does size always matter? mate choice and sperm allocation in panulirus guttatus, a highly sedentary,...

Does size always matter? Mate choice and spermallocation in Panulirus guttatus, a highly sedentary,

habitat-specialist spiny lobster

Erika Magallón-Gayón, Patricia Briones-Fourzán1)

& Enrique Lozano-Álvarez(Unidad Académica de Sistemas Arrecifales, Instituto de Ciencias del Mar y Limnología,

Universidad Nacional Autónoma de México. Prol. Av. Niños Héroes s/n, DomicilioConocido, Puerto Morelos, Quintana Roo 77580, Mexico)

(Accepted: 3 October 2011)

Summary

Spotted spiny lobsters, Panulirus guttatus, are small, obligate reef-dwellers that exhibit ahighly sedentary lifestyle and a low tendency to aggregate with conspecifics, and that repro-duce asynchronously year-round. Individual females can produce multiple clutches per yearbut have a short receptivity per clutch. As in most spiny lobsters, females of P. guttatus mateonly once per clutch and resist further mating attempts, features that may favour developmentof female mate choice but limit the potential for sperm competition. We separately examinedmate choice by large and small mature females through laboratory experiments that con-trolled for effects of male–male competition, quality of shelter, and mere social attraction.Only large females expressed preference for larger males relative to their own size, suggest-ing that only large females that mate with small males risk sperm limitation on fecunditysuccess. In couples that mated, males deposited rather small, thinly spread spermatophoreson the sterna of females. Spermatophore area (considered as a proxy measure of sperm con-tent) increased with male size and showed no relationship with female size, suggesting thatmales of P. guttatus have a short sperm-recovery period or do not exhibit strategic spermallocation in a non-competitive context. A comparison of average sperm allocation betweenP. guttatus and its sympatric species, P. argus (a much larger, highly mobile, and highly so-cial species with more seasonal reproductive periods and a longer receptivity of females perclutch), suggests that males of P. guttatus allocate proportionally less sperm to females, onaverage, than males of P. argus do. According to predictions of across-species risk models,this result suggests that males of P. guttatus perceive lower average levels of sperm compe-tition risk than males of P. argus do, implying that different Panulirus species may exhibit

1) Corresponding author’s e-mail address: [email protected]

© Koninklijke Brill NV, Leiden, 2011 Behaviour 148, 1333-1358DOI:10.1163/000579511X605740 Also available online - www.brill.nl/beh

1334 Magallón-Gayón, Briones-Fourzán & Lozano-Álvarez

different mating strategies in accordance with their particular life-history and sociobiologicaltraits.Keywords: coral reef habitat, mate choice, mating system, Palinuridae, sperm allocation,sperm competition risk, spiny lobsters.

1. Introduction

Females of many crustaceans mate at a lower rate than males because fe-males brood the fertilized egg clutches for several weeks or months whilemales can continue to court and mate with other females (Shuster, 2008). Tocompensate for their lower mating rate, females may become more selectivein their choice of mate, generating competition for access to mates amongmales (Jennions & Petrie, 1997; Kokko & Johnstone, 2002). During any re-productive period, males that provide females with more sperm and/or betterresources are more likely to win access to females, resulting in a polygynoussystem wherein some males mate with multiple females and some males donot mate at all (Shuster, 2008; Barry & Kokko, 2010). Yet, with successivematings, males may become sperm-depleted, increasing the risk of spermlimitation on fecundity success of their female partners (Warner et al., 1995;Qvarnström & Forsgren, 1998; Shuster & Wade, 2003). To overcome thisrisk, females may mate with multiple males, generating sperm competition(competition between sperm from more than one male for fertilization ofa given set of ova, Parker, 1970) but still exert ‘cryptic’ choice by, for ex-ample, discarding some ejaculates or delaying oviposition (Eberhardt, 1996;Hinojosa & Thiel, 2003; Jennions & Kokko, 2010). However, production ofejaculates is presumably costly to males (Dewsbury, 1982); therefore, ejac-ulate traits over a male’s lifetime are usually related to the mating context,e.g., the potential for sperm competition to occur and the likely level of anysuch sperm competition (Wedell et al., 2002; Parker & Ball, 2005).

Sperm competition models usually distinguish between a ‘0 versus 1 rivalscenario’ (from the male perspective), which concerns species wherein fe-males typically mate once per clutch but sometimes mate twice, resulting inlow average levels of sperm competition or no sperm competition at all, anda ‘1 versus several rivals scenario’, which concerns species wherein typicallya female mates with several males, resulting in high average levels of spermcompetition (for recent reviews, see Ball & Parker, 2007; Parker & Pizzari,2010; Kelly & Jennions, 2011). Different models make different predictions

Mate choice and sperm allocation in P. guttatus 1335

as to how males of a given species should allocate their sperm according tothe perceived level of sperm competition (Jennions & Petrie, 1997; Engqvist& Reinhold, 2006; Kelly & Jennions, 2011). However, several models pre-dict that, across species, sperm allocation should increase in the 0 versus 1rival risk range, in particular when female resistance to mating attempts bymales reduces the variance in females’ mate numbers (Parker & Ball, 2005;Fromhage et al., 2008; Parker & Pizzari, 2010).

Spiny lobsters (Palinuridae) are large decapod crustaceans that offer avaluable opportunity to compare mate choice and sperm allocation acrossrelated species, yet few studies have addressed these issues (e.g., MacDi-armid & Butler, 1999; MacDiarmid & Sainte-Marie, 2006). Spiny lobstersexhibit a modified form of external fertilization as, during copulation, themale plasters a paired spermatophore on the female sternum; thus, there areno physical constraints to mating between males and females of different rel-ative sizes. In the clade Silentes (e.g., the temperate genus Jasus), the sper-matophores are gelatinous and short-lived, and the females must use themwithin minutes to fertilize her single annual clutch. By contrast, in the cladeStridentes (e.g., the tropical-subtropical genus Panulirus), the more complexspermatophores consist of a basal adhesive matrix that cements to the fe-male sternum, a middle matrix in which the highly convoluted tubule con-taining the sperm is embedded, and an outer protective matrix that becomeshard shortly after deposition, thus allowing females to mate well before theirovaries are ripe. When the female is ready to spawn, she scratches the sper-matophore with her pereopods to release the sperm, but the ‘eroded’ sper-matophore can remain attached to her sternum for some time. The femalethen broods the fertilized eggs on her pleopods for several weeks until hatch-ing (Matthews, 1951; Berry, 1970; Berry & Heydorn, 1970; Chitty, 1973;George, 2005).

The substantial difference in the time-lag from mating to spawning be-tween Jasus and Panulirus suggests that the potential for female choiceshould be greater in the former than in the latter (MacDiarmid & Sainte-Marie, 2006). However, such time-lag actually varies widely among Pan-ulirus species, from a few hours to up to several weeks (review in Mac-Diarmid & Sainte-Marie, 2006). Although limited evidence for multiplematings by individual females has been found in a few species with longtime-lags (e.g., P. argus and P. laevicauda from Brazil, Mota-Alves & Paiva,


1976; P. cygnus from Australia, Melville-Smith et al., 2009), most Pan-ulirus females typically mate only once per clutch and resist further mat-ing attempts (Berry, 1970; Lipcius et al., 1983; Lipcius & Herrnkind, 1985;Robertson, 2001; MacDiarmid & Sainte-Marie, 2006), suggesting that mostPanulirus males perceive a level of sperm competition in the 0 versus 1 ri-val range (Engqvist & Reinhold, 2005, 2006; Fromhage et al., 2008). There-fore, a comparison of average sperm allocation and other relevant life-historytraits may provide insights into the relative perceived levels of sperm com-petition risk across Panulirus species.

Panulirus argus and P. guttatus co-occur across the wider Caribbean re-gion. Although in Florida (USA) and Mexico no evidence of multiple sper-matophores has been found in hundreds of examined females of these species(Chitty, 1973; Briones-Fourzán & Contreras-Ortiz, 1999; MacDiarmid &Butler, 1999; Bertelsen & Matthews, 2001), the two species differ in manyreproductive traits that may impact female choosiness and the perceived levelof sperm competition risk in males. In P. argus, females can mate up to onemonth before spawning (Sutcliffe, 1953), large males are capable of adjust-ing the area of their spermatophores to the size of the female partners, andspermatophore area has a greater influence than female size on subsequentfecundity success, suggesting that P. argus females in general should pre-fer to mate with large males to avoid sperm limitation and that males per-ceive a high level of sperm competition risk (MacDiarmid & Butler, 1999).In contrast, P. guttatus females appear to mate only within 24–48 h beforespawning and fecundity success is only impacted in large females that matewith considerably smaller males, suggesting that only large females shouldseek to mate with large males to avoid sperm limitation on fecundity suc-cess (Robertson, 2001). These findings, however, also suggest that males ofP. guttatus do not tend to adjust the area of the spermatophore to the sizeof the female, which may reflect a low perceived level of sperm competitionrisk. Therefore, the aims of the present study were (1) to examine femalemate choice in P. guttatus in laboratory experiments to test the predictionthat only large females express preference for large males, (2) to examine therelationship between spermatophore area, male size, and female size in mat-ing events occurring during these experiments to determine whether malesadjust the area of their spermatophores to the size of the female, and (3) tocompare relative average sperm allocation between P. guttatus (estimated inthis study) and P. argus (estimated using data from MacDiarmid & Butler


(1999)) to provide insight into the potential levels of sperm competition riskperceived by males of these sympatric species.

2. Material and methods

2.1. Study species

Panulirus guttatus is a small-sized (�90 mm in carapace length), strictlynocturnal, and highly cryptic spiny lobster. These lobsters remain deeplyhidden in reef crevices during the day and exhibit a low tendency to cohabitwith conspecifics (Sharp et al., 1997; Briones-Fourzán & Lozano-Álvarez,2005, 2008). They only emerge for a brief interval at night to forage close totheir dens and not necessarily on a daily basis (Lozano-Álvarez & Briones-Fourzán, 2001; Segura-García et al., 2004; Wynne & Côté, 2007). Giventheir highly sedentary nature and limited home range, individuals of P. gutta-tus possibly remain on the same reef patch for their entire benthic life, poten-tially resulting in highly fragmented populations of variable sizes (Lozano-Álvarez et al., 2002; Negrete-Soto et al., 2002; Robertson & Butler, 2009).Consequently, all reproductive cycles of a female occur on the same reefpatch (Briones-Fourzán & Contreras-Ortiz, 1999; Robertson, 2001). Malesmature at a slightly larger size than females (Chitty, 1973; Robertson & But-ler, 2003), but the size at onset of maturity and the extent of the breedingseason vary geographically. In the Caribbean coast of Mexico, where thepresent study took place, breeding occurs year-round with no discernablepeaks but shows a nadir for a short period of one to two months, usually dur-ing late summer. Ovigerous females occur from 36.0 mm carapace length(CL) but are rare below 42.0 mm CL, and females >50 mm CL can pro-duce four (and possibly more) clutches per annual cycle (Briones-Fourzán &Contreras-Ortiz, 1999; Negrete-Soto et al., 2002).

2.2. Collection and holding conditions

Lobsters were captured by hand during nocturnal SCUBA divings along thePuerto Morelos coral reef system (Mexican Caribbean coast) and transportedimmediately to our research facilities at Puerto Morelos, where they weresegregated by sex in several holding tanks (3 m in diameter with approx.5600 l seawater) fitted with multiple shelters. The holding tanks (and all


experimental units mentioned below) received seawater from an open-flowsystem and were in the open but under shade. We measured CL of eachlobster to the nearest 0.1 mm, then tied a nylon thread holding a uniquelynumbered flag tag around the carapace. Individual lobsters were held for 1–2 weeks on average and fed to satiation every other day with a mixture offresh/frozen shrimp, clams, mussels, squid and fish.

2.3. Pilot experiment: testing for female receptivity and social attraction

Spiny lobsters depend on available structured shelter for survival and ex-hibit variable degrees of gregarious den sharing, a social behaviour that ismediated by conspecific chemical attraction (review in Childress, 2007).Although P. guttatus lobsters in particular exhibit a low tendency to sharedens, they do show some degree of conspecific chemical attraction (Briones-Fourzán & Lozano-Álvarez, 2005). Therefore, it was critical for our matechoice experiments to determine the extent of the receptivity period of fe-males, because non-receptive females might still ‘choose’ a male for socialreasons other than mating (Roughgarden & Akçay, 2010). Between Marchand August 2006, we conducted a pilot experiment to determine (1) the timeof mating as related to the degree of ovarian maturation and (2) the ten-dency, or lack of tendency, of females with ovaries in different stages ofmaturation to cohabit with males. As maturation progresses, the ovaries —visible through the transparent membrane between the cephalothorax andabdomen — increase in volume while changing in colour from off-white(stage 1, undeveloped) to salmon pink (stage 2) to orange (stage 3) to brick-red (stage 4, fully ripe) (Chitty, 1973). Also, stage-4 ovaries fill all availablespace within the cephalothoracic cavity and their posterior lobes extend wellinto the abdominal region. Twenty females (45.0–62.5 mm CL) with ovariesin stages 1–3 were individually placed in separate small tanks (1 m in diame-ter with approx. 250 l seawater) together with one mature male (size rangeof males: 44.0–72.4 mm CL). The female and the male were individuallypositioned inside one of two identical shelters located at opposite parts ofthe tank. The shelters were inverted clay pots with a low lateral entranceand sufficiently large (0.28 m in diameter × 0.32 m tall) so as to looselyaccommodate both lobsters. Because P. guttatus lobsters tend to cling to theinterior walls or ceiling of reef crevices, we lined the pots interiorly with awire mesh slightly separated from the walls to provide gripping surface for


the lobsters (Lozano-Álvarez & Briones-Fourzán, 2001). The shelters had alarge hole at the top that remained covered during the trials. Twice a day (at10:00 h and 18:00 h) for up to 30 days, we recorded the location of the maleand female in each tank by uncovering and looking through the shelter holes.The shelter containing the female was carefully turned upside-down so thatwe could monitor the female, always underwater, for ovarian maturation andfor the presence of a spermatophore on her sternum. The feeding regime wasthe same as for the holding period.

In 11 of the 20 females used for the pilot experiment, ovarian maturationdid not progress beyond stage 3. Not one of these females mated and theyshowed a similar tendency to cohabit with the male (44%, N = 126 record-ings) as to occupy a separate shelter (56%) at any recording time (Yates-corrected χ2

1 = 2.04, p = 0.153). The other nine females eventually reachedovarian stage 4. Two spawned without mating and the rest mated between 1and 3 days after reaching ovarian stage 4 and spawned between 0 and 1 dayafter mating. These females consistently cohabited with the male for 24 to 36h before mating. Therefore, we only used females with stage-4 ovaries in ourmate choice experiments to reduce the chances that females might ‘choose’a male for social reasons other than mating.

2.4. Experimental design of the mate choice experiments

To test the prediction that only large females would prefer to mate with largemales, females with stage-4 ovaries (42.2–68.7 mm CL) were categorizedinto large (�53 mm CL, N = 48) and small (�52 mm CL, N = 48)based on the median size of the local female population (52.5 mm CL),and subjected to separate mate choice experiments. Each experiment con-sisted of three treatments. In two treatments (N = 12 trials each) the femalewas offered a choice between two equally-sized males (difference in CL be-tween the males: −0.1 ± 0.1 mm, mean ± SE), both of which were eitherLarge (treatment LL) or Small (treatment SS), and in the other treatment(N = 24 trials) the female was offered a choice between one large maleand one small male (treatment LS) (Underwood et al., 2004; Underwood &Clarke, 2005). Large males were substantially larger than the correspondingtest female (10.8 ± 0.20 mm CL, mean ± SE) whereas small males weresmaller than or, due to size constraints in a few trials, approximately equal insize to the female (from −8 mm to +1.5 mm CL the size of the test female;


−2.0 ± 0.25 mm CL on average). To avoid potentially confounding effectsof season and behavioural changes through the moult cycle, we interspersedtrials from the three treatments of the two experiments across the experimen-tal period (February to July 2007) and used exclusively males in intermoult,as determined by microscopic examination of the tip of a pleopod (see Lyle& MacDonald, 1983). All 96 test females were used only once but 33 of the192 experimental males were used twice also due to size constraints. Thesemales, however, were returned to the holding tanks for at least eight daysbefore being used again. Lobsters were not fed for the duration of the trials(see below) but were held and fed for a few days after the end of the trialsbefore being returned to the reef habitat.

The experimental units were four opaque fiberglass Y-mazes, each withtwo head tanks (Briones-Fourzán & Lozano-Álvarez, 2005). The Y-maze(2.0 m long × 0.8 m wide × 0.6 m tall) contained approx. 500 l seawaterwhen filled to a standpipe height of 0.3 m. A sealed panel measuring 1.0 mlong × 0.6 m tall divided half the length of the Y-maze into two equal arms.Seawater flowed into the head tanks and then from each head tank to an armof the Y-maze at a rate of 2.0 l/min. The water then mixed in an open areabefore flowing out through the standpipe (Figure 1).

Large spiny lobster males defend high-quality shelters and can hinder themating attempts of smaller males through aggressive behaviour (Berry, 1970;Lipcius & Herrnkind, 1985; Lozano-Álvarez & Briones-Fourzán, 2001;Robertson, 2001), but even their mere proximity may induce behaviouralor chemical changes in nearby males that may help females to indirectlyassess dominance (Raethke et al., 2004). Therefore, it was important to con-trol for the potentially confounding effects of shelter quality and male–malebehavioural or chemical interactions on female choice (Crespi, 1989; Back-well & Neville, 1996; Taborsky et al., 2009; Roughgarden & Akçay, 2010).To control for shelter quality, we provided the test female and each of thetwo males with identical shelters of the same type used in the pilot experi-ment. To isolate the males out of reach of physical and sensory contact witheach other (and to preclude them from pursuing the female), the male shel-ters were positioned in the upstream parts of the arms of the Y-maze, withthe entrances facing downstream, and one male was tethered to each shel-ter with a nylon thread (0.4 m in length) (Díaz & Thiel, 2003). One end ofthe thread was secured with a swivel clip to a tie around the male carapacewhile the other end was tied to the protruding section of a PVC pipe (2 cm


Figure 1. Panulirus guttatus. Schematic representation of the three treatments (LL, SS, LS)used in mate choice experiments: LL: both males were large; SS: both males were small; LS:one male was large and the other male was small. The males were tethered to their sheltersand the female was left unrestrained in her shelter. The large circles represent the sheltersviewed from above and the small circles represent the ‘observation holes’, which remained

covered during the trials. The arrows denote the sea water flow.

in diameter) perpendicularly fixed to the top of the shelter (Figure 1). Thistethering technique restrained the distance moved by the males but allowedthem to move around, enter, and exit their shelters without entanglement.The female shelter was placed in the open area (i.e., the downstream part) ofthe Y-maze with its entrance facing upstream so that the unrestrained femalecould receive chemical cues from both males, and we positioned a semicir-cular wire screen behind her shelter to prevent her from using the corners ofthe open area as refuge (Figure 1). To ensure that no chemical cues remainedafter each trial, the Y-mazes were drained and thoroughly brushed, and waterwas allowed to flow at a high rate until the beginning of the next trial.


All trials began 1 h before sunset. First, the males were randomly allocatedand tethered to their shelters and then the female was placed inside hershelter, which remained enclosed in a mesh cylinder for an acclimatizationperiod of 3 h, after which the cylinder was removed. Originally, we had setthe duration of the trials for 12 h because we expected the receptive femalesto choose a male at some time during the night and to remain in that male’sshelter for the rest of the night as occurred in the pilot experiment. During thefirst few trials, however, most females were still in their own shelters after12 h with no signs of having mated. Therefore, we ran these trials until thesecond morning (i.e., a 36-h trial). By then, all females were found in a maleshelter and those that were already in a male shelter after 12 h were still inthat same shelter. These findings were consistent with the pilot experimentand with other experiments in which 24-h video recordings of the activitypattern of P. guttatus lobsters during up to six consecutive days revealed thatno individuals ever left their shelters during the daytime and only a few didso for a short period during any night (Lozano-Álvarez & Briones-Fourzán,2001; Segura-García et al., 2004). Therefore, we ran all trials for 36 h andconsidered that a female made a choice if she was found paired with one ofthe males in his shelter even if mating had not yet occurred. If she also hadan intact spermatophore, or an eroded spermatophore and a clutch of eggs,we considered that she had mated with that male. If a female was found inher own shelter with no traces of spermatophore, she was considered as nothaving made a choice. Females that mated during the trials were transferredto a separate tank to determine the time lag to spawning. For females thatwere already brooding eggs at the end of their trials, the time lag to spawningwas conservatively estimated as 1 day.

Table 1 shows the layout of the data from the experimental design. Thenull hypothesis of random mate choice is q1 = θp1, q2 = θp2, where p1 andp2 are the proportions of females choosing a male in treatments LL and SS,respectively; q1 and q2 are the proportions of females choosing a large maleor a small male, respectively, in treatment LS, and θ is a constant, unknownparameter (Underwood & Clarke, 2005). If females showed a preference formales of a given size, q1 and q2 would not be equal to p1 and p2. However,because the experimental results conform an asymmetrical contingency table(Table 1), the expected proportions have to be estimated using a maximumlikelihood procedure. This procedure was originally developed by Under-wood & Clarke (2005: Appendix A, Section A3) and is reproduced here forconvenience (the terms M1, m1, M2, m2, n1 and n2 are defined in Table 1).


Table 1. Asymmetrical contingency table for Panulirus guttatus derivedfrom the mate choice experimental design.

Treatment Females that chose a male Females that did not choose a male Total

LL m1 M1 − m1 M1SS m2 M2 − m2 M2LS n1 (large) n2 (small) N − n1 − n2 N

Large and small mature females were subjected to separate experiments consisting of threetreatments in which individual females were allowed to choose between two males. In treat-ment LL (N = 12 trials) both males were large, in treatment SS (N = 12 trials) both maleswere small, and in treatment LS (N = 24 trials) one male was large and the other was small.Expected numbers were estimated using a maximum likelihood procedure.

For p1, the proportion of females that chose a (large) male in treatmentLL:

ap̂31 + bp̂2

1 + cp̂1 + d = 0

where:

a = (M1 + M2)(M1 − n2)

b = m1n2 + m2n1 + M1(M1 − M2) + M1(n1 + n2 + m2 − 2m1)

+ M2(2n2 − m1)

c = −[m1n1 + m1n2 + 2m2n1 + m1m2 − m21

+ M1(2m1 + m2 + n1) + M2(n2 − m1)]d = (m1 + m2)(m1 + n1)

This can be solved iteratively for p̂1 using:

p̂1(1) = p̂1(0)−[a(p̂31(0))+b(p̂2

1(0))+c(p̂1(0))+d]/[3a(p̂21(0))+2b(p̂1(0))+c]

where p̂1(0) = m1/M1 as the initial value and replacing p̂1(0) by p̂1(1) foreach iteration, which quickly converges.

The other parameters are then estimated as:

p̂2 = m1 − M1p̂1 + m2(1 − p̂1)

m1 − M1p̂1 + M2(1 − p̂1)

q̂1 = θ̂ p̂1 = p̂1(n1 + n2)

(p̂1 + p̂2)N

q̂2 = θ̂ p̂2 = p̂2q̂1

p̂1


The observed and expected numbers are then subjected to an accurate χ2

test with 1 degree of freedom, which gives a more realistic answer thana naïve χ2 test because it properly controls Type I error rates, especiallyfor small sample sizes (e.g., numbers of replicates) (Underwood & Clarke,2005).

2.5. Sperm allocation

In P. argus, spermatophore area is a reasonable proxy measure of spermallocation because there are strong relationships between spermatophorearea and weight, and between spermatophore weight and number of sperm(MacDiarmid & Sainte-Marie, 2006). In P. guttatus, the potential for count-ing sperm from spermatophores is limited due to the short time lag betweenmating and spawning. Thus, out of 850 females examined in Florida (Chitty,1973) and 891 examined in Mexico over several years (Briones-Fourzán &Contreras-Ortiz, 1999; Negrete-Soto et al., 2002), only 6 and 11 females,respectively, carried intact spermatophores. However, because the fecunditysuccess of large females of both P. argus and P. guttatus was negatively im-pacted when they mated with considerably smaller males (MacDiarmid &Butler, 1999; Robertson, 2001), and spermatophore area in P. argus waspositively related with male size (MacDiarmid & Butler, 1999), we consid-ered that if the relationship between spermatophore area and male size inP. guttatus was also positive, then spermatophore area could also be used asa reasonable proxy measure for sperm allocation in this species. From allmating events that took place in the mate choice experiments and the pilotexperiment, we traced the outlines of intact and eroded spermatophores ona transparent acetate sheet with a fine-point indelible marker and scannedthem as digital images to calculate their total area (in mm2) using commer-cial software (MacDiarmid & Butler, 1999; Rubolini et al., 2006; Galeottiet al., 2009). First, we examined the relationship between spermatophorearea and male size (CL) with a simple linear regression. Then, we did a fac-torial regression analysis to examine a potential interaction effect betweenmale size and female size (independent predictors) on spermatophore area(dependent variable). To reduce co-linearity between the predictors and theinteraction term, the factorial model was fitted to data with centred predictors(i.e., by subtracting the mean of the predictor from each datum) (Quinn &Keough, 2002). Finally, we examined the relationship between spermato-phore area and the difference in size between mating partners (male CL mi-nus female CL).


2.6. Average sperm allocation: P. guttatus versus P. argus

We used two methods to compare average sperm allocation between P. gutta-tus and P. argus. First, we qualitatively compared spermatophore ‘coverage’relative to the five segments of the female sternum. For P. guttatus, cover-age was examined in all mating events that took place during our experi-ments. For P. argus, coverage was examined in 20 females with intact sper-matophores observed in situ at a local fishing pier and in several photographsof sterna of recently mated females provided by Dávila et al. (2007). For amore quantitative comparison that would control for the differential sizes be-tween the smaller P. guttatus (maximum size 90 mm CL) and the larger P.argus (200 mm CL), we devised a dimensionless ‘sperm allocation index’defined as the ratio of the square-root of the spermatophore area to the sizeof the female (CL). For P. guttatus, this index was averaged across matingevents. For P. argus, the index was estimated using the equations for sperma-tophore area versus female size provided by MacDiarmid & Butler (1999).

3. Results

3.1. Female mate choice

In the experiment with large females (Figure 2a), 11 of the 12 test femaleschose a male in treatment LL, as did 11 of the 12 females in treatment SS.In treatment LS, however, 13 of the 24 test females chose the large male,four chose the small male, and seven were found in their own shelter with nosigns of having mated. The result of the exact test was marginally significant(χ2

1 = 4.445, p = 0.035), indicating that large females showed preferencefor large males.

In the experiment with small females (Figure 2b), 10 of the 12 test femaleschose a male in treatment LL and 11 of the 12 test females chose a male intreatment SS. However, of the 24 females in treatment LS, 11 chose the largemale, 10 chose the small male, and three were found in their own shelter.The result of the exact test was not significant (χ2

1 = 0.170, p = 0.680),indicating that small females did not show preference for males of eithersize class.


Figure 2. Panulirus guttatus. Observed versus expected numbers of (a) large females and(b) small females that chose a large male (Large), a small male (Small), or no male (None) ineach of three treatments constituting the mate choice experiments. In treatment LL (N = 12trials), both males were large, in treatment SS (N = 12 trials), both males were small, and intreatment LS (N = 24 trials), one male was large and the other was small. Expected numbers

were estimated using a maximum likelihood procedure.

3.2. Mating and spawning events

In addition to the seven females that mated during the more extended pilotexperiment, 17 females mated during the shorter mate choice trials (Table 2).


One died of unknown causes shortly after, and another one completely re-moved the spermatophore and did not spawn. The other 15 females spawnedfrom one to five days after mating (Table 2). The overall mean ± SE time lagbetween mating and spawning was 1.3±0.23 days (N = 22, Table 2). Sevenadditional females (44.1–54.5 mm CL, not shown) spawned during their tri-als without evidence of having previously mated and subsequently lost theirunfertilized clutches.


In five of the 24 mating events, the females left insufficient spermatophoretraces to reliably estimate its area (Table 2), and in another event the sperma-tophore was so large (135.0 mm2) relative to the rest (51.6–91.7 mm2) thatit stood out as an extreme outlier (Table 2). Data from this event were ex-cluded from the regression analyses to avoid influencing the results (Quinn& Keough, 2002). Spermatophore area significantly increased with male size(R2 = 0.293, β = 0.541 ± 0.210 (mean ± SE), F1,16 = 6.617, p = 0.020)(Figure 3a). In contrast, the result of the factorial regression was not signifi-cant (F3,14 = 2.347, p = 0.117, R2

adj = 0.192, intercept = 70.259 ± 2.741(mean ± SE)) because, although spermatophore area increased significantlywith male size (partial correlation: β = 0.651 ± 0.273, t = 2.381, p =0.031), it was not affected by female size (β = 0.016 ± 0.239, t = 0.068,p = 0.947) (Figure 3b) or by the interaction term (t = 0.928, p = 0.369).Results were qualitatively unchanged if sperm area was square-root trans-formed to homogenize dimensionality of variables. The relationship betweenspermatophore area and difference in size between mates was also not sig-nificant (R2 = 0.154, F1,16 = 2.923, p = 0.107).


The spermatophores of P. guttatus were small, typically covering only a por-tion of the surface of the fifth segment of the female sternum but occasion-ally, when the male was substantially larger than the female, a narrow por-tion of the fourth segment as well. Regardless, in all cases, the paired sper-matophores were thinly spread and distinctly separated, one on each side ofthe midline of the female sternum. In contrast, the paired spermatophores ofP. argus were thickly spread over the entire surface of the fifth, fourth, and


Table 2. Relevant data from mating events of Panulirus guttatus duringthe pilot experiment and the mate choice experiments for small and large

females.

Female CL Male CL Male CL minus Spermatophore Time lag to(mm) (mm) female CL (mm) area (mm2) spawning (days)

Pilot experiment45.5 53.7 8.2 53.31 148.1 58.2 10.1 62.44 148.5 72.1 23.6 – 052.6 59.5 6.9 – 056.4 72.0 15.6 80.80 158.4 61.2 2.8 70.53 162.5 57.0 −5.5 – 0Mate choice experiments

Small females42.2b 55.9 (LL) 13.7 68.82 246.8b 47.2 (LS) 0.4 59.82 248.4a 48.0 (LS) −0.4 – 148.5a 60.1 (LS) 11.6 86.50 150.2b 64.7 (LS) 14.5 134.99e 550.3a 48.6 (LS) −1.7 51.98 151.0b 50.8 (LS) −0.2 60.97 351.5b 51.3 (SS) −0.2 91.66 251.8a 61.9 (LS) 10.1 72.04 1

Large females54.3a 48.2 (SS) −6.1 51.58 154.7c 65.0 (LS) 10.3 76.7555.4a 66.5 (LS) 11.1 – 156.0d 64.0 (LL) 8.0 74.0756.1b 48.4 (SS) −7.7 71.75 258.3a 52.4 (SS) −5.9 61.90 158.6a 57.6 (SS) −1.0 71.88 160.4a 70.8 (LL) 10.4 75.73 1

Letters in parentheses after male size denote the corresponding mate choice treatment: (LL)both males were large, (SS) both males were small, (LS) one male was large and one malewas small. In LS, the chosen male is underlined. CL, carapace length; –, spermatophore tracesinsufficient to reliably estimate area.a Female spawned during trial.b Female spawned after trial.c Female died 2 days after mating without spawning.d Female removed spermatophore without spawning.e Extreme outlier.


Figure 3. Panulirus guttatus. Scattergrams showing the relationships between spermato-phore area (mm2) and (a) male size (carapace length, mm), (b) female size. The regression

equation is indicated for (a).

occasionally third, sternal segments of the female, merging or even overlap-ping over the midline of the female sternum (e.g., see Dávila et al., 2007).The sperm allocation index was 0.16 for P. guttatus (average female sizeand spermatophore area: 52.6 mm CL and 72.5 mm2) and 0.31 for P. argus(average female size and spermatophore area: 95.0 mm CL and 861.0 mm2,respectively).

4. Discussion

4.1. Female choice

The pilot experiment confirmed that females of P. guttatus have a shorttemporary dissociation between ovarian maturity, receptivity, mating, andspawning, which should increase the potential for female mate choice. Yet,by subjecting large and small receptive females to separate experiments con-trasting three different treatments in which females were given a choice be-tween equally-sized or differentially-sized males while controlling for thepotentially confounding effects of shelter quality, male–male competition,and mere social attraction, the prediction was supported that only largefemales would tend to choose large males. This result is consistent withRobertson’s (2001) finding that in P. guttatus male size did not generally af-fect the fecundity of females but that fecundity success was impacted in largefemales (>55 mm LC) that mated with substantially smaller males (�45 mmCL).


However, mate choice occurs at the level of populations, not species(Crespi, 1989). This consideration is particularly relevant for P. guttatus be-cause this species is distributed in highly fragmented populations with vary-ing socio-sexual contexts (e.g., lobster density, size distribution of malesand females, sex ratio) (Lozano-Álvarez et al., 2007; Wynne & Côté, 2007;Robertson & Butler, 2009), which may affect the degree of choosiness oflocal females (Jennions & Petrie, 1997; Wong & Candolin, 2005). That is,a female may be able to choose a suitable male when there are many malesto choose from, but not when males are scarce (Kokko & Rankin, 2006).Moreover, in Panulirus females, failing to mate in time will result in theextrusion of the unfertilized clutch or in extensive resorption of ova in theovaries (Chitty, 1973; Lipcius & Herrnkind, 1985; MacDiarmid & Sainte-Marie, 2006). These time constraints may further override female choosiness(Backwell & Neville, 1996; Taborsky et al., 2009), especially if the time lagbetween receptivity, mating, and spawning is short, as is clearly the case forP. guttatus. Thus, large females of P. guttatus about to spawn will probablynot forgo an opportunity to mate with a small male if large males are notavailable, potentially explaining why most of the large females in treatmentSS readily paired with a small male.

On the other hand, our visual estimation of ovary ripeness was not a pre-cise indicator of the degree of female receptiveness. Thus, 83 of the 96 testfemales paired with one male but only 17 actually mated, while an additionalseven spawned without having mated. Although not all receptive crustaceanfemales necessarily mate in experimental conditions (e.g., MacDiarmid &Butler, 1999; Díaz & Thiel, 2003; Dávila et al., 2007), these results sug-gest that some test females were not yet ready to mate throughout the trialswhile others had already reached the critical time for spawning early dur-ing the trials. And yet, one female that did mate completely removed thespermatophore and did not spawn. MacDiarmid & Butler (1999) and Dávilaet al. (2007) also recorded removal of spermatophores by females of P. ar-gus, while Chitty (1973) observed that two females of P. guttatus did notspawn after mating and underwent extensive resorption of ova. It remains tobe determined whether this behavior in Panulirus females is associated withstress due to captivity or indicates some type of cryptic choice (e.g., Thiel &Hinojosa, 2003; Sainte-Marie, 2007).



The relationship between ejaculate size, male size, and female size exhibitsdifferent patterns in different crustacean species (e.g., MacDiarmid & Butler,1999; Hinojosa & Thiel, 2003; Brockerhoff & McLay, 2005; Rubolini et al.,2006; Sainte-Marie, 2007), probably because ejaculate traits are generallyassociated with the risk of sperm competition in any species (Williams et al.,2005; Ball & Parker, 2007; Parker & Pizzari, 2010). In experiments in whichindividual males of P. argus were allowed to mate with several females ofdifferent sizes in the absence of rivals, spermatophore area increased withboth male and female size and had a greater impact on fecundity successthan female size, indicating that large males in particular exhibit strategicsperm allocation (MacDiarmid & Butler, 1999). In our experiments, whereinindividual males of P. guttatus had the opportunity to mate with only onefemale in the absence of rivals, spermatophore area increased with male sizebut showed no apparent relationship with female size. This pattern agreeswith Robertson’s (2001) finding that, even in a competitive context (i.e.,multiple males and females confined in individual tanks), only large femalesthat mated with considerably smaller males showed a reduction in fecundity.

In conjunction, these findings suggest that large males of P. guttatus do notexhibit strategic sperm allocation. This contention, which relies on the as-sumption that spermatophore area reflects sperm content, needs to be furthertested by, for example, confronting individual males with several females ofdifferent sizes (e.g., MacDiarmid & Butler, 1999) because the rationale formale strategic allocation is that the amount of sperm reserves will affect thetrade-off between fertilization success in present and future matings. In na-ture, however, the mating rate of males of a given species depends on thedistribution of female receptivity in time and space, the presence/absenceof potential rivals, and the time required to replenish sperm reserves (Barry& Kokko, 2010), features that are likely to differ between P. argus and P.guttatus (see below). Also, the physiological task of adjusting ejaculate sizepresumably has some associated costs which for some species may be sec-ondary to the cost associated to lost mating opportunities due to insufficienttime to recover sperm stores, but for other species can make strategic spermallocation maladaptive (Kelly & Jennions, 2011). For example, in labora-tory experiments, males of the freshwater crayfish Austropotamobius itali-cus were unable to adjust the area of their spermatophores in the presence of


one or more rivals (Galeotti et al., 2009). Further studies are needed to un-ravel these still little-explored issues in decapod crustaceans in general andPanulirus lobsters in particular.

Interestingly, a recent meta-analysis of empirical studies revealed that,contrary to predictions of classical within-species sperm competition mod-els (but see Ball & Parker, 2007), males in a 0 versus 1 rival scenario actu-ally transfer larger ejaculates to non-mated females that are unlikely to mateagain than to females that are likely to mate again (Kelly & Jennions, 2011).Thus, males of P. guttatus may attempt to transfer as large a spermatophoreas possible at any mating opportunity they can obtain because the availabilityof receptive females is probably low at any given time (see below), femalesare unlikely to mate more than once per clutch, and males are unlikely tomate in rapid succession with another female. But if large males of P. gut-tatus retain sperm reserves better than small males do, as occurs in manycrustacean species (Jivoff, 1997; MacDiarmid & Butler, 1999; Hinojosa &Thiel, 2003; Sato et al., 2006; Sato & Goshima, 2007), then these males maybe able to deposit proportionally larger spermatophores than those of smallmales even after successive matings.


Female and male reproductive structures occur in three combinations in thegenus Panulirus: (1) female sternum fully calcified and male penile processwith a curved smooth crest with no ‘hairbrush’; (2) female sternum with oneor more pairs of soft decalcified areas and male penile process as in combina-tion 1, and (3) female sternum as in combination 2 and male penile processwith a pointed serrated crest with a distinct hairbrush (George, 2005). De-calcified areas of the female sternum possibly serve as a guide for males todeposit appropriately sized spermatophores, but both P. guttatus and P. argusexhibit combination 1, which is considered the plesiomorphic condition forthe genus (George, 2005). However, males of P. argus produce rather thickspermatophores that cover the entire surface of the last two to three seg-ments of the female sternum (e.g., see MacDiarmid & Sainte-Marie, 2006;Dávila et al., 2007), whereas males of P. guttatus produce remarkably thinspermatophores, as previously noted by Chitty (1973), that typically covera portion of the last female sternal segment only. Even accounting for thelarge difference in size between the two species, their sperm allocation in-dices (0.31 versus 0.16) indicate that P. argus males allocate proportionally


more sperm to individual females, on average, than P. guttatus males do. Ac-cording to across-species sperm competition models (Parker & Ball, 2005;Fromhage et al., 2008; Parker & Pizzari, 2010), this would suggest that malesof P. argus perceive a higher level of sperm competition risk than males ofP. guttatus. This notion agrees with the differential life-history and sociobi-ological traits between these sympatric species.

In P. guttatus, the distribution of female receptivity is likely to be highlydispersed in time due to asynchronous, year-round reproductive activity, po-tentially resulting in a low availability of mates for males at any given time.For these small, highly cryptic and rather asocial lobsters, the cost associatedwith mate searching is probably high because they are quite vulnerable topredators (Sharp et al., 1997; Briones-Fourzán et al., 2006; Lozano-Álvarezet al., 2007), partially explaining why even at night many individuals remainin their shelters (Lozano-Álvarez & Briones-Fourzán, 2001; Segura-Garcíaet al., 2004). Under these conditions, males should attempt to mate at anyopportunity they can obtain (Dewsbury, 1982; Kokko & Johnstone, 2002;Barry & Kokko, 2010). However, their highly sedentary lifestyle and lim-ited home range possibly constrain the rate of encounters with mates andalso with rivals. In conjunction with the short receptivity per clutch of fe-males, these traits may reduce the perceived level of sperm competition riskfor males (Engqvist & Reinhold, 2005; Parker & Pizzari, 2010), leading to asmall average sperm allocation in this species.

In P. argus, by contrast, the distribution of female receptivity is probablymore concentrated in time because this species has more seasonal breed-ing periods during which individual females rarely produce more than twoclutches (Lipcius, 1985; Cruz & de León, 1991; Padilla-Ramos & Briones-Fourzán, 1997; Bertelsen & Matthews, 2001). Also, adults of P. argus arefar less vulnerable to predators because they grow to considerable sizes, arehighly mobile and gregarious, and express coordinated antipredator groupbehavior (Herrnkind et al., 1975, 2001; Briones-Fourzán et al., 2006). Thesetraits may increase the potential for simultaneous encounters with mates, butalso with rivals (Kokko & Johnstone, 2002; Wong & Candolin, 2005; Barry& Kokko, 2010), which in conjunction with the long receptivity per clutchof P. argus females (up to one month, Sutcliffe, 1953), may increase the per-ception of sperm competition risk among males, leading to a high averagesperm allocation in this species.


In conclusion, large females of P. guttatus expressed preference for largemales in controlled laboratory experiments, suggesting that large femalesthat mate with small males risk sperm limitation on fecundity success. How-ever, in nature, the degree of choosiness of P. guttatus females may be con-strained by their short receptivity period and the abundance and size of lo-cally available mates. Spermatophore area (considered as a proxy measurefor sperm allocation) increased with male size with no apparent relationshipwith female size, suggesting that males of P. guttatus do not exhibit strategicsperm allocation and/or have a short sperm-recovery period. By comparingseveral traits between P. guttatus and its sympatric species, P. argus, we pro-pose that males of P. guttatus perceive a lower level of sperm competitionrisk than males of P. argus, and that different Panulirus species may exhibitdifferent mating strategies in accordance with their particular life history,reproductive traits, and socio-sexual contexts.

Acknowledgements

We thank Fernando Negrete-Soto and Cecilia Barradas-Ortiz for their invaluable technicalsupport in all field and laboratory activities. Edgar Escalante-Mancera, Marisol Pérez-Ortiz,and David Placencia-Sánchez provided additional help. A graduate scholarship to E.M.G.was granted by Consejo Nacional de Ciencia y Tecnología (Mexico). Annual permits tocollect lobsters from the Puerto Morelos reef system were issued by Comisión Nacional deAcuacultura y Pesca, México.

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does size always matter? mate choice and sperm allocation in panulirus guttatus, a highly sedentary,...

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