Behav Ecol Sociobiol (1993) 32:283-291 Behavioral Ecology and Sociobiology © Springer-Verlag 1993

Male reproductive success in two midwife toads, Alytes obstetricans and A. cisternasii Rafael Mfirquez

Departamento de Biologia Evolutiva, Museo Nacional de Ciencias Naturales (C.S.I.C.), Jos6 Guti6rrez Abascal 2, E-28006 Madrid, Spain

Received July 7, 1992 / Accepted December 19, 1992

Summary. One population of the midwife toad species Alytes obstetricans and one of A. cisternasii were studied in Spain for two consecutive reproductive seasons. Males that were most successful at hatching a high proportion of their clutch did not obtain more matings. On the other hand, in both species larger body size conferred a significant reproductive advantage on males. These results are explained mainly by the increased number of mates obtained by larger males, probably as a result of female choice. The selection gradients for body size in males (regressions of reproductive success on body size) were not significantly different within species be- tween years nor between species within the same period of time. Hatching success (proportion of the eggs hatched) was not correlated with male body size in A. obstetricans. Hatching success in A. cisternasii was weak- ly negatively correlated with male body size in 1988.

Introduction

The distribution of male reproductive and mating suc- cess may be interpreted as a consequence of sexual selec- tion through intrasexual competition, mate choice or both. Anuran systems have attracted particular attention and their study has contribued significantly to the re- emergence of the controversy on the role of female choice as an evolutionary mechanism (e.g. Ryan 1985, 1991). In particular, the adaptiveness of female choice is a subject of considerable debate in sexual selection theory (Bradbury and Andersson 1987). The debate takes place between those who believe that adaptive fe- male choice is the most likely form of choice and those who believe that a Fisherian "runaway" process may lead to female choices that appear to be maladaptive in light of natural selection. This debate mainly concerns cases where males contribute only gametes.

When males provide a resource that directly increases female fitness, there seems to be general agreement that females should maximize their reproductive success by

choosing "optimal" males. This hypothesis, advanced by Trivers (1972) and reiterated by Searcy (1982) and others, is particularly applicable to the case of uniparen- tal male care. Halliday (1983, p. 6) states that " In species in which males carry out some or all of the parental care of the young, females may choose males on the basis of their capacity to do so". Male parental care therefore has been considered to be a trait which females can use in making an adaptive selection of a mate (Wil- liams 1975). Instances of such care are plentiful among fish (Blumer 1979; Gross and Sargent 1985) and also occur among frogs (Lamotte and Lescure 1977; McDiar- mid 1978; Ridley 1978; Salthe and Mecham 1974; Wells 1981; Townsend and Stewart 1986a, b; Summers 1989; Townsend 1989). However, in the majority of these in- stances, male parental care is often limited to tending the eggs and/or larvae in a given territory. In a few cases paternal care is provided independently of site at- tachment (Fricke 1975; Gross and Shine 1981). In these cases adaptive female choice would be expected to be focused on the quality of the male as a caretaker, thereby excluding other confounding factors such as territory quality (e.g. Berglund et al. 1986, 1988). It is therefore of interest to study the relationship between male mating success and parental quality in the midwife toads, a group of anurans with male parental care of the eggs.

In the Iberian Peninsula there are two species of mid- wife toads, Alytes obstetricans and A. cisternasii (Anura, Discoglossidae). Information on the breeding biology of these toads has been provided by several authors (e.g. de l'Isle 1873, 1876; Boulenger 1912; L6pez Jurado et al. 1979; Heinzmann 1970; Crespo 1982; Rodriguez Jim6n- ez 1984, 1988; M/trquez and Verrell 1991; Mfirquez 1992). Male midwife toads call on land to attract fe- males, usually near a body of water. When a female approaches the male, they enter amplexus on land. After an elaborate sequence of movements, females extrude a string of large eggs which are fertilized by the male while in amplexus. The male then proceeds to tangle the egg string around its ankles. The string that connects the eggs is elastic but resistant, and egg transfer from

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the female to the male usually involves 100% of the eggs (de l'Isle 1876; pers. observation). The male remains on land with the eggs throughout larval development (20-35 days). Males can have eggs from more than one female in a single batch (de l'Isle 1876; Boulenger 1912; L6pez-Jurado et al. 1979; pers. observation). In the pop- ulations studied, males matured only one batch of eggs (from one to three females) per season (M/trquez 1992). When the tadpoles are fully formed inside the eggs, males walk to the water and release the whole egg mass. After some minutes in the water the tadpoles hatch, each tadpole leaving an empty egg capsule.

Female mate choice in anurans has been documented in several species. Often the phenotypic characteristic on which choice is based is male size, sometimes assessed through some characteristic of the advertisement call (e.g. Arak 1983; Ryan 1980, 1985). Male size is often considered as an important trait, not only because it can be easily measured in the field, but also because it is likely to be associated with age (Hemelaar 1983; Hemelaar and Van Gelder 1980; but see Halliday and Verrell 1988). If rapid growth or survival ability are her- itable and positive components of fitness, females may pass on a selective advantage to their offspring (Trivers 1972). Moreover, sexual size dimorphism is a subject of interest in itself in the evolutionary literature and in sexual selection (Arak 1988; Halliday and Verrell 1986).

Evidence for female choice in anuran mating systems is not always easy to obtain (Arak 1983). However, mid- wife toads appear to have suitable matching system where such evidence might be found. The two popula- tions of midwife toads studied show no clear evidence for any form of direct intrasexual competition. Territor- iality or fighting between males or females have never been reported in natural conditions. Two unpublished cases of observations in captivity of female Alytes fight- ing for access to males (A. muletensis, S. Bush, pers. comm., and A. obstetricans, L. Brown and P. Verrell, pers. comm.) occurred in laboratory conditions with fe- male-biased sex ratios, which have never been reported in the field. Therefore, the most likely form of intrasex- ual competition is indirect acoustical competition among males to attract females (Mfirquez and Verrell 1991; Mfirquez 1992). The advertisement call of Alytes is a short (100-160 ms long), extremely soft tonal note re- peated at long intervals (0.5-10 s) (Heinzmann 1970; Crespo 1981 ; Crespo et al. 1989). It is therefore unlikely to be highly costly energetically. Furthermore, midwife toads appear to have relatively low risks associated with calling that might impose a strong selective pressure on males (Alytes remains are rarely found in owl pellets, the most likely acoustically oriented predator; Martin and L6pez 1990; B. Sanehiz pers. com.). Furthermore, there is a correlation between male size and call funda- mental frequency, and, at least for A. obstetricans, there is evidence for selective phonotaxis in females towards low-frequency calls (MArquez 1990). The whole picture conforms with the kind of anuran mating system de- scribed by Arak (1983) where, if sexual selection is oper- ating, mate choice by females if the likely mechanism.

In this paper I tested two hypotheses: (1) that males that are successful at hatching a large proportion of their clutches obtain more matings than those that are less successful, and (2) that larger males obtain more mat- ings. To answer these questions I sought to determine:

1. Distributions of female clutch size (number of eggs produced by a female in a single mating) and male clutch size, (number of eggs in an egg mass carried by a male from one or more matings) 2. Relationships among male body size, numbers and mates and number of eggs obtained 3. Variance in male parental success in producing viable tadpoles (proportion of the male clutch hatched) 4. Relationships among male size, reproductive success, and male parental success.

Methods

I visited two populations of midwife toads in Spain, one of Alytes obstetricans, and one ofA. cisternasii, after sunset for two consecu- tive reproductive seasons (1988 and 1989). The A. obstetricans field site, in the Pyrenees, was visited in summer over a total of 108 nights (41 in 1988 and 67 in 1989). The A. cisternasii field site, in M6rida, Extremadura (Central Spain), was visited in autumn, over a total of 58 nights (35 in 1988 and 23 in 1989). During these visits, I explored the sites and collected adult individuals. The sites and field methodology are described in detail in Mhrquez (1992).

Males were captured on their way to release their egg masses in the water. I placed each male individually in a marked zip-lock bag. Snout-vent length (SVL) was measured by pressing the toad flat against a ruler. After wiping off any excess water, I weighed each individual with its load of eggs with a Quantum or an "A & C" portable electronic balance (accuracy _0.01 g). A Pesola spring scale (with accuracy +0.25 g) was used in summer 1989. I then removed the egg mass, weighed it on the same balance, and placed it overnight in an individually marked small vial of water. The next morning, the vials contained free-swimming newly emerged tadpoles and the remainder of the egg masses. The remains of the egg masses contained many empty egg capsules and a number of unviable eggs. These were preserved in a 4% solution of formalin. I released the tadpoles in their natural habitat the same morning. At this time any empty egg masses of Alytes found on the shore were collected and preserved. I marked all specimens of A. obstetricans individually by toe clipping; for A. cisternasii, only those individuals captured in an area (50 x 70 m) along 50 m of shoreline of the stream were marked. I released all toads at their original capture sites on the night of capture.

I measured male clutch size and hatching success directly by counting unviable eggs and empty egg capsules in hatched egg masses. Data on the proportion of the clutch that hatched was square root arcsin transformed for statistical analysis (Sokal and Rohlf 1981, p. 427).

Neither female clutch size nor number of matings per male could be assessed directly by observing the collected (hatched) egg masses. When a male had several clutches, all the egg strings were tangled together forming a single bundle where clutches from dif- ferent females were not distinguishable. Therefore, a statistical analysis of the frequency distribution of male clutch sizes was used to determine the distribution of female clutch sizes (Reading and Clarke 1988). The statistical technique is known as "MIX", and is described in detail in Madonald and Pitcher (1979). MIX analy- ses histograms as mixtures of statistical distributions and finds a set of overlapping component distributions that gives the best fit to the histogram. The number and characteristics of the distribu- tions can be set in the model as initial assumptions. The statistical method used to fit the mixture distribution to the data is maximum-

likelihood estimation for grouped data. In contrast to the method used by Reading and Clarke (1988), the model was set for normal distributions, with three equidistant modes and proport ional vari- ances (the variance of the second mode was twice the variance of the first, and so on). The MIX simulation yields standard errors for the estimates and, assuming that the distributions of female clutch sizes are normal, the significance of differences in clutch size between years can be tested statistically. These numbers were contrasted with oviductal egg counts from a lower number of fe- males of each species. In order to minimize the impact of the collec- tion of these females on the study populations, additional observa- tions of females from other populations were included to increase the data.

Zero classes, of males and females that had not secured any matings, could not be assessed because it was impossible to deter- mine if males encountered without eggs had recently hatched a batch or would mate after being released. Consequently all the conclusions reached only apply to individuals with one or more clutches.

Univariate size-selection gradients were calculated using the method of Lande and Arnold (1983) as the linear regression of relative reproductive success on log(SVL), where relative reproduc- tive success is individual clutch size divided by the mean number of eggs (hatched and unhatched) per sex. For calculation of the size selection gradients for males (fi), I included only those individ- ual males for which data on reproductive success were available for either of the two years.

The correlation between male body size (SVL) and reproductive success may be affected by two components of selection: number of mates and number of eggs per female. The importance of number of mates can be assessed by estimating the relative contri- bution of number of mates to the correlation between male body size and reproductive success. This contr ibution could not be ad- dressed directly because the actual number of mates for each male could not be precisely assessed. A bootstrap simulation (Efron 1982) was used to estimate the sexual selection gradient for male body size (gradient between SVL and number of mates) using the distributions fitted by the maximum likelihood program (MIX). For each male clutch, a probability was assigned to the likelihood

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of that number of eggs being the result of one, two, or three or more female clutches. The probabilities were determined from the amount of overlap of the three distributions (Fig. 1). The simula- tion was conducted separately for all conspecific males recorded in each year and for the combined results for both years. The number of iterations was set at 1000. In each iteration, a number of males equal to the actual sample size of males for that period was randomly sampled, with replacement. Data recorded for each sampled male in this simulation include the log(body size), the number of eggs in male clutch and the number of mates (1, 2 or 3). A value of three was arbitrarily assigned to the "more than two mates" class. The number of mates was assigned at random, with the probability determined by the male clutch size value in relation to the three curves generated by MIX (Fig. 1). At the end of each iteration, the simulation program calculated a regres- sion coefficient for the effect of log(body size) on relative reproduc- tive success (total selection gradient for body size) and relative mating success (which I will call sexual selection gradient for body size: fit)- Averages for the total selection gradient and for the sexual/ selection gradient were estimated from the 1000 iterations. Thes~ estimates were normally distributed, and hence the variances of the estimates permitted statistical comparisons.

Resu l t s

Male and female clutch size

The frequency distributions of egg numbers in male clutches and the normal curves fitted by the MIX pro- gram are shown in Fig. 1. The modes of the first fitted normal distributions are the estimates of female clutch size (number of eggs). These estimates were highly simi- lar between years for A. obstetricans (41.67 vs. 39.53 eggs, for 1988 and 1989 respectively), while the similarity is slightly less striking for A. cisternasii (38.13 vs. 44.07

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Alytes obstetricans

41 82 124

Mean: 72.72

Alytes cisternasii

Mean: 86.5

(a) 1988

gt 165

>. 40 f..) z 3o

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10-

z

Alytes obstetricans

k : , . : ,

40 80 I20 160

Mean: 61.96

Alytes cisternasii

4Z~l~ 881 13~2

Mean: 64.80

(b) 1989

Fig. 1 a, b. Distribution of number of eggs per male clutch. Normal curves (fitted by MIX) represent the distribution of the number of female clutches (one to three) corresponding to each male clutch size

286

eggs, for 1988 and 1989 respectively). For compar i son of average female clutch size between species it is best to look at the combined data for bo th years as the esti- mate is more precise due to the larger sample size. The difference in estimated number o f eggs per female clutch (41.14 for A. obstetricans and 40.55 for A. cisternasii) was no t statistically significant (P > 0.05). Oviductal egg counts were obtained f rom a total o f 45 females. The results for 26 A. cisternasii females were: 2 = 47.77, SD = 12.5, range: 19-74. Only eight o f these females were collected f rom the study popula t ion ; the rest came f rom different sites o f the Iberian Peninsula. The 19 females o f A. obstetricans had a mean o f 42.37 eggs (SD = 11.06, range: 20-62). These females included seven individuals f rom the study popula t ion while the rest were obtained f rom specimens collected in other popula t ions in Spain and Portugal.

The means and s tandard deviations for male and fe- male clutch sizes for each species each year and the com- bined data for bo th years are shown in Table 1. Average male clutch size was significantly different between years for bo th species (Mann-Whi tney U-test, P _ 0.0001). In 1988 average male clutch size was significantly larger for A. eisternasii than for A. obstetricans (Mann-Whi t - ney U-test, P_<0.001). In 1989, on the other hand, male clutch sizes were not significantly different between spe- cies (Mann-Whi tney U-test, P<0 .4779) . The combined data for bo th years do show, however, that A. cisternasii

had a significantly larger average male d u t c h size than A. obstetricans (Mann-Whi tney U-test, P < 0.0001).

Male size selection gradients

Figure 2 shows the linear regressions o f number o f eggs on male size (SVL). In each case, the regression was significant (P < 0.05) indicating that larger males carried more eggs. Figure 3 shows the linear regressions o f the number o f tadpoles hatched on male size. Here too, the correlat ion was significant in each case (P<0 .05 ) . The correlat ions were still significant if recalculated omit t ing extreme outliers. Therefore, larger males not only carried more eggs, but also successfully fathered larger numbers o f tadpoles.

For bo th species, males calling with eggs were no t significantly larger than those wi thout eggs (pooled re- sults for 1988 and 1989: A. obstetricans with eggs n = 12,

= 42.21 mm, SD = 4.707; A. obstetricans without eggs n = 1 2 , )?=42.92 ram, S D = 4 . 2 1 , M ann-Whi tne y U-test, P > 0 . 6 2 . A. cisternasii with eggs n = 2 0 , 2 = 3 5 . 6 5 m m , S D = 4 . 7 1 ; A. cisternasii without eggs n = 2 2 , 2 = 36.57 ram, SD = 2.91, Mann-Whi tney U-test P > 0.14.)

The values for total selection gradients (tim), estimat- ed sexual selection gradients for males (flmt), selection

Table 2. Values describing selection gradients for male body size (snout-vent length, SVL)

Table 1. Average and standard deviations for male and female clutches in Alytes obstetricans and A. cisternasii

Data for 1988

A. obstetricans A. cisternasii (n = 219) (n = 175)

Male clutch (SD) 72.72 eggs (27.11) ~b 86.53 eggs (34.20) ab Female clutch (SD) 41.67 eggs (10.94) 38.13 eggs (9.21)

Data for 1989

A. obstetricans A. cisternasii (n = 372) (n = 86)

Male clutch (SD) 61.96 eggs (25.38) b 60.80 eggs (25.94) b )? Female clutch (SD) 39.53 eggs (10.37) 44.07 eggs (9.60)

Combined data for 1988 and 1989

A. obstetricans A. cisternasii (n = 591) (n = 261)

Male clutch (SD) 65.21 eggs (26.42) 4 78.05 eggs (33.91)" Female clutch (SD) 41.14 eggs (11.14) 40.55 eggs (9.68)

Average male clutches were calculated using all the hatched egg- masses collected (n), including those hatched in the laboratory and those collected in the field (of unknown father). Average female clutch sizes were estimated from the fitted distributions in Fig. 1

a Significantly different means between species for the same year(s) P<0.001 b Significantly different within species between years P _< 0.001

A. obstetricans A. cisternasii

1988 n 101 162 fl 4.017 3.262 fi* 3.960 (0.836) 3.432 (0.796) fis ~ 3.015 (0 .858) 2.722(0.765) (fi~*)/(fl~) 0.766 (0.183) 0.795 (0.139) S 0.0055 0.0038 fl'=S' 0.1486 0.1110

1989 n 159 84 fi 4.420 4.615 fit 4.478 (0.921) 4.550 (0.748) fis t 2.919 (0.855) 3.607 (0.714) (fit)/(fit) 0.648 (0.123) 0.794 (0.097) S 0.0057 0.0036 fl'=S' 0.1591 0.1292

1988-1989 n 260 246 fl 4.320 4.480 fit 4.316 (0.628) 4.555 (0.772) fl~ 2.984 (0.601) 3.671 (0.716) (fix)/(fit) 0.691 (0.097) 0.807 (0.092) S 0.0059 0.0048 fl'=S' 0.1599 0.1484

Size selection gradients (fl), estimated male size selection gradient as recalculated by the bootstrapping simulation (fit), male size sex- ual selection gradient estimated by the bootstrapping simulation (fist), selection differentials (S), and standardized selection gra- dients [fl '=flx erLog(SVL)]. The ratio (fl~*)/(fl*) represents the pro- portion of male selection gradient for body size which is due to sexual selection. The standardized selection gradients are equal to the standardized selection differentials (S'=S/aLog(SVL)). All values are based only on successfully reproductive males. Values in parentheses are the standard deviations of the values estimated through the bootstrapping simulation

180

160

140

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Alytes obstetricans y = 3 . 0 4 6 x - 59.68, r2 = 0.148 n = 1 0 1

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(b) 1989 Fig . 2 a, b. L i n e a r r e g r e s s i o n : n u m b e r o f eggs in m a l e c l u t c h ve r sus m a l e b o d y size ( s n o u t - v e n t l eng th , SVL) in a 1988 a n d b 1989

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32 34 36 38 40 42 44 46 48 50

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Number of Tadpoles H a t c h e d

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160

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A lytes cisternasii y = 2.698x - 23.366, r2 = 0.049 n = 162

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SVL (mm)

(b) 1989

288

differentials (5), and standardized selection differentials (S') for size in each species for 1988, 1989, and both years combined, are shown in Table 2. It should be noted again that the calculated sexual selection gradient (]~m t) only includes the effect of relative mating success and not of female quality. The standard deviations generated by the iterations of the simulation process allow compar- ison of the estimated values. Only reproductive males were considered for the calculation of the gradients and differentials. No statistically significant difference was found between male size selection gradients for 1988 and 1989 in either species (ANCOVA, homogeneity of slopes, P > 0.8).

In all cases, the sexual selection gradients estimated through the bootstrapping simulation were significantly different from zero. This suggests that the relationship between number of mates and male size was significant and positive. Furthermore, the estimated sexual selection gradients were not statistically different from the total selection gradients (whether re-estimated by the simula- tion or measured directly). Hence, the correlation ob- served between male size and number of eggs obtained was explained mainly by the number of matings. These results indicate that most of the observed selection gra- dients for size (65 81%) can be accounted for by the positive correlation between male body size and number of mates. Other factors such as size-assortative mating may account for the remaining variance. It is difficult to assess the relevance of size-assortative mating. In the present study the number of cases of amplexus for which data on size of males and females was obtained was very limited because handling males immediately after amplexus often caused the males to lose their clutch of eggs. The correlation between male and female body size of amplectant pairs (pooled results for 1988 and 1989) was not significant for A. obstetricans (n= 13, r= 0.22, P>0.46) while it was significant for A. cisternasii (n=31, r=0.41, P<0.02, slope=0.39). Since the slope of the linear regression is not very high for A. cisternasii, it is safe to say that sexual selection (represented by number of female mates obtained) is responsible for most of the observed large-male reproductive advantage.

Male parental success

The distribution of the proportion of the clutch success- fully hatched by males of both species, is shown for both years (Fig. 4). The average hatching proportion was lower for A. obstetricans (n=591, 2=0.740, SD= 0.235) and was also more variable than for A. cisternasii (n=261, 2=0.885, SD=0.117). The hatching percent- ages were significantly higher for A. cisternasii (Mann- Whitney U-test, P<0.0001). In order to make a mean- ingful comparison between the variances in hatching success for the two species, special consideration had to be given to the fact that the values compared were variances of proportions. The binomial distribution of the probability of hatching was used to generate an esti- mate of the expected variance. The expected variance of a binomial distribution depends on its parameters.

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A lytes obstetricans

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Proportion ofthe eggs hatched

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140

120

100

80

60

40

Alytes cisternasii

20

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Proportion o f the eggs hatched

Fig. 4. Frequency distr ibution o f the propor t ions of the eggs in a male clutch that hatched successfully. Da ta for 1988 and 1989 combined

In this case the differences in average hatching success between species had to be considered. Therefore, I com- pared the observed variances with the expected variance of a binomial distribution characterized by two parame- ters, p and n. For each species p is equal to the ratio of all the eggs in male clutches that hatched, over the sum of all eggs in all male clutches. For this binomial distribution, the variances is Var=pq/n where q= 1 - p and n is the number of binomial trials (in this case, n is the harmonic mean of the number of eggs carried by a male). The expected variance for the hatching suc- cess of A. obstetricans, thus calculated, was 0.00354, and the observed variance divided by the expected variance is 16.07. Thus the observed variance is 16 times larger than expected if hatching of the egg masses were purely the result of the combined probabilities of hatching of individual eggs, given that each egg had the same proba- bility p of hatching. The same calculations were per- formed for A. cisternasii. The observed variance was also higher than expected but only 7.69 times higher

Table 3. Mean and variance (var) of proportion of eggs in male clutches that hatched and associated probabilities based on the binomial distribution

A. obstetricans A. cisternasii

Number of egg masses 591 261 Mean (proportion eggs 0.740 0.885

hatching/male) Variance (proportion eggs 0.055 0.0134

hatching/male) Harmonic mean (eggs/male) 58.214 62.655 p(= all eggs hatched/all eggs) 0.70875 0.8796 q(= 1 -p) 0.29125 0.1204 Observed var (proportion of 0.057 0.013

clutch hatched) Expected var (=pq/harmonic mean) 0.0035459 0.0016903 Observed var/Expected var 16.0749 7.6909

(Table 3). Although no statistical test is available to com- pare the deviation of the observed variances from the expected, A. obstetricans appears to have twice as high a deviation, possibly indicating higher variance in hatch- ing success among male A. obstetricans. These results suggest that if A. obstetricans females are able to select more efficient males they are likely to obtain larger bene- fits in direct fitness than A. cisternasii females.

Male size, parental success, and reproductive success

In order to separate the effect of the size of the male clutch (number of eggs) and body size of the male on hatching percentage, a multiple regression was con- ducted. In A. obstetricans both the effect of male body size and egg number were found to be non-significant (P>0.05) . In 1988 male size and hatching success were significantly negatively related for A. cisternasii ( P < 0.01) though the correlation coefficient was extremely low (r=0.20). The number of eggs in the male clutch, on the other hand, was not significantly correlated with hatching success (P > 0.05).

289

Male size selection gradients

The observed selection gradients are caused mainly by an increase in the upper limit of the number of eggs carried by larger males. At first sight this result could be interpreted as a direct consequence of the inability of small males to carry large clutches. However, in both species, males recorded calling with eggs were not signifi- cantly larger than those without eggs. These results sug- gest that smaller males did not cease to call after obtain- ing the first clutch. Hence, it is difficult to predict how the size-selection gradients would change if unmated males were included since we have no indication that would suggest that unmated males would be smaller than males found carrying clutches. Moreover, the lack of temporal trends in the size of males captured through the breeding seasons, in both species, and the short dura- tion of the mating season of A. cisternasii (Mfirquez 1992) suggest that there are no significant size-related differences in the tendency of males to remain at the breeding grounds in either species. Therefore, it is im- plausible that the observed male size-selection gradients could be accounted for by large males remaining longer at the breeding grounds. Besides, given the necessary synchronization of the development times of the eggs from different females in a single male egg mass, it is unlikely that time spent at the breeding grounds could be as important in Alytes as in other species of anurans without male parental care. Taken together, the evidence supports the hypothesis that the observed trends are like- ly to be a consequence of female choice. The preliminary observations of female A. obstetricans selecting lower frequency calls in two-speaker playback tests (Mfirquez 1990) support this explanation. Similar trends in other anurans have been interpreted as adaptive choice (e.g. Wilbur et al. 1978; Woodward 1982, 1986) or as mere mechanistic coupling of the vocal and hearing organs (Ryan et al. 1990; Mfirquez and Tejedo 1990). Further research on pre-copulatory behaviour in Alytes may de- termine the validity of these explanations as well as the possible role of additional mechanisms such as male choice, male-male competition through acoustic spacing, or even female-female competition.

Discussion

Male and female clutch size

The estimated number of eggs per female clutch is simi- lar for both species. The estimates obtained are in agree- ment with the numbers found for A. obstetricans by Reading and Clarke (1988) (means 39.1-51.6) and results of oviductal egg counts.

In both species male clutch size was more variable between years than female clutch size. Particularly, in A. cisternasii the total number of eggs per male batch was 29.7% higher in 1988 than in 1989. This may be due to the weather in 1989 which either caused some individuals to mate in summer, or selected against those males that carried larger clutches (see M/Lrquez 1992).

Male size, parental success, and reproductive success

Variability in hatching success was higher in A. obstetri- cans than in A. cisternasii, beyond the difference due to the fact that an overall lower mean hatching percent- age (closer to 0.5) in A. obstetricans allowed for wider fluctuations. Hatching success did not correlate signifi- cantly with sire size in A. obstetricans whereas in A. cisternasii a very slight, though significant, negative cor- relation between male size and hatching percentage was found in 1988 (n= 162, r2=0.041, P<0.05) . Given that male size was correlated with number of eggs in the clutch (Fig. 2) this trend could be interpreted at first sight as a limitation of the male's ability to fertilize large numbers of eggs (e.g. Verrell 1986, 1988, 1989). How- ever, multiple regression analyses indicate that male size,

290

and not clutch size, accounted for the observed trend. The existence of a large-male mating advantage in Alytes, in the absence of intense male-male competition, is probably due to female mate choice.

Hatching success, however, was not correlated with male clutch size in either species. Thus, male reproduc- tive success and male parental ability do not covary. Since number of mates accounts for a sizeable percent- age of clutch size, it appears that number of mates is not correlated with parental ability. These results are relevant in view of the different optimizing strategies that could be expected in either sex. Females would bene- fit f rom maximizing hatching success of their clutch of eggs, whereas males would maximize their fitness by hatching more tadpoles. This would be achieved by opti- mizing both the number of eggs received and hatching success, where the former may be more important .

The hypothesis of mate choice being based on pater- nal quality does not rely on the assumption of a genetic basis of paternal quality if female preference per se is heritable. The immediate gain in reproductive success through the choice of a " g o o d fa ther" may be enough to account for the selection of an otherwise neutral fe- male preference. Even if male hatching success if not repeatable or heritable, if females were able to assess the quality of a male in a given season, their ability to select such males would be adaptive. However, the results clearly indicate that, if females are expressing a choice, they are not selecting better caretakers for their eggs. I f this discussion was extended to the field of anur- an communication, it could be hypothesized that female choice would be reflected in the nature of the acoustical parameters preferred in the male call. Directional selec- tion for static or dynamic properties of the male call could be demonstrated (sensu Gerhardt 1991). Female preferences for non-repeatable, non-heritable compo- nents of the vocalization would be reflected in direction- al selection on dynamic properties of the calls. On the other hand, if static properties of the calls were selected it would be more likely that a repeatable and/or heritable male trait would be selected by females. Such hypotheses remain to be tested by bioacoustical experiments.

Speciation within Alytes is estimated to have occurred 15-16 million years ago (Maxson and Szymura 1984). Paternal care appears to have been retained in the differ- ent species, given the similarities of the breeding behav- iour of the two species (M/trquez and Verrell 1991 ; Mfir- quez 1992). Therefore ample time should have been available for the evolution of optimization strategies such as female choice of good caretakers. Such a trend was not found in this study. This result could be ac- counted for by low predictability of male quality before mating, or low heritability of male parental ability, or by a strong selection pressure for other strategies (such as a male strategy of maximization of mate number) that obscures the expression of female choice of good caretakers.

Larger males, however, obtained more matings. Is there a relationship between male size and female fit- ness? As larger males do not hatch a larger proport ion of the clutch, female preferences for larger males would

only have an adaptive explanation if male survival abili- ty or growth rate had significant heritabilities. Halliday and Verrell (1988) argued that large size in amphibians and reptiles was more related to juvenile growth than to age, and evidence for heritability of growth rate re- mains to be obtained in anurans. Therefore, the fact that larger males obtained more matings should not be interpreted as support for the resurrection fo the "sexy son" hypothesis (Weatherhead and Robertson 1979; Kirkpatr ick 1985). A more simple mechanistic explana- tion may be in order: larger males may have the ability to maximize their reproductive success by increasing the number of mates (by emitting more attractive calls or being physically able to carry larger egg masses), while females may not have the ability to maximize it because mate parental quality cannot be assessed prior to mat- ing.

Acknowledgements. I thank S.J. Arnold, L.D. Houck, R. Lande, M. Lloyd, and M.J. Wade, for their assistance and advice in the analysis of the data. I am especially indebted to P. Phillips who developed the bootstraping simulation software. I am also grateful to J.L. Alvarez, J. Fabo, M. Kenstaff, J.-P. Martinez-Rica, A. Rodriguez Jim6nez, M. Tejedo, and particularly to G. Rosas, who helped me in the field and laboratory. A. Berglund, S. B611, T. Halliday, M. Lizana, A. Salvador, N. Sanderson, M. Tejedo, P. Verrell, and two anonymous reviewers provided useful comments for the improvement of this manuscript, hence my gratitude. E.G. Crespo provided data of oviductal counts of female Alytes from Portugal. The" Diputaci6n General de Arag6n", and the "Agencia Medio Ambiente, Junta de Extremadura" granted the collecting permits. Funding for field work was provided by N.S.F. Doctoral Dissertation Improvement Grant BSR-8714956 (S.J. Arnold, P.I.) and Hinds Fund Grants (U. of Chicago), additional funding was provided by project CYCIT PB 89-0045C (P. Alberch, P.I.) Min- isterio de Educaci6n y Ciencia, Spain.

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