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Effects of sperm storage and male colour on probability of paternity in a polychromatic lizard
Animal Behaviour 77 (2009) 419–424
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Effects of sperm storage and male colour on probability of paternity in a polychromatic lizard Mats Olsson*, Tonia Schwartz, Tobias Uller 1, Mo Healey School of Biological Sciences, University of Wollongong
a r t i c l e i n f o Article history: Received 14 December 2007 Initial acceptance 3 March 2008 Final acceptance 31 October 2008 Published online 29 November 2008 MS. number: 07-20026R Keywords: alternative mating tactics Australian painted dragon lizard Ctenophorus pictus sexual selection
Sexual selection may take place before or after mating and may involve a large number of different mechanisms, for example, overt male aggression, mate choice, sperm competition and cryptic female choice. In most species, males show similar reproductive tactics and, hence, achieve their reproductive success in the same or a similar way. Sometimes, however, males evolve alternative reproductive tactics. One such example is the polychromatic Australian painted dragon lizard, Ctenophorus pictus, in which red males beat yellow males in staged contests for females and show different emergence patterns posthibernation in the wild with red males emerging to establish territories before yellow males do (at least in some years). Here we show that yellow males have significantly larger testes in relation to body size and condition than red males and copulate for a shorter period of time. Our mating experiments further showed that sperm storage played a significant role in male reproductive success (i.e. males sired offspring in later ovarian cycles than the one in which they actually mated). Furthermore, yellow males had a three times higher probability of paternity in some situations of sperm competition than red males, suggesting that male polymorphism may be associated with different reproductive tactics. 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd.
Sexual selection is a formidable force that sometimes exerts its power in processes before copulation, sometimes after, resulting in a diverse array of trait differences not only between males and females, but sometimes also between males of different categories within the same species and population (Andersson 1994; Rui et al. 2008). An example of this is males of the sneaker morph of the ruff, Philomachus pugnax (a shorebird) which have larger testicles and a higher rate of sperm production than the more dominant morph (Jukema & Piersma 2006). Another such example is the sideblotched lizard, Uta stansburiana, in which three colour morphs cycle through time in a negative frequency- and context-dependent manner; orange-throated males are ‘ultraterritorial’ and defend large territories, but can be invaded by yellow-throated males (rock–paper–scissors game; Sinervo & Lively 1996). Blue males are ‘cooperators’ and can be invaded by orange-throated males (Zamudio & Sinervo 2000; Calsbeek & Sinervo 2002; Sinervo et al. 2006). Thus, in both these species, males have different reproductive tactics with fitness benefits arising from overt male aggression in one morph and from competition in the female reproductive tract after copulation in another. We studied similar phenomena in the Australian painted dragon lizard, Ctenophorus pictus, a species with three different male * Correspondence: M. Olsson, School of Biological Sciences, University of Wollongong, Northfields Avenue, Wollongong, NSW 2522, Australia. E-mail address: [email protected] (M. Olsson). 1 T. Uller is currently at the Edward Grey Institute, Department of Zoology, University of Oxford, Oxford OX1 3PS, U.K.
morphs with red, orange and yellow head colour which are distinct to the human eye and stable throughout life. Females are monomorphic with respect to head colour (camouflaged; Cogger 2000; Healey et al. 2007; independent scoring of >200 males showed 100% congruence between M.O. and M.H.; Olsson et al. 2007a). We report elsewhere that red males defeat yellow males significantly more often in staged contests for females (Healey et al. 2007). When those laboratory trials were conducted, however, the frequency of orange males in the natural population was so low that they did not allow experimentation and were therefore subject to no further analysis (Olsson et al. 2007a). Intuitively then, one may expect red and yellow males to conform to the ‘dominant versus sneaker’ strategy scenario in the wild. We therefore designed a two-step study in which we first compared the gonadosomatic indices (testes size in relation to body mass and body condition) of the three morphs to assess whether the sneaker morph (subordinate yellow morph) had larger testes. Thereafter, we staged laboratory experiments in which we allowed females to be ‘cuckolded’ by males of both morphs, in (1) direct competition (within the same ovarian cycle) and (2) indirect competition (with competition between stored and freshly inseminated sperm). The rationale for this was that, if males of the different morphs coexist because yellow males are better at sperm competition than red males, and red males are better at defending territories (sneaker versus dominance strategies), then the relative success of each morph in sperm competition (including sperm storage) needs to be evaluated under a competitive scenario that mimics processes in the wild
0003-3472/$38.00 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. doi:10.1016/j.anbehav.2008.10.017
M. Olsson et al. / Animal Behaviour 77 (2009) 419–424
METHODS Field Protocol The Australian painted dragon is a small (adult snout–vent length (SVL) 65–95 mm, mass 8–16 g), diurnal lizard occurring in sandy habitats with a range covering central and western New South Wales to Western Australia. It is short-lived and only ca. 10% live to a second year. At our study site (Yathong Nature Reserve, NSW; 145 350 E, 32 350 S), females were allowed to mate with their territorial male for ca. 2 weeks after emergence from hibernation. They were thereafter captured by noosing, transported in cloth bags to the University of Wollongong inside a large insulated box (‘Esky’), and kept in captivity for mating experiments as outlined below to assess effects of male colour, mating order and ‘cuckoldry’ on probability of paternity. Relative testes mass in red and yellow males was estimated by noosing and humanely killing (by injection of Brietal) a sample of males (N ¼ 19: nine red, 10 yellow). The testes were then dissected out and weighed (0.001 g) and the carcass weighed thereafter. Husbandry and Mating Protocol Females, their observed wild partners and additional wildcaught males were noosed and brought to facilities at the University of Wollongong where we staged matings with multiple or single males in consecutive ovarian cycles to allow the sperm from males to compete under natural storage circumstances in the female reproductive tract. Thus, we staged copulations with females that had been allowed to mate in the wild with their natural partner(s), who were then ‘cuckolded’ by males offered to the female in copulations in the laboratory, and then returned to their home cages. Female follicles are palpable and reach a size of about 4–6 mm during the receptive part of the ovarian cycle, at which stage males were introduced to the female cages over the following 4 months (September–December), corresponding to their natural mating season. Therefore, we mated females as soon as they became receptive in an ovarian cycle according to the procedure illustrated in Fig. 1. Depending on interindividual differences between females (e.g. how quickly they reyolked follicles, what condition they were in upon capture, etc.), the number of ovarian cycles they went through (and consequently clutches they laid) varied between two and five with a total of 80 paternityassessed clutches. Fourteen females were mated once each in the laboratory with two randomly selected males of different head
Capture and transport to lab facility Egg formation
3
1
1
1
Copulations in the wild
3
2
Emergence from hibernation
1
during a prolonged breeding season. This effect may be particularly important when males mate early in the season and their sperm is stored for subsequent fertilizations. Thus, males may, in this way, sire offspring regardless of whether they are dead or alive (Olsson et al. 2007b). To assess such paternity dynamics, we backtracked the paternity of each offspring from copulations in a series of ovulatory cycles using molecular genetics (microsatellites; Schwartz et al. 2007). Importantly, this mimics ‘real-time’ ongoing processes of sexual selection in the wild as opposed to single-clutch comparisons of probability of paternity for males in staged sperm competition experiments with virgin females, which would generate results of limited importance for strong inferences about evolution in the wild (Uller & Olsson 2008). We then tested the accumulated data statistically in two ways: (1) we assessed clutchspecific differences in siring success between male categories, ensuring that a particular female/male pairing was only used once per clutch (while controlling for multiple testing), and (2) we selected one data point at random to ensure independence of data and to avoid pseudoreplication and then assessed male morphspecific reproductive tactics (‘Reduced data set’).
Time through breeding season (in weeks, approximate)
420
Ovulation and egg laying
Once receptive, mating with 1 or 2 lab males
Egg formation
Ovulation and egg laying
Figure 1. Description of mating protocol and approximate temporal separation in time during the mating season of female ovarian cycles and receptivity periods. Note that this is an approximate description of overall patterns, since individual females went through between two and five cycles, depending on how quickly they reyolked follicles in a new cycle. Each ovarian cycle is approximately 3 weeks on average.
colour within 1 h of each other before they produced their second clutch. An additional 20 females were mated once only in the laboratory. Copulations were timed (1 s) with a stopwatch (since sperm transfer is likely to be continuous throughout the copulation; Olsson 2001) whenever penetration and withdrawal of the hemipenis (and hence interruption of the copulation) were clearly visible. Once mating trials ended, an additional 45 clutches were laid by the laboratory mated females without a prior copulation and, hence, sired from stored sperm only. Mean clutch size SD was 3.6 1.2 (N ¼ 80) and was not correlated with increasing clutch number per female (Spearman rank correlation: rS ¼ 0.17, P ¼ 0.13) or date in the season (rS ¼ 0.04, P ¼ 0.82). The low availability of males (N ¼ 26) made it impossible to mate each female with a unique male in each ovarian cycle. However, our clutch-specific analyses and sacrifice of data following randomization ensured independence of data and no pseudoreplication. No male was mated more than once per day. All females were mated with males in rotation, making sure that a given female was only mated once with a given male except in one single case. All lizards were weighed and measured (SVL and total length, 1 mm). In captivity, the lizards were held separately in (600 600 mm and 500 mm high) cages with a 40 W spotlight at one end to allow thermoregulation to their preferred body temperature (ca. 36–37 C as per cloacal temperatures of freeranging territorial, displaying males; M. Olsson & E. Wapstra, unpublished data). They were fed crickets and mealworms dusted with calcium and multivitamins ad libitum every second day and sprayed with a mist of water twice daily.
M. Olsson et al. / Animal Behaviour 77 (2009) 419–424
Female cages were checked for recently laid eggs at least twice daily, which were immediately removed and placed in moist vermiculite (mixed with water in a 1:7 ratio) and incubated at ca. 30 C for ca. 60 days until hatching, when the offspring had the tail tip (3 mm) removed and stored in 95% ETOH for DNA analysis and subsequent paternity assignment.
Molecular Paternity Assignment For a combination of permanent marking and sampling of DNA, two toe tips (ca. 2 mm from different feet) and ca. 5 mm of the tail tip were removed from adult lizards cooled to ca. 5 C, which prevents bleeding and minimizes pain in these ectotherms (Sinervo & DeNardo 1996). This did not affect lizard health or behaviour. DNA was isolated from toe and tail clip tissue samples using phenol/chloroform extraction (Sambrook et al. 1989). Using fluorescently labelled primers, individuals’ DNA was PCR amplified at four microsatellite loci: CP01, CP02, CP10 and CP17 (Schwartz et al. 2007). All loci were amplified individually in 7 ml reactions using 10–20 ng of DNA, 0.22 mM dNTP, 0.3 mM of each primer, 1 PCR buffer (Qiagen Inc., Valencia, CA, U.S.A.) containing Tris–HCL, KCL (NH4)2SO4, 15 mM MgCl2 pH 8.7 (final concentration of MgCl2 was 1.5 mM) and 0.05 U of HotStart Taq (Qiagen). Cycling conditions included a hot start denaturation at 95 C for 15 min, 30 cycles of 95 C for 30 s, annealing temperature for 30 s, 72 C for 30 s, and a final extension at 72 C for 30 min. Fluorescent amplifications were electrophoresed on a genetic analyser (ABI3130xl: locus CP17; ABI3900: loci CP01, CP02, CP10: Applied Biosystems, Inc., Foster City, CA, U.S.A.) using the LIZ500 size standard (Applied Biosystems). Alleles were scored using GeneMapper (Applied Biosystems) and confirmed visually. Genotypes from the adults were used to calculate allele frequencies, observed and expected heterozygosites, frequency of null alleles and the polymorphic information content (PIC: a measure of informativeness related to expected heterozygosity; in CERVUS 2.0) (Marshall et al. 1998; Table 1). Mother–offspring genotypes were compared across clutches for the presence of null alleles, as indicated when the mother is homozygous for a particular allele and her offspring are homozygous for a different allele. The following analyses were conducted with and without potential null alleles to verify the robustness of the paternity assignments. To determine 95% and 80% confidence levels for the assignments, we conducted two simulation analyses: first, using zero genotyping error rate (complete exclusion) and, second, using a 0.01 genotyping error rate. For these analyses we assumed that 95% of potential fathers had been sampled, since most matings were staged in the laboratory with known male pairs. All males were used as potential fathers for all the offspring since they may have mated with the female in the field prior to being brought into the
laboratory. Results were compared across the different analyses and assigned fathers were assessed by eye across mother–offspring pairs. Any males mated to a particular female at any time throughout this study were specifically compared to all of her offspring to verify the male’s reproductive success. Any potential mismatches between father–offspring–mother were doublechecked with the raw data. This study was approved by The Animal Ethics Committee, University of Wollongong, and conducted in compliance with Wollongong University animal ethics protocols. A scientific licence was also issued under the National Parks and Wildlife Act 1974 by the National Parks and Wildlife Service, NSW, Australia. All lizards were released at the place of capture after the study was completed. RESULTS Male Body and Relative Testes Size Red and yellow males did not differ significantly in mean body mass (red males: X SE ¼ 5:9 0:42 g; yellow males: 6.0 0.26 g; t test: t17 ¼ 0.27, P ¼ 0.79) or SVL (red males: X SE ¼ 62:3 1:7 mm; yellow males: 64.3 0.93 mm; t17 ¼ 1.33, P ¼ 0.20). Testes mass, however, differed between the two morphs (red males: X SE ¼ 0:15 0:01 g; yellow males: 0.18 0.004 g; tested with testes mass as the response variable, head colour as the factor and condition as the covariate: ANOVA: F2,16 ¼ 4.5, R2 ¼ 0.36, P ¼ 0.028; head colour: F1,16 ¼ 5.3, P ¼ 0.035; condition: F1,16 ¼ 3.9, P ¼ 0.07). Red and yellow males also differed in how their testes size was related to body size (heterogeneity of slopes test: F3,18 ¼ 4.6, R2 ¼ 0.48, P ¼ 0.017; head colour: F ¼ 7.2, P ¼ 0.017; SVL: F ¼ 0.79, P ¼ 0.39; head colour*SVL: F ¼ 6.7, P ¼ 0.02; Fig. 2). Paternity Analysis All of the loci used had a high polymorphic information content (range 0.819–0.957) with the overall exclusionary power of 0.996 (Table 1). The high exclusionary power allowed us to exclude sampled males as potential fathers when the mother was known. Thereby we could effectively determine whether offspring had been fathered by an unsampled field male (Table 1). Simulations estimated that with the known mother’s genotype, paternity could be successfully assigned at 95% confidence in 87% of cases with complete exclusion, and 27% of cases with a 0.01 error rate. The paternity assignments were consistent across the different analyses and the assigned father never had more than one allele mismatch
0.225
Locus
No. of alleles
PIC
Excl
HO
HE
Null frequency
CP01 CP02 CP10 CP17
15 27 41 28
0.819 0.914 0.957 0.937
0.518 0.722 0.847 0.784
0.843 0.888 0.965 0.961
0.840 0.924 0.964 0.949
0.004 þ0.0163 0.036 0.011
Mean
27.75
0.906
0.718
0.914
0.920
Total
0.996
PIC represents the polymorphic information content; Excl represents the power of the locus to exclude a randomly selected male from being the father of a randomly selected offspring based only on the offspring genotype. HO and HE represent the observed and expected heterozygosities; none of these loci were out of Hardy–Weinberg equilibrium. Null frequency represents the predicted frequency of null alleles at that locus based on mother–offspring mismatches. N ¼ 34 females, and 26 males.
Testes mass (g)
0.205 Table 1 Details of microsatellite markers used for parentage analysis
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Red Yellow
0.185 0.165 0.145 0.125 0.105 0.085 57
59
61
63
65
67
69
71
SVL (mm) Figure 2. Testes mass of red and yellow males in relation to body size (snout–vent length, SVL).
M. Olsson et al. / Animal Behaviour 77 (2009) 419–424
Table 2 Descriptive data (X SE) of male size and copulation duration for males mating first or second and for red and yellow males
Snout–vent length (mm) Time in copula (s)
First
Second
Red
Yellow
65.10.65 26.15.1
65.81.02 24.74.7
64.81.04 32.89.8
64.70.96 12.81.9
and always fell within the 95% confidence level, or the 80% confidence level with siblings assigned the same father at the 95% confidence level. Overall, 225 of the 310 offspring were assigned a father from the males we had genotyped. Offspring for which a father could not be assigned were excluded from the analyses of determinants of paternity.
4 Difference in no. of young from field and laboratory sperm
422
3 2 1 0 -1 -2 -3 -4 -5
Mating Order Siring success at an individual level Data from 13 males selected at random that had been used more than once in different clutches and with different females were used to test for covariation between the proportion of sired young in their first and second copulations. These paternity scores were not correlated (rS ¼ 0.03, P ¼ 0.91), suggesting that some males did not do consistently better (or worse) in terms of siring success. Reduced data set We conducted pairwise t tests, that is, we assessed differences between categories of males in the probability of paternity within female clutches. This procedure thus identifies explicit differences in the average probability of paternity between categories of males (e.g. analysing differences between males mating first and second in the laboratory while ignoring offspring arising from stored sperm). There was no difference in paternity (number of sired young) between stored and first males (difference in mean number of sired young SE ¼ 0.61 0.64, N ¼ 26; t1 ¼ 0.97, P ¼ 0.34), between stored and second males (0.67 0.88, N ¼ 12; t1 ¼ 0.76, P ¼ 0.46) or between first and second males (0.50 0.73, N ¼ 12; t1 ¼ 0.68, P ¼ 0.51). Sperm Storage, Copula Duration and Body Size
2 3 Clutch number
4
Figure 4. Mean difference in number of sired offspring between sperm from field inseminations and summed laboratory inseminations SE. Thus, positive values represent more offspring from stored sperm from field matings. Pairwise t tests: clutch number (cn) 1: t1 ¼ 7.70, N ¼ 31, P < 0.0001; cn 2: t1 ¼ 0.75, N ¼ 24, P ¼ 0.46; cn 3: t1 ¼ 0.24, N ¼ 19, P ¼ 0.82; cn 4/5 (pooled 4 and 5): t1 ¼ 13.8, N ¼ 6, P < 0.0001. For cn 4/5, the test was performed on log-transformed absolute values, which successfully normalized the data.
descriptive data). A descriptive plot of the entire data set suggested that sperm stored from field matings declined in competitive ability throughout the season (rS ¼ 0.39, N ¼ 80, pseuodreplication precludes calculation of significance level; Fig. 3). When single data points were generated per individual male and female through randomization, this relationship did not reach significance (rS ¼ 0.26, N ¼ 34, P ¼ 0.14). However, when females’ last clutches were deliberately selected, thus assessing the most extreme cases of storage effects on probability of paternity, field males (i.e. those copulating first in the season) sired significantly fewer offspring later in the season (correlation between siring success and clutch number: rS ¼ 0.37, N ¼ 34, P ¼ 0.030). Furthermore, the summed number of offspring produced from field versus laboratory matings changed in sign throughout the season: early clutches were primarily sired by field males whereas later clutches were mostly fertilized by stored sperm from laboratory matings (Fig. 4). This was also suggested by a negative correlation between the fertilization success of field stored sperm and firstmale probability of paternity (rS ¼ 0.63, N ¼ 26, P ¼ 0.0005). However, negative correlations between first and second males did not reach significance (rS ¼ 0.48, N ¼ 12, P ¼ 0.11), nor did those between stored and second males (rS ¼ 0.39, N ¼ 12, P ¼ 0.21). Male Coloration Differences between red and yellow males in copulation behaviour became apparent through a comparison of time spent in
7 6 5 4 3 2 1 0 0
1
2
3
4
5
6
Clutch number Figure 3. Fertilization success of stored spermatozoa (i.e. sperm from field matings prior to female captures) in relation to clutch number. Symbol size represents number of observations (1–10).
Mean number of offspring sired
Offspring from stored sperm
Since ejaculate volume is correlated with time in copula in a closely related species (C. fordi; Olsson 2001), and possibly with body size, we looked for correlations between the difference between first and second males in SVL and time in copula and the corresponding difference in proportion of sired young, but these were not significantly correlated (P > 0.10; see Table 2 for
1
2.5 2 1.5 1 0.5 0
Red
Yellow
Figure 5. Fertilization success (X SE) of yellow and red males that mated first.
M. Olsson et al. / Animal Behaviour 77 (2009) 419–424
copula for red and yellow first males (for which we had the largest sample). Red males showed an almost three times longer copulation time on average than yellow males (Kruskal–Wallis test: c21 ¼ 4.86, P ¼ 0.029; reds: X SE ¼ 37:7 13:5 s, N ¼ 10; yellows: 13.4 2.34 s, N ¼ 14; Table 2). In spite of this, yellow males sired more than three times as many offspring on average per copulation as red males (mean number of offspring per mating in competition with stored and second males SE ¼ 0.40 0.22 versus 1.8 0.53; t17.2 ¼ 2.43, Satterthwaite’s approximation, P ¼ 0.026; Fig. 5). DISCUSSION Our approach to this study is set in a framework of sperm competition, cryptic female effects, evolution of territoriality and alternative reproductive tactics in a polymorphic lizard. We have shown that females mate multiply if offered this opportunity and that female sperm storage capacity is high enough to ensure the siring of offspring in several ovarian cycles subsequent to the one in which a target male is copulating. This further supports similar observations in a previous study (Olsson et al. 2007b). Furthermore, the fertilization success of fresh sperm was negatively related to that of stored sperm, possibly as sperm from early copulations are used up at fertilization, forced out of the oviduct at ovulation, or simply die over time and decline in competitive ability against later ejaculates. This suggests that numerical sperm competition is the underlying mechanism to the results in these mating trials (e.g. Parker 1998). Elsewhere, one of us (M.O.) has demonstrated, in the field as well as in controlled laboratory experiments, that cryptic female choice or ‘sperm selection’ could increase offspring viability in two different reptile species, adder snakes, Vipera berus, and sand lizards, Lacerta agilis (Madsen et al. 1992; Olsson et al. 1994a, b, 1996). Thus, we emphasize that our analyses in the current study have seriously considered the ‘female perspective’ as a highly viable alternative hypothesis to sperm competition between males. However, the quantitative relationships in terms of probability of paternity of early and late copulating males seem to lend much more parsimonious support to a numerical sperm competition scenario than to cryptic female choice in this species. Given the numerical aspect to sperm competition and female sperm-storing capacity, territoriality is likely to prevent rival matings (thus acting as a paternity guard) and further enhance the reproductive success of territorial males. We have shown elsewhere (Olsson et al. 2007a) that the opportunity for sperm competition (as approximated by the frequency of multiple paternity) may vary between years and be driven by the availability of perches for territorial patrolling, and that territorial defence appears to be a highly successful means of preventing loss of paternity to rivals in this species. Thus, if red males are better at defending both their territories and females, as our laboratory trials suggest, yellow male competitive ability would have to arise through some other mechanism, such as via greater postcopulatory reproductive success in a situation of sperm competition. Our experiment mimicked such a situation, in which both yellow and red males were allowed to act as sneakers that mate with the females of territorial males, originally mated under natural conditions (as verified by molecular genetics). For males mating for their first time in the laboratory, yellow males had, on average, three times the reproductive success of red males, in spite of copulating for only a third of the time (which is an important aspect, since transfer of spermatozoa and ejaculate products continues throughout copulation in squamate reptiles; Olsson & Madsen 1998). This result is therefore congruent with the observation that yellow males have larger testicles in relation to body size than red males, and they may even adopt shorter copulations as part of a sneaker strategy. Furthermore, the difference in testes size in relation to body size between the two morphs may suggest
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additional tactical differences between them, for example that sperm competition is more important for securing fitness benefits in younger, smaller yellow males competing with red males emerging earlier from hibernation to monopolize females (Olsson et al. 2007a). Female sperm storage has been argued to result from selection arising from a low encounter rate of partners (see numerous examples across taxa in Birkhead & Møller 1998). Painted dragons, however, occur in large numbers with territories that are on average less than 100 m along their longest axes (Olsson et al. 2007a). Thus, having too few mating opportunities seems a farfetched explanation for the evolution of sperm storage in this species, which may have evolved for other adaptive reasons (Olsson et al. 2007b). In summary, we have shown that painted dragons have the capacity for sperm storage over several ovarian cycles, and that male reproductive success in laboratory experiments is largely driven by these effects. We have also shown that a contributing factor to the maintenance of both red and yellow morphs in this species may be differences in components of sexual selection, with yellow males potentially having greater fitness benefits from postcopulatory processes than red males, and that this may be related to the larger relative testes size of yellow males. Acknowledgments We thank E. and G. Snaith, and G. and M. Swan for logistical support and field assistance. We are financially supported by the Australian Research Council (M.O. and T.U.), and the Wenner–Gren Foundations (T.U.). We thank C. Sherman for assistance in the laboratory and six referees for their much appreciated contributions. References Andersson, M. B. 1994. Sexual Selection. Princeton, New Jersey: Princeton University Press. Birkhead, T. & Møller, A. P. 1998. Sperm Competition and Sexual Selection. San Diego: Academic Press. Calsbeek, R. & Sinervo, B. 2002. The ontogeny of territoriality during maturation. Oecologia, 132, 468–477. Cogger, H. G. 2000. Reptiles and Amphibians of Australia, 6th Edn. Melbourne: New Holland Publishers (Australia). Healey, M., Uller, T. & Olsson, M. 2007. Seeing red: morph-specific contest success, and survival rates, in a colour-polymorphic agamid lizard. Animal Behaviour, 74, 337–341. Jukema, J. & Piersma, T. 2006. Permanent female mimics in a lekking shorebird. Biology Letters, 2, 161–164. Madsen, T., Shine, R., Loman, J. & Håkansson, T. 1992. Why do female adders copulate so frequently? Nature, 402, 34–35. Marshall, T. C., Slate, J., Kruuk, E. B. & Pemberton, J. M. 1998. Statistical confidence for likelihood-based paternity inference in natural populations. Molecular Ecology, 7, 639–655. Olsson, M. 2001. ‘Voyeurism’ prolongs copulation in the dragon lizard (Ctenophorus fordi). Behavioral Ecology and Sociobiology, 50, 378–381. Olsson, M. & Madsen, T. 1998. Sexual selection and sperm competition in reptiles. In: Sperm Competition and Sexual Selection (Ed. by T. R. Birkhead & A. P. Møller), pp. 503–578. San Diego: Academic Press. ¨ m, H., Madsen, T. & Shine, R. 1994a. Promiscuous Olsson, M., Norberg, A., Tegelstro lizard females have more viable young (under subheading: Can female adders multiply?). Nature, 369, 528. ¨ m, H., Madsen, T. & Shine, R. 1994b. Rewards of Olsson, M., Gullberg, A., Tegelstro ‘promiscuity’. Nature, 372, 230. ¨ m, H. 1996. Sperm Olsson, M., Shine, R., Madsen, T., Gullberg, A. & Tegelstro selection by females. Nature, 383, 585. Olsson, M., Healey, M., Wapstra, E., Schwartz, T., LeBas, N. & Uller, T. 2007a. Mating system variation and morph fluctuations in a polymorphic lizard. Molecular Ecology, 16, 5307–5315. Olsson, M., Schwartz, T., Uller, T. & Healey, M. 2007b. Sons are made from old stores: sperm storage effects on sex ratio in a lizard. Biology Letters, 35, 491–493. Parker, G. A. 1998. Sperm competition and the evolution of ejaculates: a theory base. In: Sperm Competition and Sexual Selection (Ed. by T. R. Birkhead & A. P. Møller), pp. 3–54. San Diego: Academic Press. Rui, R. F., Taborsky, M. & Brockman, H. J. 2008. Alternative Reproductive Strategies. Cambridge: Cambridge University Press.
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Animal Behaviour journal homepage: www.elsevier.com/locate/yanbe
Effects of sperm storage and male colour on probability of paternity in a polychromatic lizard Mats Olsson*, Tonia Schwartz, Tobias Uller 1, Mo Healey School of Biological Sciences, University of Wollongong
a r t i c l e i n f o Article history: Received 14 December 2007 Initial acceptance 3 March 2008 Final acceptance 31 October 2008 Published online 29 November 2008 MS. number: 07-20026R Keywords: alternative mating tactics Australian painted dragon lizard Ctenophorus pictus sexual selection
Sexual selection may take place before or after mating and may involve a large number of different mechanisms, for example, overt male aggression, mate choice, sperm competition and cryptic female choice. In most species, males show similar reproductive tactics and, hence, achieve their reproductive success in the same or a similar way. Sometimes, however, males evolve alternative reproductive tactics. One such example is the polychromatic Australian painted dragon lizard, Ctenophorus pictus, in which red males beat yellow males in staged contests for females and show different emergence patterns posthibernation in the wild with red males emerging to establish territories before yellow males do (at least in some years). Here we show that yellow males have significantly larger testes in relation to body size and condition than red males and copulate for a shorter period of time. Our mating experiments further showed that sperm storage played a significant role in male reproductive success (i.e. males sired offspring in later ovarian cycles than the one in which they actually mated). Furthermore, yellow males had a three times higher probability of paternity in some situations of sperm competition than red males, suggesting that male polymorphism may be associated with different reproductive tactics. 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd.
Sexual selection is a formidable force that sometimes exerts its power in processes before copulation, sometimes after, resulting in a diverse array of trait differences not only between males and females, but sometimes also between males of different categories within the same species and population (Andersson 1994; Rui et al. 2008). An example of this is males of the sneaker morph of the ruff, Philomachus pugnax (a shorebird) which have larger testicles and a higher rate of sperm production than the more dominant morph (Jukema & Piersma 2006). Another such example is the sideblotched lizard, Uta stansburiana, in which three colour morphs cycle through time in a negative frequency- and context-dependent manner; orange-throated males are ‘ultraterritorial’ and defend large territories, but can be invaded by yellow-throated males (rock–paper–scissors game; Sinervo & Lively 1996). Blue males are ‘cooperators’ and can be invaded by orange-throated males (Zamudio & Sinervo 2000; Calsbeek & Sinervo 2002; Sinervo et al. 2006). Thus, in both these species, males have different reproductive tactics with fitness benefits arising from overt male aggression in one morph and from competition in the female reproductive tract after copulation in another. We studied similar phenomena in the Australian painted dragon lizard, Ctenophorus pictus, a species with three different male * Correspondence: M. Olsson, School of Biological Sciences, University of Wollongong, Northfields Avenue, Wollongong, NSW 2522, Australia. E-mail address: [email protected] (M. Olsson). 1 T. Uller is currently at the Edward Grey Institute, Department of Zoology, University of Oxford, Oxford OX1 3PS, U.K.
morphs with red, orange and yellow head colour which are distinct to the human eye and stable throughout life. Females are monomorphic with respect to head colour (camouflaged; Cogger 2000; Healey et al. 2007; independent scoring of >200 males showed 100% congruence between M.O. and M.H.; Olsson et al. 2007a). We report elsewhere that red males defeat yellow males significantly more often in staged contests for females (Healey et al. 2007). When those laboratory trials were conducted, however, the frequency of orange males in the natural population was so low that they did not allow experimentation and were therefore subject to no further analysis (Olsson et al. 2007a). Intuitively then, one may expect red and yellow males to conform to the ‘dominant versus sneaker’ strategy scenario in the wild. We therefore designed a two-step study in which we first compared the gonadosomatic indices (testes size in relation to body mass and body condition) of the three morphs to assess whether the sneaker morph (subordinate yellow morph) had larger testes. Thereafter, we staged laboratory experiments in which we allowed females to be ‘cuckolded’ by males of both morphs, in (1) direct competition (within the same ovarian cycle) and (2) indirect competition (with competition between stored and freshly inseminated sperm). The rationale for this was that, if males of the different morphs coexist because yellow males are better at sperm competition than red males, and red males are better at defending territories (sneaker versus dominance strategies), then the relative success of each morph in sperm competition (including sperm storage) needs to be evaluated under a competitive scenario that mimics processes in the wild
0003-3472/$38.00 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. doi:10.1016/j.anbehav.2008.10.017
M. Olsson et al. / Animal Behaviour 77 (2009) 419–424
METHODS Field Protocol The Australian painted dragon is a small (adult snout–vent length (SVL) 65–95 mm, mass 8–16 g), diurnal lizard occurring in sandy habitats with a range covering central and western New South Wales to Western Australia. It is short-lived and only ca. 10% live to a second year. At our study site (Yathong Nature Reserve, NSW; 145 350 E, 32 350 S), females were allowed to mate with their territorial male for ca. 2 weeks after emergence from hibernation. They were thereafter captured by noosing, transported in cloth bags to the University of Wollongong inside a large insulated box (‘Esky’), and kept in captivity for mating experiments as outlined below to assess effects of male colour, mating order and ‘cuckoldry’ on probability of paternity. Relative testes mass in red and yellow males was estimated by noosing and humanely killing (by injection of Brietal) a sample of males (N ¼ 19: nine red, 10 yellow). The testes were then dissected out and weighed (0.001 g) and the carcass weighed thereafter. Husbandry and Mating Protocol Females, their observed wild partners and additional wildcaught males were noosed and brought to facilities at the University of Wollongong where we staged matings with multiple or single males in consecutive ovarian cycles to allow the sperm from males to compete under natural storage circumstances in the female reproductive tract. Thus, we staged copulations with females that had been allowed to mate in the wild with their natural partner(s), who were then ‘cuckolded’ by males offered to the female in copulations in the laboratory, and then returned to their home cages. Female follicles are palpable and reach a size of about 4–6 mm during the receptive part of the ovarian cycle, at which stage males were introduced to the female cages over the following 4 months (September–December), corresponding to their natural mating season. Therefore, we mated females as soon as they became receptive in an ovarian cycle according to the procedure illustrated in Fig. 1. Depending on interindividual differences between females (e.g. how quickly they reyolked follicles, what condition they were in upon capture, etc.), the number of ovarian cycles they went through (and consequently clutches they laid) varied between two and five with a total of 80 paternityassessed clutches. Fourteen females were mated once each in the laboratory with two randomly selected males of different head
Capture and transport to lab facility Egg formation
3
1
1
1
Copulations in the wild
3
2
Emergence from hibernation
1
during a prolonged breeding season. This effect may be particularly important when males mate early in the season and their sperm is stored for subsequent fertilizations. Thus, males may, in this way, sire offspring regardless of whether they are dead or alive (Olsson et al. 2007b). To assess such paternity dynamics, we backtracked the paternity of each offspring from copulations in a series of ovulatory cycles using molecular genetics (microsatellites; Schwartz et al. 2007). Importantly, this mimics ‘real-time’ ongoing processes of sexual selection in the wild as opposed to single-clutch comparisons of probability of paternity for males in staged sperm competition experiments with virgin females, which would generate results of limited importance for strong inferences about evolution in the wild (Uller & Olsson 2008). We then tested the accumulated data statistically in two ways: (1) we assessed clutchspecific differences in siring success between male categories, ensuring that a particular female/male pairing was only used once per clutch (while controlling for multiple testing), and (2) we selected one data point at random to ensure independence of data and to avoid pseudoreplication and then assessed male morphspecific reproductive tactics (‘Reduced data set’).
Time through breeding season (in weeks, approximate)
420
Ovulation and egg laying
Once receptive, mating with 1 or 2 lab males
Egg formation
Ovulation and egg laying
Figure 1. Description of mating protocol and approximate temporal separation in time during the mating season of female ovarian cycles and receptivity periods. Note that this is an approximate description of overall patterns, since individual females went through between two and five cycles, depending on how quickly they reyolked follicles in a new cycle. Each ovarian cycle is approximately 3 weeks on average.
colour within 1 h of each other before they produced their second clutch. An additional 20 females were mated once only in the laboratory. Copulations were timed (1 s) with a stopwatch (since sperm transfer is likely to be continuous throughout the copulation; Olsson 2001) whenever penetration and withdrawal of the hemipenis (and hence interruption of the copulation) were clearly visible. Once mating trials ended, an additional 45 clutches were laid by the laboratory mated females without a prior copulation and, hence, sired from stored sperm only. Mean clutch size SD was 3.6 1.2 (N ¼ 80) and was not correlated with increasing clutch number per female (Spearman rank correlation: rS ¼ 0.17, P ¼ 0.13) or date in the season (rS ¼ 0.04, P ¼ 0.82). The low availability of males (N ¼ 26) made it impossible to mate each female with a unique male in each ovarian cycle. However, our clutch-specific analyses and sacrifice of data following randomization ensured independence of data and no pseudoreplication. No male was mated more than once per day. All females were mated with males in rotation, making sure that a given female was only mated once with a given male except in one single case. All lizards were weighed and measured (SVL and total length, 1 mm). In captivity, the lizards were held separately in (600 600 mm and 500 mm high) cages with a 40 W spotlight at one end to allow thermoregulation to their preferred body temperature (ca. 36–37 C as per cloacal temperatures of freeranging territorial, displaying males; M. Olsson & E. Wapstra, unpublished data). They were fed crickets and mealworms dusted with calcium and multivitamins ad libitum every second day and sprayed with a mist of water twice daily.
M. Olsson et al. / Animal Behaviour 77 (2009) 419–424
Female cages were checked for recently laid eggs at least twice daily, which were immediately removed and placed in moist vermiculite (mixed with water in a 1:7 ratio) and incubated at ca. 30 C for ca. 60 days until hatching, when the offspring had the tail tip (3 mm) removed and stored in 95% ETOH for DNA analysis and subsequent paternity assignment.
Molecular Paternity Assignment For a combination of permanent marking and sampling of DNA, two toe tips (ca. 2 mm from different feet) and ca. 5 mm of the tail tip were removed from adult lizards cooled to ca. 5 C, which prevents bleeding and minimizes pain in these ectotherms (Sinervo & DeNardo 1996). This did not affect lizard health or behaviour. DNA was isolated from toe and tail clip tissue samples using phenol/chloroform extraction (Sambrook et al. 1989). Using fluorescently labelled primers, individuals’ DNA was PCR amplified at four microsatellite loci: CP01, CP02, CP10 and CP17 (Schwartz et al. 2007). All loci were amplified individually in 7 ml reactions using 10–20 ng of DNA, 0.22 mM dNTP, 0.3 mM of each primer, 1 PCR buffer (Qiagen Inc., Valencia, CA, U.S.A.) containing Tris–HCL, KCL (NH4)2SO4, 15 mM MgCl2 pH 8.7 (final concentration of MgCl2 was 1.5 mM) and 0.05 U of HotStart Taq (Qiagen). Cycling conditions included a hot start denaturation at 95 C for 15 min, 30 cycles of 95 C for 30 s, annealing temperature for 30 s, 72 C for 30 s, and a final extension at 72 C for 30 min. Fluorescent amplifications were electrophoresed on a genetic analyser (ABI3130xl: locus CP17; ABI3900: loci CP01, CP02, CP10: Applied Biosystems, Inc., Foster City, CA, U.S.A.) using the LIZ500 size standard (Applied Biosystems). Alleles were scored using GeneMapper (Applied Biosystems) and confirmed visually. Genotypes from the adults were used to calculate allele frequencies, observed and expected heterozygosites, frequency of null alleles and the polymorphic information content (PIC: a measure of informativeness related to expected heterozygosity; in CERVUS 2.0) (Marshall et al. 1998; Table 1). Mother–offspring genotypes were compared across clutches for the presence of null alleles, as indicated when the mother is homozygous for a particular allele and her offspring are homozygous for a different allele. The following analyses were conducted with and without potential null alleles to verify the robustness of the paternity assignments. To determine 95% and 80% confidence levels for the assignments, we conducted two simulation analyses: first, using zero genotyping error rate (complete exclusion) and, second, using a 0.01 genotyping error rate. For these analyses we assumed that 95% of potential fathers had been sampled, since most matings were staged in the laboratory with known male pairs. All males were used as potential fathers for all the offspring since they may have mated with the female in the field prior to being brought into the
laboratory. Results were compared across the different analyses and assigned fathers were assessed by eye across mother–offspring pairs. Any males mated to a particular female at any time throughout this study were specifically compared to all of her offspring to verify the male’s reproductive success. Any potential mismatches between father–offspring–mother were doublechecked with the raw data. This study was approved by The Animal Ethics Committee, University of Wollongong, and conducted in compliance with Wollongong University animal ethics protocols. A scientific licence was also issued under the National Parks and Wildlife Act 1974 by the National Parks and Wildlife Service, NSW, Australia. All lizards were released at the place of capture after the study was completed. RESULTS Male Body and Relative Testes Size Red and yellow males did not differ significantly in mean body mass (red males: X SE ¼ 5:9 0:42 g; yellow males: 6.0 0.26 g; t test: t17 ¼ 0.27, P ¼ 0.79) or SVL (red males: X SE ¼ 62:3 1:7 mm; yellow males: 64.3 0.93 mm; t17 ¼ 1.33, P ¼ 0.20). Testes mass, however, differed between the two morphs (red males: X SE ¼ 0:15 0:01 g; yellow males: 0.18 0.004 g; tested with testes mass as the response variable, head colour as the factor and condition as the covariate: ANOVA: F2,16 ¼ 4.5, R2 ¼ 0.36, P ¼ 0.028; head colour: F1,16 ¼ 5.3, P ¼ 0.035; condition: F1,16 ¼ 3.9, P ¼ 0.07). Red and yellow males also differed in how their testes size was related to body size (heterogeneity of slopes test: F3,18 ¼ 4.6, R2 ¼ 0.48, P ¼ 0.017; head colour: F ¼ 7.2, P ¼ 0.017; SVL: F ¼ 0.79, P ¼ 0.39; head colour*SVL: F ¼ 6.7, P ¼ 0.02; Fig. 2). Paternity Analysis All of the loci used had a high polymorphic information content (range 0.819–0.957) with the overall exclusionary power of 0.996 (Table 1). The high exclusionary power allowed us to exclude sampled males as potential fathers when the mother was known. Thereby we could effectively determine whether offspring had been fathered by an unsampled field male (Table 1). Simulations estimated that with the known mother’s genotype, paternity could be successfully assigned at 95% confidence in 87% of cases with complete exclusion, and 27% of cases with a 0.01 error rate. The paternity assignments were consistent across the different analyses and the assigned father never had more than one allele mismatch
0.225
Locus
No. of alleles
PIC
Excl
HO
HE
Null frequency
CP01 CP02 CP10 CP17
15 27 41 28
0.819 0.914 0.957 0.937
0.518 0.722 0.847 0.784
0.843 0.888 0.965 0.961
0.840 0.924 0.964 0.949
0.004 þ0.0163 0.036 0.011
Mean
27.75
0.906
0.718
0.914
0.920
Total
0.996
PIC represents the polymorphic information content; Excl represents the power of the locus to exclude a randomly selected male from being the father of a randomly selected offspring based only on the offspring genotype. HO and HE represent the observed and expected heterozygosities; none of these loci were out of Hardy–Weinberg equilibrium. Null frequency represents the predicted frequency of null alleles at that locus based on mother–offspring mismatches. N ¼ 34 females, and 26 males.
Testes mass (g)
0.205 Table 1 Details of microsatellite markers used for parentage analysis
421
Red Yellow
0.185 0.165 0.145 0.125 0.105 0.085 57
59
61
63
65
67
69
71
SVL (mm) Figure 2. Testes mass of red and yellow males in relation to body size (snout–vent length, SVL).
M. Olsson et al. / Animal Behaviour 77 (2009) 419–424
Table 2 Descriptive data (X SE) of male size and copulation duration for males mating first or second and for red and yellow males
Snout–vent length (mm) Time in copula (s)
First
Second
Red
Yellow
65.10.65 26.15.1
65.81.02 24.74.7
64.81.04 32.89.8
64.70.96 12.81.9
and always fell within the 95% confidence level, or the 80% confidence level with siblings assigned the same father at the 95% confidence level. Overall, 225 of the 310 offspring were assigned a father from the males we had genotyped. Offspring for which a father could not be assigned were excluded from the analyses of determinants of paternity.
4 Difference in no. of young from field and laboratory sperm
422
3 2 1 0 -1 -2 -3 -4 -5
Mating Order Siring success at an individual level Data from 13 males selected at random that had been used more than once in different clutches and with different females were used to test for covariation between the proportion of sired young in their first and second copulations. These paternity scores were not correlated (rS ¼ 0.03, P ¼ 0.91), suggesting that some males did not do consistently better (or worse) in terms of siring success. Reduced data set We conducted pairwise t tests, that is, we assessed differences between categories of males in the probability of paternity within female clutches. This procedure thus identifies explicit differences in the average probability of paternity between categories of males (e.g. analysing differences between males mating first and second in the laboratory while ignoring offspring arising from stored sperm). There was no difference in paternity (number of sired young) between stored and first males (difference in mean number of sired young SE ¼ 0.61 0.64, N ¼ 26; t1 ¼ 0.97, P ¼ 0.34), between stored and second males (0.67 0.88, N ¼ 12; t1 ¼ 0.76, P ¼ 0.46) or between first and second males (0.50 0.73, N ¼ 12; t1 ¼ 0.68, P ¼ 0.51). Sperm Storage, Copula Duration and Body Size
2 3 Clutch number
4
Figure 4. Mean difference in number of sired offspring between sperm from field inseminations and summed laboratory inseminations SE. Thus, positive values represent more offspring from stored sperm from field matings. Pairwise t tests: clutch number (cn) 1: t1 ¼ 7.70, N ¼ 31, P < 0.0001; cn 2: t1 ¼ 0.75, N ¼ 24, P ¼ 0.46; cn 3: t1 ¼ 0.24, N ¼ 19, P ¼ 0.82; cn 4/5 (pooled 4 and 5): t1 ¼ 13.8, N ¼ 6, P < 0.0001. For cn 4/5, the test was performed on log-transformed absolute values, which successfully normalized the data.
descriptive data). A descriptive plot of the entire data set suggested that sperm stored from field matings declined in competitive ability throughout the season (rS ¼ 0.39, N ¼ 80, pseuodreplication precludes calculation of significance level; Fig. 3). When single data points were generated per individual male and female through randomization, this relationship did not reach significance (rS ¼ 0.26, N ¼ 34, P ¼ 0.14). However, when females’ last clutches were deliberately selected, thus assessing the most extreme cases of storage effects on probability of paternity, field males (i.e. those copulating first in the season) sired significantly fewer offspring later in the season (correlation between siring success and clutch number: rS ¼ 0.37, N ¼ 34, P ¼ 0.030). Furthermore, the summed number of offspring produced from field versus laboratory matings changed in sign throughout the season: early clutches were primarily sired by field males whereas later clutches were mostly fertilized by stored sperm from laboratory matings (Fig. 4). This was also suggested by a negative correlation between the fertilization success of field stored sperm and firstmale probability of paternity (rS ¼ 0.63, N ¼ 26, P ¼ 0.0005). However, negative correlations between first and second males did not reach significance (rS ¼ 0.48, N ¼ 12, P ¼ 0.11), nor did those between stored and second males (rS ¼ 0.39, N ¼ 12, P ¼ 0.21). Male Coloration Differences between red and yellow males in copulation behaviour became apparent through a comparison of time spent in
7 6 5 4 3 2 1 0 0
1
2
3
4
5
6
Clutch number Figure 3. Fertilization success of stored spermatozoa (i.e. sperm from field matings prior to female captures) in relation to clutch number. Symbol size represents number of observations (1–10).
Mean number of offspring sired
Offspring from stored sperm
Since ejaculate volume is correlated with time in copula in a closely related species (C. fordi; Olsson 2001), and possibly with body size, we looked for correlations between the difference between first and second males in SVL and time in copula and the corresponding difference in proportion of sired young, but these were not significantly correlated (P > 0.10; see Table 2 for
1
2.5 2 1.5 1 0.5 0
Red
Yellow
Figure 5. Fertilization success (X SE) of yellow and red males that mated first.
M. Olsson et al. / Animal Behaviour 77 (2009) 419–424
copula for red and yellow first males (for which we had the largest sample). Red males showed an almost three times longer copulation time on average than yellow males (Kruskal–Wallis test: c21 ¼ 4.86, P ¼ 0.029; reds: X SE ¼ 37:7 13:5 s, N ¼ 10; yellows: 13.4 2.34 s, N ¼ 14; Table 2). In spite of this, yellow males sired more than three times as many offspring on average per copulation as red males (mean number of offspring per mating in competition with stored and second males SE ¼ 0.40 0.22 versus 1.8 0.53; t17.2 ¼ 2.43, Satterthwaite’s approximation, P ¼ 0.026; Fig. 5). DISCUSSION Our approach to this study is set in a framework of sperm competition, cryptic female effects, evolution of territoriality and alternative reproductive tactics in a polymorphic lizard. We have shown that females mate multiply if offered this opportunity and that female sperm storage capacity is high enough to ensure the siring of offspring in several ovarian cycles subsequent to the one in which a target male is copulating. This further supports similar observations in a previous study (Olsson et al. 2007b). Furthermore, the fertilization success of fresh sperm was negatively related to that of stored sperm, possibly as sperm from early copulations are used up at fertilization, forced out of the oviduct at ovulation, or simply die over time and decline in competitive ability against later ejaculates. This suggests that numerical sperm competition is the underlying mechanism to the results in these mating trials (e.g. Parker 1998). Elsewhere, one of us (M.O.) has demonstrated, in the field as well as in controlled laboratory experiments, that cryptic female choice or ‘sperm selection’ could increase offspring viability in two different reptile species, adder snakes, Vipera berus, and sand lizards, Lacerta agilis (Madsen et al. 1992; Olsson et al. 1994a, b, 1996). Thus, we emphasize that our analyses in the current study have seriously considered the ‘female perspective’ as a highly viable alternative hypothesis to sperm competition between males. However, the quantitative relationships in terms of probability of paternity of early and late copulating males seem to lend much more parsimonious support to a numerical sperm competition scenario than to cryptic female choice in this species. Given the numerical aspect to sperm competition and female sperm-storing capacity, territoriality is likely to prevent rival matings (thus acting as a paternity guard) and further enhance the reproductive success of territorial males. We have shown elsewhere (Olsson et al. 2007a) that the opportunity for sperm competition (as approximated by the frequency of multiple paternity) may vary between years and be driven by the availability of perches for territorial patrolling, and that territorial defence appears to be a highly successful means of preventing loss of paternity to rivals in this species. Thus, if red males are better at defending both their territories and females, as our laboratory trials suggest, yellow male competitive ability would have to arise through some other mechanism, such as via greater postcopulatory reproductive success in a situation of sperm competition. Our experiment mimicked such a situation, in which both yellow and red males were allowed to act as sneakers that mate with the females of territorial males, originally mated under natural conditions (as verified by molecular genetics). For males mating for their first time in the laboratory, yellow males had, on average, three times the reproductive success of red males, in spite of copulating for only a third of the time (which is an important aspect, since transfer of spermatozoa and ejaculate products continues throughout copulation in squamate reptiles; Olsson & Madsen 1998). This result is therefore congruent with the observation that yellow males have larger testicles in relation to body size than red males, and they may even adopt shorter copulations as part of a sneaker strategy. Furthermore, the difference in testes size in relation to body size between the two morphs may suggest
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additional tactical differences between them, for example that sperm competition is more important for securing fitness benefits in younger, smaller yellow males competing with red males emerging earlier from hibernation to monopolize females (Olsson et al. 2007a). Female sperm storage has been argued to result from selection arising from a low encounter rate of partners (see numerous examples across taxa in Birkhead & Møller 1998). Painted dragons, however, occur in large numbers with territories that are on average less than 100 m along their longest axes (Olsson et al. 2007a). Thus, having too few mating opportunities seems a farfetched explanation for the evolution of sperm storage in this species, which may have evolved for other adaptive reasons (Olsson et al. 2007b). In summary, we have shown that painted dragons have the capacity for sperm storage over several ovarian cycles, and that male reproductive success in laboratory experiments is largely driven by these effects. We have also shown that a contributing factor to the maintenance of both red and yellow morphs in this species may be differences in components of sexual selection, with yellow males potentially having greater fitness benefits from postcopulatory processes than red males, and that this may be related to the larger relative testes size of yellow males. Acknowledgments We thank E. and G. Snaith, and G. and M. Swan for logistical support and field assistance. We are financially supported by the Australian Research Council (M.O. and T.U.), and the Wenner–Gren Foundations (T.U.). We thank C. Sherman for assistance in the laboratory and six referees for their much appreciated contributions. References Andersson, M. B. 1994. Sexual Selection. Princeton, New Jersey: Princeton University Press. Birkhead, T. & Møller, A. P. 1998. Sperm Competition and Sexual Selection. San Diego: Academic Press. Calsbeek, R. & Sinervo, B. 2002. The ontogeny of territoriality during maturation. Oecologia, 132, 468–477. Cogger, H. G. 2000. Reptiles and Amphibians of Australia, 6th Edn. Melbourne: New Holland Publishers (Australia). Healey, M., Uller, T. & Olsson, M. 2007. Seeing red: morph-specific contest success, and survival rates, in a colour-polymorphic agamid lizard. Animal Behaviour, 74, 337–341. Jukema, J. & Piersma, T. 2006. Permanent female mimics in a lekking shorebird. Biology Letters, 2, 161–164. Madsen, T., Shine, R., Loman, J. & Håkansson, T. 1992. Why do female adders copulate so frequently? Nature, 402, 34–35. Marshall, T. C., Slate, J., Kruuk, E. B. & Pemberton, J. M. 1998. Statistical confidence for likelihood-based paternity inference in natural populations. Molecular Ecology, 7, 639–655. Olsson, M. 2001. ‘Voyeurism’ prolongs copulation in the dragon lizard (Ctenophorus fordi). Behavioral Ecology and Sociobiology, 50, 378–381. Olsson, M. & Madsen, T. 1998. Sexual selection and sperm competition in reptiles. In: Sperm Competition and Sexual Selection (Ed. by T. R. Birkhead & A. P. Møller), pp. 503–578. San Diego: Academic Press. ¨ m, H., Madsen, T. & Shine, R. 1994a. Promiscuous Olsson, M., Norberg, A., Tegelstro lizard females have more viable young (under subheading: Can female adders multiply?). Nature, 369, 528. ¨ m, H., Madsen, T. & Shine, R. 1994b. Rewards of Olsson, M., Gullberg, A., Tegelstro ‘promiscuity’. Nature, 372, 230. ¨ m, H. 1996. Sperm Olsson, M., Shine, R., Madsen, T., Gullberg, A. & Tegelstro selection by females. Nature, 383, 585. Olsson, M., Healey, M., Wapstra, E., Schwartz, T., LeBas, N. & Uller, T. 2007a. Mating system variation and morph fluctuations in a polymorphic lizard. Molecular Ecology, 16, 5307–5315. Olsson, M., Schwartz, T., Uller, T. & Healey, M. 2007b. Sons are made from old stores: sperm storage effects on sex ratio in a lizard. Biology Letters, 35, 491–493. Parker, G. A. 1998. Sperm competition and the evolution of ejaculates: a theory base. In: Sperm Competition and Sexual Selection (Ed. by T. R. Birkhead & A. P. Møller), pp. 3–54. San Diego: Academic Press. Rui, R. F., Taborsky, M. & Brockman, H. J. 2008. Alternative Reproductive Strategies. Cambridge: Cambridge University Press.
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