May 1998 along coasts of Anglesey and the Lleyn Peninsula, North Wales (Table 1, Fig. 1). A site was determined as sheltered if the exposure grade was more than 5.0 in Ballantine s (1961) biologically-defined exposure scale, and a site was expressed as an intermediate grade (i.e. 6.5 in Table 1) if the fauna and flora of a site showed a biologically intermediate condition. At each site, adult and subadult individuals were taken randomly from mid- to sub-littoral areas,

2 490 M.H. SON & R.N. HUGHES Table 1. Nucella lapillus. List of sampling sites. St. Locality UK grid Exposure scale Substratum condition No. of specimens No. reference (Ballantine, 1961) examined 1 Colman Beach SH Rocky shore Porth Nyfyn SH Rocky shore with boulders (ø cm) Trevor SH Boulder shore (ø cm) Plas Menai SH Rocky shore with boulders (ø cm) Aberffraw SH Sand beach with boulders (ø cm) Llanfwrog SH Sand beach with boulders (ø cm) Cemaes Bay SH Rocky shore Bull Bay SH Rocky shore Point Lynas SH Rocky shore Dulas Bay SH Rocky shore Moelfre SH Rocky shore Red Wharf Bay SH Sand beach with boulders (ø cm) Penmon SH Sandy mud-flat with boulders (ø cm) Beaumaris SH Sandy mud-flat with boulders (ø cm) Menai Bridge SH Boulder shore (ø cm) Bangor SH Sandy mud-flat with boulders (ø cm) 156 so avoiding excessive depletion in any one place. Samples ranged from individuals per site, and were fixed immediately with 10% neutralised formalin solution. Individuals less than 15 mm in shell length (SL), heavily bored by spionid polychaetes (Polydora sp.), and parasitised by cercaria larvae of trematode flukes (Parorchis sp.) were discarded to avoide possible effects of ontogenetic variation (Moore, 1938; Crothers, 1977), underestimation of shell weight (SWt), and overestimation of SWt (Rothschild, 1941; Feare, 1970), respectively. Sex determination was based on the presence of the sperm-ingesting gland in the female, observed under a stereomicroscope. This gland remains fully developed except at very high TBT concentration levels (> 100 ng/l) (Gibbs, Pasoce & Burt, 1988) and so is a reliable indicator of sex on most shores. After identification of sex, the empty shell was boiled in 10% KOH solution for an hour and shaken throughly in water to dislodge any remaining tissue. The shell was then washed and cleaned to discard any fouling organisms (e.g. barnacles and polychaetes) and dried in an oven for an hour (ca. 80 C). Seven linear shell characters (SL, SW, SD, BWL, AL, SCL & SCW) were measured using vernier callipers (Table 2, Fig. 2) and SWt was measured using an electronic balance. External shell volume (SV) was measured using a displacement technique, after plugging the shell with commercial filler. After submersion of the shell, displaced water volume was recorded in a syringe (Fig. 3) and this measurement was taken as the SV. Relative shell thickness (STH) was calculated as SWt/SV. Apical angle (AA) was measured directly using field callipers and protractor. Shell-lip condition (SLcon) of individuals was examined and categorised into 3 stages: stage 1 (sharp lip), stage 2 (thickening lip) and stage 3 (thickened lip). Tooth rows (TR) of individuals were counted. Statistical analysis Multivariate methods were used mainly to detect and characterise subtle size and shape differences between male and female Nucella lapillus. Extreme outliers, detected by protocols in MINITAB, were omitted from analysis (usually < 5%). Principal component analysis (PCA) was applied to data standardised as z-scores in order to equalise variances and to remove scaling effects (Tissot, 1988) resulting from different units of measurement. These procedures satisfy the assumption of PCA that all samples be drawn from a multivariate, normal distribution (Black & Reyment, 1971). Hartley s Fmax-test (P<0.0001) and Bartlett s X 2 -test (P<0.0001) were used to check homogeneity of variances and a normal probability plot was used to check the distribution of residuals. Interpretation of each principal component (PC) was achieved by examination of the eigenvector. This provides a series of loadings showing the correlation of the original variables with the PC (Kuris & Brody, 1976). Ratio variables, measuring relative shell shape,

3 SEXUAL DIMORPHISM OF NUCELLA LAPILLUS 491 N Anglesey Lleyn Peninsula U.K. Figure 1. Nucella lapillus. Map of Anglesey and the Lleyn Peninsula, North Wales showing sampling sites for studying for sexual dimorphism (for the localities see Table 1). Arabic numerals represent the station numbers in Table 1. were subject to arcsine (ASIN) transformation prior to bivariate least squares linear regression on SL. Differences in ratio variables (e.g. SW/SL) were examined by analysis of covariance (ANCOVA) of arcsine-transformed values on SL for shells identified as males and females, with sex as the factor and SL as the covariate, using the SPSS PC +. RESULTS Shell size and shape of Nucella lapillus differed significantly between males and females (Fig. 4). PCA produced first two components that accounted for 75.2% of the total variation

4 492 M.H. SON & R.N. HUGHES Apical angle Siphonal canal length Shell length Body whorl length Aperture length Shell width Siphonal canal width Shell depth Figure 2. Nucella lapillus. The seven linear shell measurements and apical angle used for comparing shell morphology.

5 SEXUAL DIMORPHISM OF NUCELLA LAPILLUS 493 among 13 variables. The first principal component (PC 1) accounted for 62.0% of the total variation, with positive loadings for all shell characters (Table 2). High loadings were observed for some (70%) of the original variables (eigenvector: ). Consistently positive loadings across variables indicate that PC 1 should be interpreted as representing overall size (e.g. Johannesson, Johannesson & Rolan-Alvarez, 1993; Prenter, Montgomery & Elwood, 1995). The mean value of PC 1 scores for females (mean 0.080) was significantly greater than that for males (mean 0.075) (two sample t-test; p<0.0001) (Table 4). The second principal component (PC 2) accounted for 13.1% of the total variation, with positive loading for 38% of the shell characters (5/13) and negative for 62% (8/13) (Table 3). PC 2 therefore could be most reasonably interpreted as representing overall shape. The mean PC 2 score for females ( 0.146) was significantly less than that for males (mean 0.130) (two sampled student t-test; p<0.0001) (Table 4). Subsequent principal components were not interpreted, as they described relatively small amounts of variation. Relative shell shape, expressed in terms of SW/SL and AL/SL, was significantly different between males and females (Fig. 5). Slopes of linear regressions for males and females significantly differed from zero for both SW/SL (ANCOVA; df 1, 2726; F ; P<0.0001) and AL/SL (ANCOVA; df 1, 2726; F ; P<0.0001). The slopes and intercepts Table 2. Nucella lapillus. Abbreviations of shell characters used in the morphological analysis of sexual dimorphism. Character Abbreviation Character Abbreviation Shell length SL Shell width SW Shell depth SD Body whorl length BWL Aperture length AL Siphonal canal length SCL Siphonal canal width SCW Tooth rows TR Shell-lip condition Slcon Shell weight SWt Shell volume SV Relative shell thickness STH Table 3. Nucella lapillus. Results from principal component analysis (PCA). This table represents the first two principal components. Samples were collected from the 16 sheltered sites around coasts of Anglesey and the Lleyn Peninsula, North Wales. Thirteen shell characters were used for the correlation matrix of the PCA after z-score transformation. See Table 2 for abbreviations. Component 1 2 Eigenvalue % of variation Cumulative % Eigenvectors: Character SL SW SW SD BWL AL SCL SCW SLcon SWt SV STH AA

6 494 M.H. SON & R.N. HUGHES Transparent glass tube Water mixed with ink and detergent Shell volume Initial volume Plugged Nucella lapillus Coarse sieve Stand Air cock Syringe Shell volume Initial volume Figure 3. Nucella lapillus. Diagramatic illustration of volume measuring apparatus used in the present study (modified from D.G. Reid, personal communication). for females were significantly greater in absolute value than those for males in the case of SW/SL (ANCOVA; df 1, 2726; F for slope 4.11; P<0.05) and AL/SL (F for slope 5.01; P<0.05). Values of SW/SL and AL/SL declined as SL increased in both sexes (Fig. 5). There was no heterogeneity between slopes for males and females SD/SL, SCL/SL, SCW/SL, TR/SL, SWt/SL, SV/SL, STH/SL and AA/SL (ANCOVA; df 1, 2726; P>0.05). DISCUSSION Despite the general contention that there is little sexual dimorphism in Nucella lapillus (e.g. Crothers, 1985), significant levels of sexual dimorphism were detected in samples from the sheltered sites both in respect of size and shape, size being relatively more contrasted than shape. Sexual dimorphism is a complex trait, potentially influenced by any factors affecting body

7 SEXUAL DIMORPHISM OF NUCELLA LAPILLUS 495 size in either or both sexes, and thus intimately connected to phylogeny and life history (Fairbairn, 1990). Nevertheless, a possible explanation for the observed difference may concern fecundity selection. Relatively larger size of females may serve to maximise space available for reproductive materials (e.g. eggs and egg capsules). In accordance with fecundity selection, larger Nucella lapillus produce more eggs (Fretter & Graham, 1994) and more capsules (Crothers, 1985). Of course, confirmation that sexual size dimorphism in N. lapillus is due to fecundity selection in females would require precise quantification of the relationship between Table 4. Nucella lapillus. Comparison of means of principal component (PC) scores and results of two-sample student t-test for the mean PC scores of male and female. Female Male Significance of sexual dimorphism Mean SD Mean SD df t-value Probability PC PC Female Male Male 1.0 2nd Principal Component Female st Principal Component Figure 4. Nucella lapillus. Plot of the samples pooled from the 16 sheltered sites for the first two axes of the principal component analysis. Data points represent the mean value of the each sample, collected from around Anglesey and the Lleyn Peninsula, North Wales. The region occupied by the majority of the male (dotted line) and the female (solid line) has been demarcated respectively. The first principal component axis represents overall size of individual (see the text for interpretation in detail).

8 496 M.H. SON & R.N. HUGHES 0.7 Female SW/SL (ASIN) 0.65 Male 0.6 Female: SW/SL (ASIN) = SL (R = 0.25; P<0.05) Male: SW/SL (ASIN) = SL (R = 0.20; P<0.05) 0.82 Female AL/SL (ASIN) Male Female: SW/SL (ASIN) = SL (R = 0.33; P<0.05) Male: SW/SL (ASIN) = SL (R = 0.26; P<0.05) Shell Length (mm) Figure 5. Nucella lapillus. Sexual dimorphism and change of relative shape in shell width (SW) and aperture length (AL), expressed as arcsine-transformed (ASIN) SW/SL (ANCOVA; df 1, 2726; F for slope 4.11; P<0.05) and AL/SL (ANCOVA; df 1, 2726; F for slope 5.01; P<0.05) respectively, according to shell length (SL). Number of examined individuals (male, female) 1430, 1301.

9 SEXUAL DIMORPHISM OF NUCELLA LAPILLUS 497 female size and fecundity. The present observation that females tend to be larger than males is in partial agreement with Moore (1938), who found a small, but significant, difference in mean shell height ( SL in the present study) between males and females from Plymouth Sound. Sexual shape dimorphism often corresponds to different reproductive roles. For example, Hallers-Tjabbes (1979) suggested that sexual dimorphism in aperture shape in Bucinum undatum (Buccinidae) corresponds to different requirements in the performance of sexual acts. In the present study, relative shell width(sw/sl) differed significantly between male and female N. lapillus (ANCOVA; P<0.0001), it decreased with increasing age (Fig. 5) and was nearly reversed beyond 35 mm in SL, as would be expected if shell shape follows a hypoallometric function (Adams & Funk, 1997). Higher mean value of SW/SL in females than males indicates that females are relatively broader than males (Fig. 5). This also could be explained by the fecundity-selection hypothesis, since broadness is positively correlated with larger internal capacity (Kirby, Bayne & Berry, 1994) in which to accommodate more reproductive materials. Relative aperture length (AL/SL) also decreased with increasing age in both sexes and was reversed beyond about 30 mm in SL (Fig. 5). Reversal of AL/SL may be related to energy allocation after sexual maturity. Once mature, females presumably allocate their energy to reproduction instead of shell growth and consequently aperture size may be fixed at this stage of the female s life cycle. Males, on the other hand, could allocate reserves to continued shell growth and several matings. As a result, relative aperture length, expressed as AL/SL, may be reversed at about 30 mm in SL, which presumably is the size at sexual maturation in the present study (Fig. 5). This size is similar to the mean size at sexual maturation (29.5 mm in SL) observed by Moore (1938) in a Plymouth Sound Nucella lapillus population. Examination of micro-growth bands of the shell (Ekaratne & Crisp, 1984) should be used to test this hypothesis. REFERENCES ADAMS, D.C. & FUNK, D.J Morphometric inferences on sibling species and sexual dimorphism in Neochlamisus bebbianae leaf beetles: multivariate applications of the thinplate spline. Systematic Biology, 46: ARAK, A Sexual dimorphism in body size: a model and a test. Evolution, 42: BALLANTINE, W.J A biologically-defined exposure scale for the comparative description of rocky shores. Field Studies, 1 (3): BLACK, R.E & REYMENT, R.A Multivariate morphometrics. Academic Press, New York. COLTON, H.S Sexual dimorphism in Strombus pugilis. Nautilus, 3: COLTON, H.S Variation in the dog whelk, Thais (Purpura Auct.) lapillus. Ecology, 3: CROTHERS, J.H On variation in Nucella lapillus (L.) shell shape in populations from the Bristol Channel. Proceedings of the Malacological Society of London, 41: CROTHERS, J.H Some observations on the growth of the common dog-whelk in the laboratory. Journal of Conchology, 29: CROTHERS, J.H Two different patterns of shellshape variation in the dog-whelk Nucella lapillus (L.). Biological Journal of the Linnean Society, 25: CURREY, J.D & HUGHES, R.N Strength of the dogwhelk Nucella lapillus and the winkle Littorina littorea from different habitats. Journal of Animal Ecology, 51: EKARATNE, S.U.K. & CRISP, D.J Seasonal growth studies of intertidal gastropods from shell microgrowth-band measurements, including a comparison with alternative methods. Journal of the Marine Biological Association of the UK, 64: FAIRBAIRN, D.J Factors influencing sexual size dimorphism in temperate waterspiders. American Naturalist, 136: FEARE, C.J The reproductive cycle of the dog whelk (Necella lapillus). Proceedings of the Malacological Society of London, 39: FRETTER, V Prosobranchs. In: The Mollusca, 7: Reproduction (A.S. Tompa, N.H. Verdonk & A.M. van den Biggelaar, eds), Academic Press, London. FRETTER, V. & GRAHAM, A British prosobranch molluscs. Ray Society, London. GIBBS, P.E., PASOCE, P.L. & BURT, G.R Sex change in the female dog-whelk, Nucella lapillus, induced by Tributyltin from antifouling paints. Journal of the Marine Biological Association of the UK, 68: HALLERS-TJABBES, C.C Sexual dimorphism in Buccinum undatum L. Malacologia, 18: HEDRICK, A.V. & TEMELES, E.J The evolution of sexual dimorphism in animals: hypothesis and tests. Trend in Ecology and Evolution, 4: HUGHES, R.N A functional biology of marine gastropods. Johns Hopkins University Press, Baltimore. HUGHES, R.N. & ELNER, R.W Tactics of a predator, Carcinus maenas, and morphological responses of the prey, Nucella lapillus. Journal of Animal Ecology, 48: JOHANNESSON, K., JOHANNESSON, B. & ROLAN- ALVAREZ, E Morphological differentiation

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