• drobiologia 183 : 1 5 7 -164, 1989.•

1989 Kluwer Academic Publishers . Printed in Belgium . 157

Loading constraints, body size and mating preference in Gammarusspecies

Jonathan Adams, Penelope J . Watt, Caroline J . Naylor & Paul J . Greenwood'Department of Pure & Applied Biology, University of Leeds, Leeds LS2 9JT, England; 'Department ofAdult & Continuing Education, University of Durham

Received 17 November 1987 ; in revised form 4 August 1988 ; accepted 29 September 1988

Key words : loading constraint, mating, sex, sexual dimorphism, Crustacea

Abstract

Size relationships among pairing Gammarus were examined with reference to two hypotheses for sexualsize dimorphism and assortative mating among aquatic Peracarida (Crustacea) . The sizes of pairingand non-pairing animals were compared in different experimental conditions where the size of one or bothsexes was controlled . The experimental results present a complex picture which suggests that both sexualselection and loading constraints are likely to play a role in determining mating decisions in these animals .

Introduction

Females are usually larger than males in all groupsof animals, except the higher vertebrates, andamong dioecious plants (Darwin, 1871) . Againstthis trend, males are not only the larger sex amongspecies of freshwater and marine Peracarida inwhich the male carries his mate but mating is alsoassortative for size (Ridley, 1983 ; Adams et al.,1985 ; Adams & Greenwood, 1987). Size dif-ferences may be the product of natural or sexualselection or their interaction . It has been inferredthat male-male competition is responsible forboth assortative mating and larger male size inGammarus (Elwood et al., 1987 ; Ward, 1987) .These are not, however, inevitably a result of nornecessarily evidence for sexual selection(Greenwood & Adams, 1987) .

Large size should always be advantageous tofemales because of egg production (e.g . Kolding• Fenchel, 1981) and males should prefer thelargest available female (Elwood et al., 1987 ;

Birkhead & Clarkson, 1980 ; Ward, 1983). Whatare the advantages of large size to males? First,females might prefer larger males (Ward, 1987 ;Hunte et al., 1985 ; Robinson & Doyle, 1985) .Second, larger males could pre-empt smallermales by pairing faster (Elwood et al ., 1987) ortakeover females in precopula from smaller males,as occurs in Asellus (Ridley & Thompson, 1979) .Third, there may be constraints on mate choiceunrelated to sexual selection, such as loadingrestrictions (Adams & Greenwood, 1987) . Whatis the advantage of small size to a female? If amale's choice is a compromise between maximis-ing reproductive success and minimising loadingpenalties (energetic costs, displacement by currentand predation) then a small female may bepreferred . Pairs with high male/female size ratiosmove faster (Adams & Greenwood, 1983 ; Adamset al., 1985 ; Naylor & Adams, 1987), and pair sizeratios increase with current speed in laboratorytrials and in nature (Adams & Greenwood, 1987) .

Work on peracarids has concentrated on the

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advantage of large size to males compared withother males due to sexual selection . Little atten-tion has hitherto been paid either to female size orto the relative sizes of males and females and noattention has been paid to the behaviour of smallmales. We report the results of experiments whichexamine the consequence of controlling male andfemale size in mate-choice trials and the rate atwhich different sizes of males and females becomepaired .

Methods

Trials were carried out with both freshwaterGammarus pulex and brackish-water G. duebeni .

The sizes of these species are similar, as are size-ratios of precopula pairs . It is implicit that datafrom one are relevant to hypotheses about both(Adams & Greenwood, 1987). All animals used intrials were taken from pre-formed pairs . Sincethese animals are self-evidently available forpairing, this procedure controls for extreme dif-ferences related to the female's time to moult . Alltrials were carried out over a few weeks at con-stant temperature to remove season and tempera-ture as variables . Since individual experienceaffects mating expectations (Hunte et al., 1985)

the males and females in each experiment weredrawn from the same stock and so expectationsof mate size range should be similar . The remain-ing variable is size difference within and betweenthe sexes . Size was assessed visually before trialsand was corroborated by weighing afterwards(blotted dry weight is highly significantly corre-lated with length : Greenwood & Adams, 1984).

One type of trial was carried out in a 100 mlpot. At the start of the trial two individuals of onesex were added to the pot and then one individualof the other sex . In some trials animals were addedwithout consideration of size . In others the size ofone or both sexes was controlled as `large' or`small' compared to the total stock range (seeResults for quantification). Trials were stoppedwhen a pair formed in the pot or after 24 h if nopair formed. All animals were weighed . Pot trialswithin a series were run simultaneously. These

trials are subsequently referred to as experiments Iand II (excess of males) and IV and V (excess offemales) .

In an alternative procedure, batches of animalssorted into single-sex containers were added to alarge tray filled to 2 cm depth with aerated water .Twenty animals of one sex were added and then,at the start of the trial, ten animals of the other sex .The sex ratio was thus the same as in the pottrials . Pairs were removed to individual pots forweighing. Fresh animals were not added tomaintain numbers and trials were completedwhen all of one sex had paired or after 24 hours .This type of experiment is extremely difficult toanalyse and interpret, since as soon as one pair isremoved it obviously alters the constitution of theavailable pool . These trials are subsequentlyreferred to as experiment III (excess of females)and VI (excess of males) .

A third procedure investigated the rate of pairformation (experiment VII) . Batches of animalswere added to a large tray, as above, but the sexratio was balanced . Thirty animals of one sexwere placed in the tray and then, at the start of thetrial, thirty animals of the opposite sex wereadded . As pairs formed they were removed toindividual pots . No new animals were added .Each trial was timed and the rate of pair formationwas recorded for the first fifteen pairs to form (i .e .for the density of animals to fall to half its initialvalue) . Typically, animals in these trials were veryactive and pairs formed rapidly . Thus we canmake an informative comparison of rates of pairformation for different size groups . Pair formationwas usually so rapid that it became important toestablish a criterion for a `properly formed pair',at which point the animals could be removed . Wehave standardised this as the point where the malebegan to swim forward holding a passive femalein typical precopula position .

Tray trials were usually run in pairs or smallbatches where large numbers of animals wereavailable. In nine out of 30 experimental runsanimals were sluggish and pairs formed only veryslowly although males and females continuallycontacted one another. We cannot account forthis behaviour in terms of time, temperature or

procedure. To standardise the results we haveexcluded trials where there were fewer than15 pairs in the first 15 minutes . The exclusionswere very much slower than this . The result issurprising, in view of Hunte et al .'s (1985) con-clusion that prior experience is important indecision making, since the test animals had beenkept in single-sex containers for up to an hourbefore each trial. In excluded trials there was norelationship between the absolute or relative sizesof the sexes and their failure to pair .

Results

Experiments I-III . Do males preferentially pairwith the largest available female (Table 1)?

In experiment I a single randomly sampled maleGammarus duebeni was offered two females . Inonly 35 of 60 trials did the male choose the largerof the two (chi2 = 1 .66, n.s .) and there was nosignificant difference between the pooled sizes ofpaired and unpaired females. Thus, in agreementwith other studies, there appears to be no evidencefor consistent male discrimination in favour of thelarger of two females presented to him .

In experiment Ila (size-controlled : large maleand two large females) the male paired with

Table 1 . Experiments with an excess of females . Mean size (mg ± 95% c.l .) of male, paired and unpaired female Gammarusduebeni in pot trials in which a single male (experiment I = random size selection ; experiment II a-d = size-controlled) was offereda choice of two females (I = random ; II = size-controlled) Experiment III : ten size-controlled male G. pulex presented withtwenty randomly selected females in trays (see text for details) . (L), (S) = large and small in population range ; significancetests = Wilcoxon signed ranks on female size .

neither female in 18 of 47 pots. Among the 29 potswhere pairing occurred there was no significanpreference (large = 17, small = 12, chi2 = 0 .92,n.s .). The females in the pots where no pairingoccurred, however, were significantly larger thanthe females in the pairing replicates (preselectedsize range non-normal, Mann-Whitney U-test,U = 629, P < 0.01). In IIb (large male and twosmall females) the males paired significantly moreoften with the larger of the two (large = 28 .5,small = 11 .5, chi2 = 7.23, P < 0.01 ; Table 1) . InIIc (small male and two large females) the malepaired with neither female in 19 of 46 replicates .Among the 27 pots where pairing occurred themales expressed a significant prefrence for thesmaller of the two (large = 8, small = 19,chi2 = 4.48, P < 0.05). There was no significantsize difference between the pools of animals in thepairing and non-pairing pots . In lid (small maleand two small females) the males expressed nosignificant size preference (large = 13,small = 23, chi2 = 2 .32, n.s . ; Table 1) .

In experiment III, ten large or ten small maleG. pulex were presented with twenty females in atray. There were ten replicates in each series . Inneither series was there a significant differencebetween the mean sizes of the groups of femalestaken into precopula and those left unpaired .However, in two of the ten replicates with small

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N Male size Female size Significance ofdifference

Paired Unpaired

I 60 78.0+2.4 34.7 + 2.1 31.6+2 .4 P > 0.1IIa 29 107.9 + 4 .9 (L) 40.6 + 1 .9 38.7 + 2.7 (L) P > 0.1

18 111 .3 + 4 .3 45 .1 + 3 .8IIb 40 97.6 + 5 .3 (L) 24.1 + 1 .4 22.5+1.7(S) P<0.0211c 27 69.6+ 6.1 (S) 37 .8 + 2.2 39 .9 + 2 .3 (L) P < 0 .02

19 71 .5 + 7 .1 41 .5 +4.1IId 36 79.4 + 2 .9 (S) 24.0+2.2 24.8 + 2.3 (S) P>0.1IIIa 10 30.0 + 0 .9 (S) 19.0+1.3 20.9+1.1 P>0.05Illb 10 47.2 + 1 .6 (L) 20.2+ 1 .0 19 .6 + 1 .1 P>0.05

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males the pairing females were significantlysmaller than those unpaired while in two of the tenreplicates with large males the pairing femaleswere significantly larger (Mann-Whitney U test,P < 0.05 in each case) .

Experiments IV- VI. Are larger males more likelyto pair (Table 2)?

In each of 40 pot replicates a single randomlyselected female G. duebeni was offered to twomales. The male which paired with the female waslarger than the single male significantly more often(large = 28, small = 12, chi2 = 6 .4, P < 0.02) andpaired males were significantly larger.

In experiment V(a-d) two large or two smallmale G. pulex were offered a single size-controlledfemale. There was no significant differencebetween the sizes of the paired and unpairedmales in any series. However, in experiment Vb(large males and a small female) there were 14 of44 replicates in which neither male chose to pairwith the female available . These males were sig-nificantly larger than the males in trials wherepairing did take place (Mann-Whitney U test,U = 514, t = 2.9, P < 0.01). In Vc (two smallmales and a large female) there were 29 of 53 potsin which neither male paired . There was no signifi-cant size difference between the sizes of males inthe pairing and non-pairing trials, or of the poolsof females .In experiment VI, 20 male G. pulex were

presented with either 10 large females (ten repli-cates) or 10 small females (eleven replicates) intrays . In replicates with large females the pairingmales were not significantly larger than theunpaired males, but in the replicates with smallfemales the mean sizes of pairing males were sig-nificantly smaller than those left unpaired .

Experiment VII. Do larger males pair faster thansmaller males?

The twenty-one experiments on pairing ratecovered a wide range of male sizes . The data ontime to pairing were used to calculate a pairingindex. In every case a regression of cumulative

pair number (not size) against log-time of pairingproduced a highly significant fit to a straight line .The time for the median (8th) pair was calculatedfrom the regression. This standard time was com-pared with the pooled male and female sizes andthe size ratio of the pairs which formed. There isa significant correlation with all these variables .Larger males pair faster than smaller males ; largerfemales became paired faster than smallerfemales ; and pairing is more rapid where there isa greater disparity between the sizes of the pairedanimals (Fig. 1) .

Effects due to size differences and due toabsolute size are confounded but which is causallysignificant? We used reduced major axes to calcu-late an `expected' female size for the pool malesize in each trial and compared this with observedfemale size. If relative size is important thenpairing should be faster when observed femalesize is much smaller than expected . Female sizevariation is limited, but there is no correlationbetween female size differences and pairing rate(N = 21 ; r = 0 .22 ; P = 0.65) and we concludethat there is no significant evidence that relativemale-female size is the factor which determinespairing rate in these trials . Larger males appear tobe absolutely faster than smaller males .

In these trials we also have a series of com-parisons between the sizes of males and femalesamong the first fifteen pairs to form and the restof the pool. Paired males are not significantlylarger (Wilcoxon ranked test on mean paired andmean unpaired males in each trial : N = 21,T = 85, P > 0.05) but paired females are signifi-cantly larger than those unpaired (N = 21,T = 23, P < 0.005). If we subdivide the trials intogroups with small, medium and larger males theresult stands : all size classes of males significantlyprefer larger females .

Discussion

The results might be taken individually to supportany of the contentions that sexual selection is oris not and that loading constraints are or are notimportant in determining sexual size dimorphism

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in aquatic Peracarida. But a unifying explanationcannot rely solely on any one experiment and webelieve these contentions are not mutually exclu-sive. We interpret the evidence as reflecting a rolefor both sexual and natural selection .

In Experiments IIb and IIIb large males pre-ferred large females (Table 1) and in VII largerfemales were universally more likely to be paired,

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Fig. 1 . Rate of pair formation in Gammarus pulex compared with size parameters for animals in each of 21 trials . Weight is theaverage for all 30 males and females selected for the trial ; size ratio is the average weight ratio of the first fifteen pairs to form ;index of rate of formation is the calculated time for the 8th of the fifteen pairs, taken from the regression of cumulative timeof formation (see text) .Kendall rank correlation of size parameters against rate index :1) male weight, Tau = 0 .44, is = 2.78, P < 0.012) female weight, Tau = 0.33, is = 2.12, P < 0.053) size ratio, Tau = 0 .44, is = 2 .81, P < 0 .01

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but in IIc and Ma smaller males preferred smallerfemales . An hypothesis to explain both the non-significant results (here and in other work) andthe behaviour of small males is that loadingconstraints interact with male choices . If thechoices of small males are limited by their ownsize relative to the females, then in trials wheresize is not controlled (e .g . experiment I) a non-

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significant pattern would be expected . Supportfor this hypothesis can be found in Vc where manylarge females remained unpaired with either oftwo small males . By contrast, in Vb large maleschose not to pair with small females : their strategycould be interpreted as a discrimination, throughprior experience and expectation, against lowrewards (Hunte et al., 1985).

There are two alternative explanations for thepattern of choices made by males in experimentsIV-VI, where there appears to be no consistentadvantage to large size for males compared toother males (Table 2). First, large males pair moreoften only where small males are restricted bytheir small size relative to the female : this supportsthe loading hypothesis. Second, small males areonly successful where large males discriminatenegatively due to low rewards ; this supportssexual selection . As noted above, large males doindeed discriminate against very small mates .Experiment VII (Fig. 1) also reveals that pairingis faster when the pool male size is larger,although the paired males in each trial are notsignificantly larger than those left unpaired .

The advantage of large size may be due to thepre-experimental conditions : animals kept in sex-restricted groups experience a biased local sexratio which could predispose them to pair withlittle discrimination. Larger males would be un-

restricted by loading and face lower costs, asElwood et al. (1987) have discussed . Smallermales are more selective (Table 1) and initiallytake longer to accept a female . Alternatively,larger males may be absolutely faster in findingmates than small males. Adams & Greenwood(1983) found no evidence that larger animalscould swim faster, but they tested animals onlyunder stream conditions and not in static trays .We have not been able to quantify female

resistance although we have attempted to carryout trials on this . We have, however, observed nolevel of activity that could account for the pairingtime difference in experiment VII in such terms .The level of both female resistance and takeovershas been a consistent contrast between our ownwork and that of Ward (e.g . 1987) who has identi-fied these as critical factors . Whatever their sig-nificance we suggest they cannot be a universalexplanation for the widespread observed dimor-phism .Among species in which males carry their

partner before mating, individuals benefit bybeing male when large or by being female whensmaller. Small paired males are further penalisedby smaller growth increments than single animals(Robinson & Doyle, 1985) . This has implicationsfor the population dynamics of these animals (seealso Ridley & Thompson, 1986) . Long lived

Table 2 . Experiments with an excess of males . Mean sizes (mg ± 95 % c .l .) of paired and unpaired male Gammarus offered accessto only a single female (experiment IV = G. duebeni, random sizes ; experiment V a-d = G. pulex, size-controlled) Experiment VI :twenty randomly sampled male G. pulex presented with ten size-controlled females in trays . There is no significant differencebetween the pool of males in any trial (see text for details) . (L), (S) = large and small in population range ; tests = Wilcoxon signedranks on male size .

N Male size Female size Significance ofdifference

Paired Unpaired

IV 40 95.4+5.1 87.1+4.7 30 .5 + 3 .1 P < 0.05Va 35 58.8+2.1 60.4 + 3 .1 (L) 30 .8 + 1 .5 (L) P > 0.05Vb 30 52.3 +2 .2 54.5 + 2 .6 (L) 12 .4 + 0 .8 (S) P > 0.05

14 59.6+3.6 12.4+1.9Vc 24 31.6+ 1 .0 31 .1 + 1 .9 (S) 28 .6 + 1 .7 (L) P > 0.05

29 29.9+ 1 .1 28 .1 +2.2Vd 35 29.6+1.1 28 .8 + 0 .9 (S) 11 .8 + 0 .9 (S) P>0.05VIa 11 38.8 + 2 .0 43.6+1 .9 13.4 + 0.6 (S) P < 0.005VIb 10 43.8+2.6 40.5 + 1 .8 26.8 + 1 .0 (L) P = 0.053

Crustacea change sex between breeding seasonsto maximise their fitness (Charnov, 1982) butGammarus are more or less annual . In somespecies, including G. duebeni, breeding is highlyseasonal and there is little scope for growth duringthe breeding period . Sex determination is cued bydaylength in this species. Males are producedearly in the season since they will benefit most bythe large size offered by a long growth period .Females lose least by small size and come later(Naylor et a1., 1988) .

It is increasingly obvious, from this study andthose of other groups, that the pattern of sizedimorphism and assortative mating in peracaridscannot be simply ascribed to sexual selection norindeed solely to loading constraints . Elsewhere itis recognised that rigid distinction or attributionof effects solely to sexual or natural selection isunprofitable and may be misleading (Williams,1966; Otte, 1979 ; Banks & Thompson, 1985 ;Sutherland, 1985) .

Summary

1) Males are larger than females and pairing issize-assortative in aquatic Crustacea where malescarry their mates prior to copulation

2) In trials where size is controlled larger maleGammarus prefer larger, more fecund females butsmaller males prefer smaller mates . Larger malesdiscriminate against the smallest females .

3) In random size trials larger males becomepaired more frequently than small males but thispattern disappears where size is controlled andsmall males are just as successful when no largefemales are available .

4) In timed trials larger males are faster to pairup than small males .

5) These results do not consistently support orcontradict either a sexual selection hypothesis forsexual size dimorphism or a theory involvingloading constraints . It appears likely that bothfactors play a role in individual decisions aboutmating .

Acknowledgements

We thank Geoff Parker, Sally Thompson, PaulWard and two anonymous referees for help andcomments . PW is in receipt of Natural Environ-ment Research Council studentshipGT4/86/ALS/20 and CN was in receipt ofstudentship GT4/82/ALS/28 .

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