SUMMARY
Sexual selection is well recognized as a driver of reproductive isolation between lineages. However, selection for increased reproductive isolation could reciprocally change the outcomes of sexual selection, when these processes share a genetic basis. Direct selection for reproductive isolation occurs in the context of ‘reinforcement’, where selection acts to increase prezygotic barriers to reduce the cost of heterospecific matings. Many studies of reinforcement focus on premating reproductive barriers, however postmating traits-such as conspecific sperm precedence (CSP)-can also respond to reinforcing selection. We tested whether i) CSP responded to reinforcing selection, and ii) this response in sympatric populations altered intraspecific sperm competition (ISC) and the strength of sexual selection, with the sister species Drosophila pseudoobscura and D. persimilis. We used sperm competition experiments to evaluate differences in CSP and ISC between two sympatric and two allopatric populations of D. pseudoobscura. Using multiple genotypes for each population allowed us to estimate not only patterns of phenotype divergence, but also the opportunity for sexual selection within each population. Consistent with a pattern of reinforcement, the sympatric populations had higher mean CSP. Moreover, ISC was altered in sympatric populations, where we observed decreased average offensive sperm competitive ability against conspecific males, allowing less opportunity for sexual selection to operate within these populations. These data demonstrate that strong reinforcing selection for reproductive isolation can have consequences for sexual selection and sexual interactions within species, in these important postmating sperm competition traits.
Introduction
When closely-related species comes into contact, the presence of heterospecifics can influence sexual interactions and therefore alter patterns of selection on reproductive traits. In cases where these species have the potential to interbreed, selection can favor divergence in sexual traits to avoid costs of heterospecific mating, a type of reproductive character displacement commonly called reinforcement [1–3]. The frequency at which reinforcement contributes to speciation is still under debate [3–4] although several recent examples provide strong evidence for reinforcement acting on mating traits [5–10]. Regardless, the mating trait changes that evolve in response to reinforcement can have collateral effects on intraspecific sexual dynamics [6]. This can in turn alter the magnitude and efficacy of sexual selection specifically within populations exposed to heterospecifics. These potential reciprocal interactions between sexual selection and reproductive isolation remain relatively untested [6–7], but can have important consequences for how we interpret evolution of sexual traits and interactions. For example, patterns of reproductive trait evolution in rapid radiations, where sexual selection is thought to be the primary driver, may be misinterpreted if they do not take into account species interactions.
For reinforcement and sexual selection to reciprocally affect the evolution of sexual traits, these traits must be involved in both processes and share a genetic basis. Currently the best example of a shared genetic basis for sexual selection and reproductive isolation comes from Drosophila sperm competition genes, several of which have been shown to mediate both sexual selection through intraspecies sperm competition (ISC) and reproductive isolation via conspecific sperm precedence (CSP) [11]. Conspecific sperm precedence occurs when a female mates with both heterospecific and conspecific males yet most of the progeny are sired by the conspecific male; this precedence can occur either through competitive mechanisms (including male sperm competition and cryptic female choice) or non-competitive mechanisms (resulting mainly from gametic incompatibilities). CSP has proven to be a strong reproductive isolation barrier among species in Drosophila [4,12–13] and in many other plant and animal species [4, and references therein]. Although ubiquitous, CSP can be overlooked as a reproductive isolating barrier because it involves inconspicuous phenotypes that are not readily observed in the field [14]. Moreover, although reinforcement studies have overwhelmingly focused on pre-mating traits, postcopulatory prezygotic traits including CSP can also be the target of reinforcement [15–17]. Previous empirical studies have been equivocal about whether heterospecific interactions and reinforcement select for increased CSP specifically in sympatry, with no single study simultaneously estimating and comparing levels of CSP in allopatric and sympatric populations [13, 18–25]
While reinforcing selection (acting on CSP) and sexual selection (acting on ISC) could interact to influence evolutionary change in post-copulatory traits, the outcomes of this interaction clearly will depend upon whether these forces act in concert or in opposition. When sexual selection and reinforcing selection act in concert, trait evolution can proceed faster than otherwise expected, but the direction of trait evolution remains unchanged. In contrast, the potential feedback between sexual selection and reproductive isolation can generate complex evolutionary outcomes when these forces act at cross-purposes. For example, sperm competition is shaped by sexual conflict between males and females (i.e. antagonistic pleiotropy [26–28]) and genotype-genotype interactions (male-male [29–30] and male-female: [31–33]. Both are expected to maintain high variance in the affected traits and, indeed, sperm competition genes are often highly variable both in terms of molecular and phenotypic variation [30, 33–34]. In contrast, under models of speciation by sexual selection—where isolation is generated by strong disruptive selection between populations and directional selection within a population [35–36]—genetic variance of traits that act as barriers to reproduction is expected to be reduced and the overall trait mean shifted. The net effect of selection imposed by intrapopulation sexual interactions and by reinforcement can together produce phenotypic and genetic variation in sperm competition traits/genes that is different from the optimal variation when sexual selection acts alone.
One way these potentially antagonistic optima could play out is when reinforcement-mediated changes in the mean and variance of sperm competition traits alter the opportunity for sexual selection among conspecifics [7]. Sperm competition contributes to variance in reproductive success because male genotypes that can disproportionately sire offspring increase their fitness compared to the fitness of rival male conspecifics [37–38]. Strong sperm competition leads to greater opportunity for sexual selection because there is greater variance in reproductive success compared to scenarios where males have equal probability of siring offspring. This generates two alternative predictions of the possible effects of reinforcement on sexual selection. First, the response to strong directional selection from reinforcement on sperm competition traits could lead to greater siring ability in intrapopulation sperm competition, increasing variance in reproductive success and opportunity for sexual selection. Alternatively, strong directional selection could reduce phenotypic variation so that competitive ability is equalized among males, thus reducing the opportunity for sexual selection.
The strategy we used to evaluate the interaction between selection for increased reproductive isolation (i.e. reinforcement) and sexual selection acting on sperm competition genes was to estimate variation between genotypes in CSP and ISC in parallel. Both CSP and ISC are measures of postcopulatory offensive sperm competition, estimated by allowing females to mate sequentially with two different male genotypes and scoring the paternity of the resulting progeny. Here our focus was on second-male or ‘offensive’ siring success. This is typically referred to as ‘P2’ and captures the ability of the second mated male to sire offspring by displacing or disabling the sperm of the first male. For our experiments the first male was either heterospecific (to estimate CSP) or conspecific (to estimate ISC) tester male. By comparing the relative competitive success of replicate male lines against a common set of either heterospecific and conspecific male tester genotypes, we could estimate post-copulatory CSP and ISC in parallel in the same experiment. Using this design we also estimated which genotype effects (male genotype, female genotype, or the interaction) might shape CSP and ISC. Females experience the most cost of heterospecific matings [39–41] and could control CSP via cryptic female choice [42], thus we would expect strong female genotype effects on CSP. This contrasts with previous studies of ISC where both male and female genetic effects, and their interaction were significant effects [31,33]. Unlike ISC the phenotypic and genetic variance for CSP has not been empirically explored and their similarity to ISC is currently unknown.
In this study, we examine evidence for reinforcement of CSP among populations of Drosophila pseudoobscura that are allopatric or sympatric with their closely related sister species D. persimilis, and evaluate the potential consequences of these heterospecific interactions for ISC and sexual selection within D. pseudoobscura populations. One of the first clear empirical demonstrations of reinforcement on premating isolation was described in this species pair [43]. This finding suggests that heterospecific interactions and matings are frequent and sustained over evolutionary time and can act as a substantial selective agent on reproductive traits in this system. Here we determine whether there is evidence that heterospecific interactions have selected for increased CSP, by comparing this barrier among populations of D. pseudoobscura that are allopatric or sympatric with D. persimilis. A pattern of stronger CSP specifically in sympatry is consistent with reinforcement; moreover, because postcopulatory traits are less likely to be directly affected by environmental conditions, this pattern is unlikely to be explained by alternative phenomena, such as ecological selection, that could also explain character displacement in sympatry (see Discussion). Using a consistent design across all populations we could also estimate premating reproductive isolation in the same experiment, and compare its strength in sympatry and allopatry. Second, we evaluate whether selection for strong CSP in sympatry has affected ISC, and thereby post-copulatory sexual selection, as might occur when CSP and ISC have shared genetic architecture. Throughout, we test for differences in trait variation across a set of distinct genotypes which allows us to specifically evaluate which sex is playing a more critical role in determining variation in heterospecific and conspecific postcopulatory interactions.
RESULTS
No difference between allopatric and sympatric populations in premating isolation
Because our experimental assessment of CSP involved first mating with a heterospecific D. persimilis male, we were able to estimate the magnitude of premating isolation in each D. pseudoobscura population in our experiment. We did not find evidence for a pattern consistent with reinforcement of premating isolation mediated by female mate preference. The average probability of heterospecific matings ranged from 46-52% between populations, and did not differ between allopatric and sympatric populations (χ2 test of independence: χ2=1.185, df=1, P=0.2763; Wald’s Test: χ2=19, df=4, P=0.75; Table 1). In pairwise tests between each allopatric and sympatric population we also failed to reject the null hypothesis. Though we did not detect a signal of reinforcement there was ample genetic variance in heterospecific mating rate between female genotypes available for selection within each population ((Fig. 1; Supplemental Table 1). Only in one of the populations (Lamoille, which is allopatric) did the identity of the D. persimilis tester line affect variation in premating isolation (Supplemental Table 1).
Reinforcement acts on conspecific sperm precedence
Unlike premating isolation, we observed a pattern consistent with reinforcement for conspecific sperm precedence (CSP). Specifically, in sympatry we find both greater average CSP (t=−6.5898, df=210.92, P<0.001; Wilcox W=4427.5, P<0.001) and less phenotypic variation in this trait (Levene-type test χ2=22.82, P<0.0001) when data were pooled by geographic region (allopatry versus sympatry) (Table 1; Fig. 2A). These differences in both the average and variance of CSP were also observed in pairwise tests between individual allopatric and sympatric populations (Supplemental Table 2).
Reinforcement has collateral effects on intrapopulation sperm competition
ISC also differed between allopatric and sympatric populations, in both mean and variance (Table 1; Fig 2B). First, mean offensive ability for ISC was significantly lower in sympatric populations (t=3.738, df=246.55,P=0.0002; Wilcox’s W=10280, P=0.0004). This contrasts with the observed increase in offensive CSP in sympatric populations. Second, there was more variation in ISC in the sympatric populations compared to the allopatric populations (Leven-type test χ2=5.74, P=0.0172). Given the differences in ISC and CSP across populations, we used the mean CSP and ISC phenotype for each male x female genotype combination within a population (i.e., each cell within the diallel crossing design) to examine the pattern of relationship between the two phenotypes across the four populations. We observed a significant negative relationship between CSP and ISC (Pearson’s r=−0.31, P=0.01; (Fig. 5). Since each male or female genotype is represented in multiple combinations we controlled for non-independence using a linear mixed effect model, and confirmed that the negative slope of the relationship was significant as indicated by a confidence interval that did not overlap zero (Profiled CI = −0.451, −0.028).
Female genotype effects contribute to CSP and male x female genotype effects explain both CSP and ISC
Of male, female, and male x female genotype effects that could contribute to explaining the variance in CSP, we found that three out of the four populations had a significant female genotype effect on CSP (Table 2; Fig 3), and all populations had a significant male x female genotype interaction effect. The D. persimilis tester male line was also significant in three out of four populations. There was no consistent pattern among populations in which effect had the largest intraclass correlation (i.e. which explained the largest proportion of variance; see Methods); in some populations the female genotype effect had the largest intraclass correlation, while in others the male x female genotype interaction had the largest intraclass correlation (Table 2). In contrast, for ISC in all four populations we only observed significant male x female genotype interaction and a significant effect of the first-tester male genotype (Table 3; (Fig. 4). In every case, the male x female genotype effect had a larger intraclass correlation (usually two to three times greater) than the identity of the specific tester male genotype within each D. pseudoobscura population.
The opportunity for sexual selection is decreased in sympatry
Our design allowed us to describe the reproductive success of males in terms of offensive (second male) and defensive (first-tester male) success. We found that the sympatric populations had significantly lower variance for reproductive success compared to the allopatric populations (Figure 6; Supplemental Table 4). The variance in reproductive success across all male genotypes (both offensive and defensive) in the allopatric Lamoille population was significantly greater than both sympatric populations (Mt. St Helena F=1.96, Bootstrap P=0.003; Sierra F=2.08, Bootstrap P=0.008), as was the variance in reproductive success in the allopatric Zion population compared to the sympatric populations (Mt. St Helena F=2.65, Bootstrap P=0.003; Sierra F=2.83, Bootstrap P=0.004).This reduced variance in reproductive success in sympatry is a product of lower offensive sperm competition values in sympatry, that result in equalized differences in the siring success between offensive and defensive males.
DISCUSSION
Interactions with heterospecifics have the potential to drive divergent sexual selection and the evolution of reproductive isolation, via reproductive character displacement and reinforcement [6–7,44]. Using D. pseudoobscura and D. persimilis, we assessed whether there was evidence for reinforcement of species barriers in sympatry via elevated female preference or conspecific sperm precedence, traits that are known to contribute to reproductive isolation across numerous taxa [2]. Premating isolation is historically considered to be a strong barrier to isolation between these species, and one that reinforcing selection has acted on [43], but we saw no evidence for reproductive character displacement for this trait. In contrast we saw a clear signal of increased CSP in sympatric populations, consistent with a pattern of reinforcement. Specifically, the average CSP was higher, and the overall level of phenotypic variation was lower, in sympatric populations, a pattern consistent with recent or recurrent directional selection acting on CSP in these populations. We further asked whether reinforcement could have collateral effects on intraspecific sperm competition and sexual selection, given that these two traits are mechanistically and genetically linked [11,45]. We found that sympatric populations also had lower ISC ability (lower offensive ability) than allopatric populations, consistent with weakened sexual selection in sympatry.
Our results indicate that CSP can strongly contribute to reproductive isolation in response to reinforcing selection. While CSP is known to be a barrier to gene flow in Drosophila [12–13] and other taxa [2], its overall importance in nature has been difficult to ascertain [14,16]. Moreover, previous studies of reinforcement sometimes qualitatively describe variation in the target premating traits, but trait variance is typically not quantified [5,9,17] even though models of speciation by sexual selection predict that strong divergent selection will erode phenotypic variation in selected traits [46–47]. Our observations of both increased mean CSP and reduced variation specifically in sympatry provide compelling support for the inference that CSP has responded to strong selection imposed by heterospecific interactions, and underscores the important role that CSP can play in maintaining species boundaries.
The pattern of reproductive character displacement that we observed for CSP is consistent with reinforcement, but other factors have been proposed to account for reproductive character displacement including differential fusion [48] or ecological differences that have collateral effects on mating traits [44,49]. Differential fusion predicts that strong reproductive isolation evolves between species in allopatry and merely prevents species collapse upon secondary contact, so that sympatric species incidentally appear to have stronger isolation [50–51]. If differential fusion operates at the deme/lineage level within a population we would expect the sympatric CSP values to be a subset of allopatric CSP values [17]. This is not the case, however, because the sympatric values of CSP are systematically higher than in allopatry ((Figure 2). Regardless, the differential persistence of demes/lineages with strong CSP in sympatry would nevertheless be consistent with selection from standing variation leading to reinforcement [52,53]. Similarly, several lines of evidence argue that systematic ecological differences between allopatry and sympatry are unlikely to explain our observed postcopulatory differences. Although both sympatric populations are located in California, they are ecological distinct (collected from two different mountain ranges) in terms of numerous ecological factors [54]. Indeed, habitat variation between sympatric populations of D. pseudoobscura has led to differences in inversion frequencies maintained by ecological forces that differ in these locations [55]. Moreover, the ecological differences across the whole range of D. pseudoobscura are largely continuous, rather than uniquely differentiating regions of allopatry and sympatry/co-occurrence with D. persimilis. Given the ecological diversity between populations we do not expect a consistent direction of natural selection acting on either the sympatric or allopatric populations. Arguably more important, there are no established mechanisms whereby external ecological factors are expected to have a direct effect on the strength of sperm competition consistent with our observed pattern. Indirect effects of diet and nutrition can affect sperm competition outcomes [56–57], but should not persist in the lab environment. Moreover, if ecological mechanisms existed there is no reason to expect they would act in the specific direction we observed here. Given this, while the ecological alternative to reinforcement might be plausible for some premating phenotypes, it is unlikely to explain the postcopulatory phenotypes that we examine here.
Our second major inference is that the response to reinforcing selection observed in CSP has had a collateral effect on the magnitude of offensive ISC and the opportunity for sexual selection in sympatric populations. The decrease in the opportunity for sexual selection in sympatry appears to be the result of a negative genetic correlation between CSP and ISC, as well as reduced variance in post-copulatory fitness based on ISC estimates. Sperm competition strongly contributes to sexual selection in D. pseudoobscura where multiple mating is frequent in wild caught females [58], and male mating success, including sperm competition, is a major component of selection in natural populations [59]. The observed reduction in offensive sperm competition differs from both of our a priori expectations. One a priori hypothesis was that selection for increased CSP in sympatry would select for increased offensive sperm competitive ability among conspecifics, if offensive ability were a general trait that acted regardless of whether the competitor was a conspecific or heterospecific male. In contrast, we observed that ISC, was lower for sympatric populations compared to allopatric populations; that is, average offensive ability was closer to 0.5, indicating a greater equalization in sperm competitive ability among competing males. Our other a priori expectation was that strong directional selection would alter sexual selection by reducing phenotypic variation. However, the reduced phenotypic variation seen for CSP in sympatry was not mirrored by reduced phenotypic variation for ISC. This observation is also inconsistent with an alternative explanation-that selection for weaker ISC in sympatry indirectly increased CSP. This alternative is more generally implausible as it requires that there has been selection specifically to reduce ISC, solely in sympatry. Instead, we infer that selection for stronger CSP in sympatry has reduced mean ISC in sympatric populations via a negative genetic correlation between these two sperm competitive phenotypes.
For reinforcing selection to influence and interfere with sexual selection, the selection favoring increased CSP must outweigh selection acting to maximize ISC. One way CSP could have a larger effect on fitness than ISC is via a higher selective premium specifically for females. Weaker CSP results in substantial fitness deficits for females because of reproductive investment in low or no fitness hybrids, whereas weaker ISC likely has a comparatively marginal effect on female fitness outcomes. Regardless, the strength of reinforcing selection on CSP depends on the frequency of heterospecific matings. Several lines of evidence suggest that heterospecific mating rates are common between these species. First, from our data we observe a large range in the frequency with which D. pseudoobscura females accept D. persimilis males in no-choice experiments, with some genotypes on average accepting D. persimils 90% of the time. Second, while no estimates for heterospecific mating rate exist from natural populations, rare F1 progeny have been identified from wild collections [60]. Third, genetic evidence suggests there has been post-speciation gene flow (i.e., evidence of movement of alleles between species) between D. pseudoobscura and D. persimils [61–62]. Notably, these estimates of realized gene flow will systematically underestimate the rate of heterospecific matings, because they will only capture events that result in F1 progeny that themselves then successfully reproduced; for example, given the presence of strong CSP, many heterospecific matings may never produce hybrid progeny.
We were able to test the hypothesis that females face more costs of hybridization [39–41] and that choice manifests as female control of sperm use patterns [63–65] by contrasting the genotype effects (male, female, and male x female genotype effects) between CSP and ISC. We observed significant male x female genotype interactions for all populations for both CSP and ISC but, interestingly, only saw significant female genotype effects for CSP. Significant female genotype effects for CSP suggest that cryptic female choice may be operating similarly to premating isolation mechanisms where females are observed to be the more “choosy” sex and female effects control the level of reproductive isolation more so than male effects [66].
Strong female genotype effects on CSP are also consistent with the current knowledge of postcopulatory sexual selection in the obscura group. Both D. pseudoobscura and D. persimilis produce two sperm morphs: longer fertilizing eusperm and shorter non-fertilizing parasperm. In D. pseudoobscura, the female reproductive tract is spermicidal and higher proportions of parasperm help protect eusperm from these negative effects [67]. Females in sympatric populations may have evolved more effective spermicide against heterospecific males at a cost of spermicidal effectiveness with conspecific males. In this case reproductive isolation would be mediated by cryptic female choice and heterospecific male-female compatibility. This hypothesis may also be consistent with our finding that the D. persimilis male genotype contributed significantly to observed variation in CSP.
Reinforcement acting on CSP suggests that other prezygotic barriers that act before CSP are not strong enough to limit the efficacy of selection on CSP in our sympatric populations [14,16]. Indeed, our analysis of premating isolation (propensity to mate with a heterospecific in the first mating) indicated that this potential barrier was equally strong in sympatry and allopatry. This is interesting because one of the first studies demonstrating reinforcement on premating barriers used the Drosophila pseudoobscura and D. persimilis sister pair [43], although subsequent studies have found more variable patterns [68–70; but see 71]. Our observation of a strong response in CSP also suggests that the populations of D. pseudoobscura and D. persimilis we examined are not strongly isolated by non-competitive (gametic) isolation, in agreement with inferences from other studies of this specific species pair [70,72]. Though we lack data on CSP from earlier collections in this species pair, our observations here might suggest that the relative contribution of barriers to reproduction has changed in sympatry over time, from premating isolation to CSP. Both gene flow between sympatry and allopatry, or a cost to female premating preferences, might explain this shift over time. Depending on the levels of gene flow among sympatric and allopatric populations, strong premating isolation in sympatry could be lost due to “swamping effects” of allopatric gene flow [73] or could lead to greater species wide reproductive isolation [74–75]. Our data suggest that it’s unlikely that gene flow from sympatry into allopatry created greater reproductive isolation in allopatry (thereby reducing the signal of reinforcement) because the average allopatric premating isolation in our experiment is similar to previous reports [43]. This suggests that reduced premating isolation has emerged in sympatry, but it is difficult to disentangle the effects of gene flow from the cost of female choice as causes of this reduction. Both processes could contribute to the large variance we see for female preference in sympatry compared to the more uniform level of premating isolation in allopatry (Fig 1). The probability that strong female preference have been lost in sympatry also depends on the frequency of this trait and any associated costs of choosiness. When D. pseudoobscura stocks are kept in the absence of heterospecific interactions female preference against heterospecifics decreases with longer periods of experimental allopatry, suggesting that it may be costly to maintain this trait [76]. In either case, the reduction in the strength of premating isolation in sympatry suggests that this barrier to reproduction may only generate transient patterns of reinforcement.
Overall, our data suggest that strong reinforcing selection for reproductive isolation can have consequences for sexual selection and sexual interactions, in these important postmating sperm competition traits. The direction of this interaction provides an interesting inversion to standard expectations about the connection between sexual selection and speciation. Sexual selection is often thought of as a driver of sexual characteristics whose evolutionary divergence then contributes to reproductive isolation. But a direct genetic connection between these processes implies reproductive isolation also has the reciprocal potential to shape sexual selection [77]. Based on our observations of higher mean but lower variance in CSP in sympatry, a negative correlation between CSP and ISC, and reduced variance in reproductive success via ISC among sympatric conspecific males, we infer that strong selection for reproductive isolation within populations exposed to heterospecific species has reduced the efficacy of sexual selection in these populations, a collateral effect of reinforcing selection that has not previously been demonstrated.
ACKNOWLEDGEMENTS
We would like to thank E. Walburn and J. Roesener for their assistance with crosses and scoring progeny, J. Powers and the IU Light Microscopy Imaging Center for assistance with the Leica microscope, M. Noor, A. Hish, and N. Phadnis for providing strains used in this experiment, and Donn Castillo for help with collecting strains. Collections were completed with assistance from IU Biology Department travel awards to DMC. Research was supported by Indiana University Dept. of Biology funding to LCM and an AmericanSociety of Naturalists student research award to DMC. DMC was supported by a President’s Diversity Initiative Dissertation Fellowship from the Indiana University Graduate School.
LITERATURE CITED
- 1.↵
- 2.↵
- 3.↵
- 4.↵
- 5.↵
- 6.↵
- 7.↵
- 8.
- 9.↵
- 10.↵
- 11.↵
- 12.↵
- 13.↵
- 14.↵
- 15.↵
- 16.↵
- 17.↵
- 18.↵
- 19.
- 20.
- 21.
- 22.
- 23.
- 24.
- 25.↵
- 26.↵
- 27.
- 28.↵
- 29.↵
- 30.↵
- 31.↵
- 32.
- 33.↵
- 34.↵
- 35.↵
- 36.↵
- 37.↵
- 38.↵
- 39.↵
- 40.
- 41.↵
- 42.↵
- 43.↵
- 44.↵
- 45.↵
- 46.↵
- 47.↵
- 48.↵
- 49.↵
- 50.↵
- 51.↵
- 52.↵
- 53.↵
- 54.↵
- 55.↵
- 56.↵
- 57.↵
- 58.↵
- 59.↵
- 60.↵
- 61.↵
- 62.↵
- 63.↵
- 64.
- 65.↵
- 66.↵
- 67.↵
- 68.↵
- 69.
- 70.↵
- 71.↵
- 72.↵
- 73.↵
- 74.↵
- 75.↵
- 76.↵
- 77.↵
- 78.↵
- 79.↵
- 80.↵
- 81.↵
- 82.↵
- 83.↵
- 84.↵
- 85.↵
- 86.↵
- 87.↵
- 88.↵
- 89.↵
- 90.↵
- 91.↵
- 92.↵
- 93.↵
- 94.↵
- 95.↵
- 96.↵
- 97.↵