Female Alpine Newts (Triturus Alpestris) Mate Initially with Males Signalling Fertility Beneﬁts

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Blackwell Publishing LtdOxford, UKBIJBiological Journal of the Linnean Society0024-40662007 The Linnean Society of London? 2007 91? 483491 Original Article ALPINE NEWT FEMALE CHOICE FOR SIGNS OF FERTILITY P. E. A. HOECK and T. W. J. GARNER

Biological Journal of the Linnean Society, 2007, 91, 483–491. With 2 figures

Female alpine newts (Triturus alpestris) mate initially with males signalling fertility beneﬁts

PAQUITA E. A. HOECK and TRENTON W. J. GARNER*

Zoologisches Institut, Universität Zürich, Winterthurerstrasse 190, 8057, Zürich, Switzerland

Received 24 March 2006; accepted for publication 1 September 2006

Gametic asymmetry implies that females invest more per gamete than males do and thus sperm is considered to be a relatively cheap resource. However, contrary to this classic view, sperm has been shown to be frequently in short supply; hence, selection favouring females that mate for fertility benefits should occur. For this reason, we determined whether males signalling fertility are preferred by female newts of the species Triturus alpestris. We performed paired female–male trials using unmated and previously inseminated females to determine potential criteria for female interest in a courting male, to establish what factors lead to successful mating and to assess the importance of female choice for direct and indirect benefits. We found that female interest in any potential mate and mating success decreased once mating had occurred. Furthermore, we detected an increase in spermatophore deposition rate and rapid spermatophore transfer in encounters that resulted in a successful mating. The results obtained indicate that female alpine newts are attracted to males showing signs of relatively high fertility and that females exhibit a decreased propensity to mate once initial sperm reserves have been acquired. Our results support the theory of initial female choice for fertility benefits. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 483–491.

ADDITIONAL KEYWORDS: amphibian – mate preference – sexual selection.

INTRODUCTION Phillippi & Yund, 2001; Garcia-Gonzalez, 2004). The- ory thus predicts that limited reserves and circum- A classic view of the sexes is that females are frugal spect sperm allocation should select for female choice with their gametes but males take advantage of their for fertility benefits (Wedell et al., 2002; Parker, 1970). relatively unrestricted supply of sperm to inseminate For internally fertilizing species, polyandry is the as many females as possible (Trivers, 1972). Although most common example of female choice for fertility gametic asymmetry in terms of both size and number benefits. When the operational sex ratio is female- are facts, sperm are actually costly to produce and a biased and individual males lack sufficient resources limited resource (Dewsbury, 1982; Wedell, Gage & for continuous effective insemination, both fecundity Parker, 2002; Montrose, Harris & Moore, 2004). and fertility may suffer; effects that can be alleviated Demography, male physiology, polyspermy, sperm by female remating (Pitnick, 1993). Similarly, male competition, and female choice all impose demands on sperm quality varies enormously, to the point where a male’s ability to produce sperm and how many some males exhibit complete infertility due to sterility. should be allocated to a given ejaculate (Wedell et al., By mating with multiple males, females can compen- 2002; Ball & Parker, 2000). Sperm are therefore fre- sate for any one sterile male, or even for a series of rel- quently in short supply and females are often exposed atively infertile males (Sheldon, 1994; Singh, Singh & to environments where their gametes are at risk due Hoenigsberg, 2002; Garcia-Gonzalez, 2004). Sperm to the unavailability of fertile sperm (Stewart-Savage, usage may be inefficient, and large numbers of sperm are sometimes required to fertilize a single ovum (Török et al., 2003). In species with large clutch sizes *Corresponding author. Current address: Institute of Zoology, Zoological Society of London, Regent’s Park NW1 4RY, London, and/or a prolonged breeding season, polyandry may be UK. E-mail: [email protected] an ongoing requirement for no other reason than

484 P. E. A. HOECK and T. W. J. GARNER fertility demands (Jones, Adams & Arnold, 2002; Vie- choice for indirect compatibility benefits may not ites et al., 2004). It is perhaps for these reasons that describe the entire spectrum of female preferences for female choice for benefits directly affecting fertility this species. Males become more circumspect with and fecundity are as important, if not more so, than regards to spermatophore production and the success choice for indirect benefits (Møller & Jennions, 2001), rate of spermatophore transfer for the entire genus is and at least a major factor as to why polyandry is so on the order of one in four under ideal laboratory con- predominant (Garcia-Gonzalez, 2004). ditions: rates are probably poorer in the field (Halli- However, female choice strictly for fertility benefits day, 1990). Although the operational sex ratio favours is not the rule because, in the majority of cases, a male excess at most locations and early in the breed- females are balancing choice for several requirements ing season, female excesses do occur and can reach the and attempting to overcome paternity control mecha- level of two females for every male (Schnüriger, 2004). nisms used by competing males (Eberhard, 1996; Population densities may reach levels as high as 58 Gavrilets, 2000; Martin & Hosken, 2003). Variation in individuals per square meter of pond substrate but, in the demographic and genetic characteristics of the some cases, density is low enough (three per square breeding assemblage and an unpredictable insemina- meter) to negatively influence the encounter rate tion status of individual females can lead to the (Schnüriger, 2004). These demographic and behav- evolution and maintenance of multiple female prefer- ioural patterns suggest that females should encounter ences (Iwasa & Pomiankowski, 1994; Johnstone, 1995, sperm-limited situations and, in these circumstances, 1996; Pitcher et al., 2003; Mays & Hill, 2004) with the exhibit a preference for males signalling fertility ben- result that female choice can become plastic (Qvarn- efits rather than genetic compatibility benefits. strom, 2001; Mays & Hill, 2004), or may exhibit shifts The present study aimed to determine if fertility in preferences based on requirements for direct and benefits are sought by female T. alpestris. We per- indirect benefits (Gabor & Halliday, 1997; Pitcher formed paired female–male trials using naïve and et al., 2003). Even so, when fertility assurance is the experienced females to determine potential criteria for dominant force behind female choice, female promis- female interest in a courting male, to establish what cuity is evolutionarily stable and any choice for indi- factors lead to insemination, and therefore to assess rect benefits should occur postcopulation (Fishman, the importance of female choice for direct and indirect Stone & Lotem, 2003). benefits. We set male courtship vigour and body size as In amphibians, polyandry has been detected in both fitness-correlated phenotypic traits to test for female externally and internally fertilizing species (D’Orgeix choice for good genes and determined overall genetic & Turner, 1995; Laurila & Seppä, 1998; Byrne & Rob- similarity between mating partners post hoc to test for erts, 1999; Osikowski & Rafinski, 2001; Jones et al., female choice for genetic compatibility. Specifically, we 2002; Garner & Schmidt, 2003; Lodé & Lesbarrères, asked the following questions: 2004; Vieites et al., 2004) and fertility benefits have 1. Do female insemination status, male courtship been described for both fertilization strategies experience, vigour, body size, and/or genetic compo- (Osikowski & Rafinski, 2001; Jones et al., 2002; Vie- sition affect a female’s interest in a male and her ites et al., 2004), but not in all cases (Byrne & Roberts, decision to accept or reject him as a potential mat- 1999, 2000). Female choice has been implicated in ing partner? numerous amphibian mating systems, primarily in 2. Do female insemination status, male courtship the context of Fisherian processes (Halliday & Tejedo, experience, vigour, body size, and/or genetic compo- 1995; Sullivan, Ryan & Verrell, 1995; Welch, Seml- sition distinguish successful mating trials (insemi- itsch & Gerhardt, 1998), but evidence for sperm limi- nation of the female) from unsuccessful ones? tation (Osikowski & Rafinski, 2001; Jones et al., 2002; Vieites et al., 2004) suggests that female choice should MATERIAL AND METHODS not be limited to indirect benefits. In a study investi- gating the potential for multiple female preferences in Adult T. alpestris were collected on land from pit fall caudates, Gabor & Halliday (1997) showed that, until traps while migrating to their native pond near mated once, female smooth newts lack discrimination Adlikon, Kanton Zürich, Switzerland (47°35′N, for male traits signalling Fisherian quality. They 8°42′E) at the beginning of March 2001. Capture on interpreted the observed pattern as a trade-up to bet- land ensured that the females were unmated that year ter quality males once fertility had been assured before the start of the experiment (Pecio, 1992). Ani- (Gabor & Halliday, 1997). mals were maintained and sampled according to Uni- Females of our study species, the alpine newt (Tritu- versity of Zurich guidelines. All individuals were rus alpestris), preferentially bias paternity towards measured for snout–vent length (SVL) to the nearest males that are genetically dissimilar (Garner & 0.01 mm and weighed to the nearest 0.01 g using a Schmidt, 2003). Nevertheless, the presence of female digital balance. SVL was measured four times in 20

ALPINE NEWT FEMALE CHOICE FOR SIGNS OF FERTILITY 485 individuals to assess measurement error: less than 1% Courtship vigour comprised several variables: we of the total variance was due to measuring error (anal- recorded the number of courtship attempts and the ysis of variance: 0.68% variance in measuring error, start and stop time of each bout of courtship. We also repeatability = 0.99). Each newt was assigned an indi- recorded any spermatophore deposition and defined a vidual, number-coded 5-litre tub, tilted at 5° and con- successful courtship as one that resulted in spermato- taining both moistened moss and standing water. phore transfer (Table 1). If a male courted immedi- Newts were provided with Rana temporaria eggs and ately after an unsuccessful sperm transfer, we treated Daphnia spp. as ad libitum food; we subsampled frog this as a continuing courtship. If the female egg clutches from one location to minimize the impact approached the male at any time during courtship or that egg removal would have on local R. temporaria followed him, she was defined as an ‘interested’ population dynamics, in accordance with Garner, (responsive) female (Denoël, Poncin & Ruwet, 2001). Angelone & Pearman (2003). An ‘uninterested’ female was defined as one that Mating experiments took place from 23 March to 3 remained stationary or never approached or followed a May 2001 in the laboratory. This period falls within male during the trial. the breeding period measured in natural populations Males were reused in mating trials, but no male was (Nöllert & Nöllert, 1992). Opaque plastic tubs used twice on the same day. This rest interval of at (56.5 cm × 36.5 cm × 31.5 cm) containing aged tap least 1 day ensured that the males had enough time to water (approximately 10 L) were used as observation recuperate and produce new sperm and other compo- chambers for individual trials. Before the start of each nents of the spermatophore (Verrell, 1987). Although trial, a haphazardly selected female was placed in an males were reused during the course of the experi- observation chamber and allowed to habituate for 1 h. ment, all trials were unique with respect to male/ After 1 h, a haphazardly selected male was added to female combination. Females that refused to mate in the centre of the tub. Each trial was allowed to run for their first trial were excluded from subsequent tests. a maximum of 1 h with continuous observation Females that mated successfully in their first trial (Gabor, Krenz & Jaeger, 2000; Osikowski & Rafinski, were provided with a second opportunity to mate. 2001). In the event that a successful sperm transfer Because of our remating design, we were able to had occurred (i.e. the sperm cap attached to the assess the effect that experience of either sex had on female’s cloaca), the trial was discontinued. If no suc- female mating behaviour. We defined male courtship cessful mating had taken place by the end of 1 h, the experience as the sum of time spent courting in all trial was discontinued. previous trials (continuous variable) because males The general courtship behaviour of triturid newts could accrue courtship quality through the time spent and T. alpestris have been described in detail by Hal- courting any one partner. Female experience was liday (1977). Behavioural variables used in this study restricted to insemination status (unmated or insem- are defined in Table 1. The start of courtship was inated) because one of the major goals of the study was defined as the point in time where a male initiated tail to assess the effect of insemination on female mating fanning in front of the female (display phase; Halliday, behaviour. Trials involving remating attempts were 1977). Interruption or cessation of courtship was performed on average 6.5 days after the previous trial. defined as the time when the male stopped fanning or Females that mated during their first trial and when the female turned away from the courting male. were subsequently tested for remating were kept

Table 1. Summary of behavioural variables used in this study

Variable Description

Display latency* Difference in time between start of trial and the initiation of the first bout of courtship Transfer latency* Difference in time between the initiation of the first or only bout of courtship and the deposition of the first spermatophore Number of spermatophores Total number of spermatophores deposited by a test male during a single trial Number of courtships Total number of courtship bouts performed by a test male during a single trial Courtship time Total amount of time a test male spent courting in a trial: the sum of all bouts Observation time Total amount of time a trial lasted (maximum 1 h) Male courtship experience Total amount of time of previously experienced courtship: the sum of all bouts Female insemination status Unmated or inseminated Female interest Any approach or forward movement towards a courting male by a test female. Binary score.

*Denoël et al. (2001).

486 P. E. A. HOECK and T. W. J. GARNER individually and outdoors in between trials in 90-L uptakes as response variables and date as the inde- tubs containing aged tap water, egg-laying strips pendent variable. Furthermore, because spermato- (Miaud, 1993), some leaves, and a piece of wood that phore production could possibly correlate with male served as a refugium from the otherwise obligatory SVL and/or weight (Halliday, 1998), the average aquatic environment. Females were fed R. temporaria number of spermatophores deposited in a trial was tadpoles and Daphnia spp. ad libitum. calculated for each male in each trial, plotted against After the final trial, test animals were sampled for SVL and weight, and the relationship tested for sig- genetic analysis by clipping a small sample from the nificance using linear regression. All statistical anal- tail tip. Tail clips were taken using surgical scissors yses other than calculations of allele frequencies and sterilized between samples with 100% ethanol. Tissue relatedness values were performed using SAS for Win- samples were preserved at −80 °C until DNA extrac- dows, release 8.02 (SAS Institute Inc.). tion. After a suitable recovery period, all animals were released at their native pond near Adlikon. DNA was RESULTS extracted from tissue samples using affinity spin col- umns (QIAamp DNA mini kit, Qiagen). Relatedness One hundred and thirty females and 63 males were estimates among all female–male pairs used in the used for mating experiments. In eight cases, no court- mating experiment were calculated from alleles ship took place: these trials were excluded from anal- detected at five polymorphic microsatellite loci: yses. Female interest data were recorded for 141 trials Ta1Ca1, Ta1Caga4, Ta3Caga1, Ta3Ca8 and Ta4Ca4U; and mating success data for 130 trials. Females exhib- for amplification conditions, see Garner et al. (2003). ited interest in the courting male in over 60% of the Polymerase chain reaction (PCR) products were elec- 141 trials (Figs 1, 2). The number of spermatophores trophoresed on Spreadex EL-600 S-50 or, in the case of deposited by a male during a single trial was in the Ta1Caga4 products, EL-800 S-50 gels using the SEA range 0–6. Overall, 65 of 258 (25.2%) spermatophore 2000 advanced submerged gel electrophoresis appara- depositions resulted in successful sperm uptake. Sper- tus (Elchrom Scientific AG). PCR product sizes were matophores were successfully transferred in 50% of scored using the TotalLab, version 1.20 software (Non- the 130 relevant observations (Figs 1, 2). Female linear Dynamics). interest did not change significantly during the time of Allele frequencies for all alleles at each of the five the experiment (P = 0.627) and the number of success- loci were calculated using Genepop, version 3.3 (Ray- ful trials was not significantly lower towards the end mond & Rousset, 1995). Measures of genetic similarity than at the beginning of the mating experiments (r-values) among pairs for each trial were estimated (P = 0.355, total data set). Male SVL (P = 0.508) and using the program Kinship, version 1.3.1 (Queller & male weight (P = 0.425) had no significant effect on Goodnight, 1989) with trial as the group variable. We the number of spermatophores produced during a used logistic regression to test the potential effect of trial. Polymorphism at microsatellite loci ranged from the different factors using both ‘female interest’ and 6–42 alleles. Genetic similarity values (r-values) were ‘success’ as response variables. The following variables in the range −0.549 to 0.712 and were normally were used as factors: female and male SVL (mm), distributed. female and male weight (g), display latency [ln(s)], Only female insemination status (P = 0.021) signif- transfer latency [ln(s)], number of spermatophores/ icantly affected female ‘interest’ (Table 2). Insemi- total observation time, number of courtships/total nated females were less responsive to males than observation time, courtship time/total observation naïve females. Trials with interested females involved time, male courtship experience [ln(sum of time spent courting in all trials)], female insemination status (unmated or inseminated), and genetic similarity (r- 120 value). ‘Transfer latency’ and ‘number of spermato- 100 phores/total observation time’ were only included in 80 57 the analysis with ‘success’ (spermatophore uptake) as 85 Yes the response variable. In addition, for the response 60 No variable ‘success’, all trials in which no spermatophore 40 49 deposition had occurred were excluded from the anal- 20 ysis because these could not result in insemination. 26 To control for changes in the likelihood of mating 0 Interest Success success and female interest over the course of the experiment, a linear regression analysis was per- Figure 1. Frequency of female interest and mating suc- formed using all available data, the occurrence of cess in trials involving previously unmated females. The y- interested females and successful spermatophore axis comprises number of females in each category.

ALPINE NEWT FEMALE CHOICE FOR SIGNS OF FERTILITY 487

Table 2. Full logistic regression model for the response variable female interest as a categorical variable

Factor d.f. Estimate Point estimate Standard error Wald Chi squared P-value

Intercept 1 −9.086 – 8.202 1.227 0.268 Female SVL 1 0.187 1.206 0.135 1.918 0.166 Male SVL 1 0.011 1.011 0.134 0.007 0.933 Female weight 1 0.115 1.122 0.521 0.049 0.825 Male weight 1 0.381 1.464 0.914 0.174 0.677 Display latency 1 −0.341 0.711 0.284 1.448 0.229 Number of courtships/obs. time 1 −7.551 < 0.001 188.9 0.002 0.968 Courtship time/obs. time 1 0.794 2.212 1.126 0.497 0.481 Male courtship experience 1 0.006 1.006 0.056 0.010 0.921 Female insemination status 1 −1.109 0.330 0.481 5.317 0.021 Relatedness 1 1.584 4.872 0.880 3.235 0.072 d.f., degrees of freedom; SVL, snout–vent length; obs. time, observation time.

Table 3. Full logistic regression model for the response variable success

Factor d.f. Estimate Point estimate Standard error Wald Chi squared P-value

Intercept 1 −1.355 – 12.426 0.012 0.913 Female SVL 1 −0.128 0.880 0.201 0.402 0.526 Male SVL 1 −0.151 0.860 0.207 0.532 0.466 Female weight 1 0.132 1.141 0.786 0.028 0.867 Male weight 1 0.771 2.163 1.437 0.288 0.592 Display latency 1 0.767 2.152 0.434 3.125 0.077 Number of courtships/obs. time 1 −437.7 < 0.001 281.4 2.421 0.120 Courtship time/obs. time 1 −2.104 0.122 1.845 1.301 0.254 Transfer latency 1 0.886 2.425 0.340 6.797 0.009 No. spermatophores/obs. time 1 4988.5 > 999.9 1146.7 18.924 < 0.0001 Male courtship experience 1 0.093 1.098 0.086 1.184 0.277 Female insemination status 1 −0.143 0.867 0.803 0.032 0.859 Relatedness 1 0.467 1.595 1.328 0.124 0.725 d.f., degrees of freedom; SVL, snout–vent length; obs. time, observation time.

35 a higher mean courtship time/observation time value (0.387; SE = 0.02) than trials with disinterested 30 females (0.323; SE = 0.042). Although 77% of unmated 25 females showed interest in courting males, only 57% of 17 20 8 Yes inseminated females did (Figs 1, 2). 15 No Two variables had a signiﬁcant effect on mating = 10 success. Transfer latency (P 0.009) and the number 16 13 of spermatophores/observation time (P < 0.0001; 5 Table 3); both signiﬁcantly affected whether or not 0 spermatophore transfer occurred. Spermatophore Interest Success transfer success was more likely when the number of Figure 2. Frequency of female interest and mating suc- spermatophores/observation time increased and when cess in trials involving inseminated females. The y-axis transfer latency decreased. The exclusion of un- comprises number of females in each category. mated females from the analysis revealed no further

488 P. E. A. HOECK and T. W. J. GARNER significant factors. Unmated females mated 48% of the their potential as fertile mates is more feasible time, but inseminated females accepted spermato- because it can hardly be considered a fair raffle when phores in one-third of the relevant trails (Figs 1, 2). female newts exhibit mating preferences and exert ultimate control over whether or not postcopulatory sperm competition will even occur (Green, 1991a, b; DISCUSSION Gabor & Halliday, 1997; Garner & Schmidt, 2003). Only the previous mating history of an individual Producing spermatophores is costly for male newts female significantly affected female interest. Insemi- (Verrell, 1987) and a certain proportion of the avail- nated females showed a reduced interest in any poten- able male population will exhibit decreased fertiliza- tial mates as opposed to those experiencing courtship tion ability, which would select for cautious female for the first time. This decrease in female interest was mating behaviour. However, male T. alpestris often not due to temporal effects attributable to the pro- require little or no female responsiveness before sper- longed nature of our study or disease (see below) matophore deposition and may go directly into the because both female interest and mating success did predeposition behaviour (the creep), depositing a sper- not change in frequency over the course of our exper- matophore without a female ever performing the tail- iment. Instead, a decrease in female responsiveness touch which is a requirement in all other triturid mat- reflects the satisfaction of an initial female mating ing systems studied to date (Halliday, 1990, 1977). preference, a preference for fertility benefits. Once Females respond to this apparently wasteful behav- sperm reserves are initially assured and an alterna- iour by approaching and sniffing the deposited sper- tive mating preference exists (Garner & Schmidt, matophore, and frequently follow this up by 2003), females should exhibit a decrease in preference displaying interest in the male responsible for the for male signals of fertility benefits, a pattern sug- spermatophore (P. Hoeck & T. W. J. Garner, pers. gested by the decreased propensity of mated females observ.). ‘Premature’ deposition behaviour decreases to show interest in any male and the decrease in mat- with an increased number of spermatophores and, ing success associated with once-mated females. after three depositions, a tail-touch is required for the The results from mating success analyses further production of the fourth spermatophore (Halliday, support this hypothesis. Mating success was strongly 1990). From this, it seems probable that ‘preliminary’ affected by two factors. We detected an increase in spermatophores function to attract females interested spermatophore deposition rate and a decrease in in fertility benefits and may serve no purpose in terms transfer latency associated with increased mating suc- of sperm competition. As concluded by Halliday cess. Increased spermatophore production may reflect (1983), there are clear benefits to female newts that greater male investment to ensure insemination with- miss a ‘preliminary’ spermatophore. Since sperm com- out female preference being involved. This would be petition risk is high in this species (Rafinski & analogous to the fair raffle proposed by Parker Osikowski, 2002; Garner & Schmidt, 2003) and males (1990a, b) for species with postcopulatory male–male do transfer multiple spermatophores to single competition because greater ejaculate investment pos- females, we do not doubt that males adopt strategies itively affected the probability of insemination and the that are designed to secure paternity under sperm risk of sperm competition is very high in alpine newts competition, as would be expected in any case where (Garner & Schmidt, 2003). Buying more tickets for the the interests of the two sexes are not coincident sperm raffle should be selected for in this and likely all (Hosken, Garner & Ward, 2001). Determining whether triturid species because spermatophore transfer rates female preferences in this species can be overridden for the genus are generally poor (Halliday, 1990). by postcopulatory sperm competition mechanisms Moreover, transfer was completed more quickly in suc- remains to be investigated. cessful encounters. Males that deposit more spermato- Genetic compatibility had no influence on female phores have a numerical advantage that would serve mating strategy in the present study when the anal- to speed up transfer rates simply by elevating the ysis was restricted to previously mated females, in spermatophore encounter rate of females even if the contradiction to what was detected in a previous study likelihood of encounter was a random effect. Males (Garner & Schmidt, 2003) and paternity patterns from often follow-up a successful spermatophore transfer sperm competition studies involving hetero- and con- by initiating a new round of courtship and will trans- population males (S. Hangartner & T. W. J. Garner, fer a second spermatophore if the female is coopera- unpubl. data) using equivalent numbers of molecular tive, which would have obvious paternity benefits markers. However, comparisons among the studies under sperm competition (Halliday, 1977; P. Hoeck & may not be appropriate due to differences in experi- T. W. J. Garner, pers. observ.). mental design. Females in the present study were pro- However, the alternative explanation that males vided males in sequence and females may not be able attract females seeking fertility benefits by signalling to assess male compatibility without direct compari-

© 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 483–491 ALPINE NEWT FEMALE CHOICE FOR SIGNS OF FERTILITY 489 sons. More importantly, unlike the previous study, (male mate opportunity, female waiting cost, and paternity patterns were not assessed in hatched lar- female mate choice) are compatible with female choice vae due to the outbreak of disease in our study popu- for fertility benefits. Furthermore, Zonneveld & Metz lation after mating and subsequent high mortality of (1991) resolved the male mating opportunity hypoth- females before substantial egg deposition could occur esis with the female waiting time hypothesis, as would (Pérez, 1913). The potential for postcopulatory sperm be expected if sperm was a limited resource and both selection prevents us from making any final conclu- females and males adopted tactics designed to make sions on the overall affect of genetic compatibility from the most of what sperm is available. Harris & Lucas the data set presented here. (2002) show that just such situations are to be Although we do not provide conclusive evidence for expected in a salamander breeding assemblage. the trade-up hypothesis in the present study, the com- In summary, our results indicate that female alpine bination of initial preference for fertility benefits newts are attracted to males exhibiting signs of rela- shown here and overall preference for compatibility tively high fertility and that females exhibit decreased benefits previously described (Garner & Schmidt, propensity to mate once initial sperm reserves have 2003) is similar to the results obtained by Gabor & been acquired. The initial female choice for fertility Halliday (1997) and in other studies describing the benefits is the best explanation for our results, rather trade-up patterns of female preferences (Pitcher et al., than male strategies to optimize paternity under 2003). Sever (2002) used histology to show that female sperm competition. It is possible that our data, when triturid newts can stratify subsequent ejaculates in a taken in concert with other published results on manner that would allow the sperm from the last male sexual selection in this species (Halliday, 1990, 1983; to mate to be used first to fertilize eggs. Storing and Garner & Schmidt, 2003), suggest that alpine newt using sperm in this manner is especially well-suited females trade-up for compatibility benefits. This latter for trade-up in newts because females may require conclusion is speculative and awaits further experi- numerous matings over the course of the breeding sea- mental investigation before confirmation. Even so, the son. Sperm stores could be continuously replenished possibility is strong that sperm limitation is of general for either fertility assurance or genetic benefits based consequence to caudate mating systems. on what level of depletion the stores are at. Neverthe- less, it is important to note that Rafinski & Osikowski (2002) did not detect any evidence for sperm prece- ACKNOWLEDGEMENTS dence in their study and first male sperm precedence We would like to thank G. Guex for providing us access was detected in a North American newt species (Jones to his study population of T. alpestris. Thanks also to et al., 2002). H.-U. Reyer for ideas and unfailing support. Statisti- Female preferences for fertility benefits may be com- cal advice was provided by B. Schmidt, J. Van Buskirk, mon in triturid species, if not in caudate amphibians S. Krackow, W. Blanckenhorn, and K. Schiegg. S. Röth- in general. Waights (1996) showed that preovulatory lisberger aided with the molecular component of the female common newts will compete directly for court- study. A. Cunningham, H.-U. Reyer, R. 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