Inbreeding in three‐spined sticklebacks (Gasterosteus aculeatus L.): effects on testis and sperm traits

Mating between relatives often results in inbreeding depression, and is assumed to have a strong effect on fitness traits such as fertility and gonad/gamete quality. However, data concerning this topic are contradictory and particularly scarce in fishes. Three‐spined sticklebacks (Gasterosteus aculeatus L.) show inbreeding depression in fertilization and hatching success, survival rates, body symmetry and behavioural traits. To date, any knowledge of the impact of inbreeding on males' gonads and gametes is lacking in this species. In the present study, testis and sperm traits were quantified in outbred and inbred males. Overall, these traits were not generally impaired by inbreeding, and this result was not changed by a second/third generation of brother–sister matings. However, testes brightness, a potential measure of oxidative stress, was negatively correlated with sperm number. Additionally, inbred males with higher body condition had significantly brighter testes, whereas their sperm number was significantly negatively correlated with sperm quality (as estimated by head volume). Such a trade‐off did not appear in outbred males. The comparatively small impact of inbreeding on testis and sperm traits might be explained by the low number of inbred individuals that reached the reproductive phase. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 107 , 510–520.     

Inbreeding depression of sperm traits in the zebra finch Taeniopygia guttata

Inbreeding depression, or the reduction in fitness due to mating between close relatives, is a key issue in biology today. Inbreeding negatively affects many fitness‐related traits, including survival and reproductive success. Despite this, very few studies have quantified the effects of inbreeding on vertebrate gamete traits under controlled breeding conditions using a full‐sib mating approach. Here, we provide comprehensive evidence for the negative effect of inbreeding on sperm traits in a bird, the zebra finch Taeniopygia guttata. We compared sperm characteristics of both inbred (pedigree F = 0.25) and outbred (pedigree F = 0) individuals from two captive populations, one domesticated and one recently wild‐derived, raised under standardized conditions. As normal spermatozoa morphology did not differ consistently between inbred and outbred individuals, our study confirms the hypothesis that sperm morphology is not particularly susceptible to inbreeding depression. Inbreeding did, however, lead to significantly lower sperm motility and a substantially higher percentage of abnormal spermatozoa in ejaculate. These results were consistent across both study populations, confirming the generality and reliability of our findings.     

Experimental evidence that high levels of inbreeding depress sperm competitiveness

The effects of inbreeding on sperm quantity and quality are among the most dramatic examples of inbreeding depression. The extent to which inbreeding depression results in decreased fertilization success of a male’s sperm, however, remains largely unknown. This task is made more difficult by the fact that other factors, such as cryptic female choice, male sperm allocation and mating order, can also drive patterns of paternity. Here, we use artificial insemination to eliminate these extraneous sources of variation and to measure the effects of inbreeding on the competitiveness of a male’s sperm. We simultaneously inseminated female guppies (Poecilia reticulata) with equal amounts of sperm from an outbred (f = 0) male and either a highly (f = 0.59) or a moderately inbred (f = 0.25) male. Highly inbred males sired significantly fewer offspring than outbred males, but share of paternity did not differ between moderately inbred and outbred males. These findings therefore confirm that severe inbreeding can impair the competitiveness of sperm, but suggest that in the focal population inbreeding at order of a brother–sister mating does not reduce a male’s sperm competitiveness.     

Testing the phenotype‐linked fertility hypothesis in the presence and absence of inbreeding

The phenotype‐linked fertility hypothesis suggests that females can judge male fertility by inspecting male phenotypic traits. This is because male sexually selected traits might correlate with sperm quality if both are sensitive to factors that influence male condition. A recent meta‐analysis found little support for this hypothesis, suggesting little or no shared condition dependence. However, we recently reported that in captive zebra finches (Taeniopygia guttata) inbreeding had detrimental effects both on phenotypic traits and on measures of sperm quality, implying that variation in inbreeding could induce positive covariance between indicator traits and sperm quality. Therefore, we here assess empirically the average strength of correlations between phenotypic traits (courtship rate, beak colour, tarsus length) and measures of sperm quality (proportion of functional sperm, sperm velocity, sperm length) in populations of only outbred individuals and in mixed populations consisting of inbreds (F = 0.25) and outbreds (F = 0). As expected, phenotype sperm‐trait correlations were stronger when the population contained a mix of inbred and outbred individuals. We also found unexpected heterogeneity between our two study populations, with correlations being considerably stronger in a domesticated population than in a recently wild‐derived population. Correlations ranged from essentially zero among outbred‐only wild‐derived birds (mean Fisher's Zr ± SE = 0.03 ± 0.10) to moderately strong among domesticated birds of mixed inbreeding status (Zr ± SE = 0.38 ± 0.08). Our results suggest that, under some conditions, the phenotype‐linked fertility hypothesis might apply.     

Inbreeding reveals mode of past selection on male reproductive characters in Drosophila melanogaster

Directional dominance is a prerequisite of inbreeding depression. Directionality arises when selection drives alleles that increase fitness to fixation and eliminates dominant deleterious alleles, while deleterious recessives are hidden from it and maintained at low frequencies. Traits under directional selection (i.e., fitness traits) are expected to show directional dominance and therefore an increased susceptibility to inbreeding depression. In contrast, traits under stabilizing selection or weakly linked to fitness are predicted to exhibit little‐to‐no inbreeding depression. Here, we quantify the extent of inbreeding depression in a range of male reproductive characters and then infer the mode of past selection on them. The use of transgenic populations of Drosophila melanogaster with red or green fluorescent‐tagged sperm heads permitted in vivo discrimination of sperm from competing males and quantification of characteristics of ejaculate composition, performance, and fate. We found that male attractiveness (mating latency) and competitive fertilization success (P₂) both show some inbreeding depression, suggesting they may have been under directional selection, whereas sperm length showed no inbreeding depression suggesting a history of stabilizing selection. However, despite having measured several sperm quality and quantity traits, our data did not allow us to discern the mechanism underlying the lowered competitive fertilization success of inbred (f = 0.50) males.     

Male attractiveness, fertility and susceptibility to oxidative stress are influenced by inbreeding in Drosophila simulans

Inbreeding frequently leads to inbreeding depression, a reduction in the trait values of inbred individuals. Inbreeding depression has been documented in sexually selected characters in several taxa, and while there is correlational evidence that male fertility is especially susceptible to inbreeding depression, there have been few direct experimental examinations of this. Here, we assessed inbreeding depression in male fertility and a range of other male fitness correlates in Drosophila simulans. We found that male fertility and attractiveness were especially susceptible to inbreeding depression. Additionally, levels of testicular oxidative stress were significantly elevated in inbred males, although sperm viability did not differ between inbred and outbred males. Copulation duration, induction of oviposition, and the proportion of eggs hatching did not differ for females mated to inbred or outbred males. Nevertheless, our results clearly show that key male fitness components are impaired by inbreeding and provide evidence that aspects of male fertility are especially susceptible to inbreeding depression.     

Males Benefit from Mating with Outbred Females in Drosophila littoralis: Male Choice for Female Genetic Quality?

The evolution and expression of mate choice behaviour in either sex depends on the sex‐specific combination of mating costs, benefits of choice and constraints on choice. If the benefits of choice are larger for one sex, we would expect that sex to be choosier, assuming that the mating costs and constraints on choice are equal between sexes. Because deliberate inbreeding is a powerful genetic method for experimental manipulation of the quality of study organisms, we tested the effects of both male and female inbreeding on egg and offspring production in Drosophila littoralis. Female inbreeding significantly reduced offspring production (mostly due to lower egg‐to‐adult viability), whereas male inbreeding did not affect offspring production (despite a slight effect of paternal inbreeding on egg‐to‐adult viability). As inbreeding depressed female quality more than male quality, the benefits of mate choice were larger for males than for females. In mate choice experiments, inbreeding did not affect male mating success (measured as a probability to be accepted as a mate in a large group), suggesting that females did not discriminate among inbred and outbred males. In contrast, female mating success was affected by inbreeding, with outbred females having higher mating success than inbred females. This result was not explained by lower activity of inbred females. Our results show that D. littoralis males benefit from mating with outbred females of high genetic quality and suggest adaptive male mate choice for female genetic quality in this species. Thus, patterns of mating success in mate choice trials mirrored the benefits of choice: the sex that benefited more from choice (i.e. males) was more choosy.     

Why does inbreeding reduce male paternity? Effects on sexually selected traits

Mating with relatives has often been shown to negatively affect offspring fitness (inbreeding depression). There is considerable evidence for inbreeding depression due to effects on naturally selected traits, particularly those expressed early in life, but there is less evidence of it for sexually selected traits. This is surprising because sexually selected traits are expected to exhibit strong inbreeding depression. Here, we experimentally created inbred and outbred male mosquitofish (Gambusia holbrooki). Inbred males were the offspring of matings between full siblings. We then investigated how inbreeding influenced a number of sexually selected male traits, specifically: attractiveness, sperm number and velocity, as well as sperm competitiveness based on a male's share of paternity. We found no inbreeding depression for male attractiveness or sperm traits. There was, however, evidence that lower heterozygosity decreased paternity due to reduced sperm competitiveness. Our results add to the growing evidence that competitive interactions exacerbate the negative effects of the increased homozygosity that arises when there is inbreeding.     

Reduced heterozygosity impairs sperm quality in endangered mammals

Inbreeding causes increases in homozygosity and is commonly associated with reductions in fertility and embryogenesis. Although the mechanisms underlying such effects are unknown, recent work has suggested that inbred males may suffer impaired ejaculate quality, thus providing a functional explanation for reductions in reproductive function in inbred populations. However, the relationship between inbreeding and sperm quality remains controversial, particularly in wild populations where the level of inbreeding is typically estimated using neutral molecular markers. Such markers are thought to reflect genome-wide levels of heterozygosity only under restricted conditions, and rarely in outbred populations. Here we employ a comparative approach that takes account of these criticisms and evaluates the evidence linking inbreeding to reductions in sperm quality in 20 mammal species. We focus on sperm abnormalities and sperm motility, which are key determinants of male fertility in many species. We show that species with reduced mean heterozygosity have impaired ejaculated quality, although subsequent analyses revealed that these effects were confined to endangered populations. Our findings therefore support the notion that inbreeding can severely impair sperm quality while concomitantly addressing criticisms surrounding the use of heterozygosity estimates to estimate the level of inbreeding.     

No evidence of inbreeding depression in sperm performance traits in wild song sparrows

Inbreeding is widely hypothesized to shape mating systems and population persistence, but such effects will depend on which traits show inbreeding depression. Population and evolutionary consequences could be substantial if inbreeding decreases sperm performance and hence decreases male fertilization success and female fertility. However, the magnitude of inbreeding depression in sperm performance traits has rarely been estimated in wild populations experiencing natural variation in inbreeding. Further, the hypothesis that inbreeding could increase within‐ejaculate variation in sperm traits and thereby further affect male fertilization success has not been explicitly tested. We used a wild pedigreed song sparrow (Melospiza melodia) population, where frequent extrapair copulations likely create strong postcopulatory competition for fertilization success, to quantify effects of male coefficient of inbreeding (f) on key sperm performance traits. We found no evidence of inbreeding depression in sperm motility, longevity, or velocity, and the within‐ejaculate variance in sperm velocity did not increase with male f. Contrary to inferences from highly inbred captive and experimental populations, our results imply that moderate inbreeding will not necessarily constrain sperm performance in wild populations. Consequently, the widely observed individual‐level and population‐level inbreeding depression in male and female fitness may not stem from reduced sperm performance in inbred males.     

Context-dependent expression of sperm quality in the fruitfly

In most species, females mate multiply within a reproductive cycle, invoking post-copulatory selection on ejaculatory components. Much research has focused on disentangling the key traits important in deciding the outcomes of sperm competition and investigating patterns of covariance among these traits. Less attention has focused on the degree to which such patterns might be context-dependent. Here, we examine whether the expression of sperm viability—a widely used measure of sperm quality—and patterns of covariance between this trait and male reproductive morphologies, change across distinct age classes and across naturally occurring genotypes, when expressed in both heterozygotic (extreme outbred) and homozygotic (extreme inbred) states in the fruitfly Drosophila melanogaster. Older males, and heterozygous males, generally exhibited higher sperm viability. The male age effect seems at least partly explained by a positive association between sperm numbers and viability. First, old males possessed more stored sperm than young males, and second, sperm numbers and viability were also positively associated within each age class. Furthermore, we found a positive association between sperm viability and testis size, but only among heterozygous, old males. These results suggest that sperm quality is a labile trait, with expression levels that are context-dependent and shaped by multiple, potentially interacting, factors.     

Close‐kin mating,but not inbred parents,reduces hatching rates and offspring quality in a threatened tortoise

Inbreeding depression, the reduction in fitness due to mating of related individuals, is of particular conservation concern in species with small, isolated populations. Although inbreeding depression is widespread in natural populations, long‐lived species may be buffered from its effects during population declines due to long generation times and thus are less likely to have evolved mechanisms of inbreeding avoidance than species with shorter generation times. However, empirical evidence of the consequences of inbreeding in threatened, long‐lived species is limited. In this study, we leverage a well‐studied population of gopher tortoises, Gopherus polyphemus, to examine the role of inbreeding depression and the potential for behavioural inbreeding avoidance in a natural population of a long‐lived species. We tested the hypothesis that increased parental inbreeding leads to reduced hatching rates and offspring quality. Additionally, we tested for evidence of inbreeding avoidance. We found that high parental relatedness results in offspring with lower quality and that high parental relatedness is correlated with reduced hatching success. However, we found that hatching success and offspring quality increase with maternal inbreeding, likely due to highly inbred females mating with more distantly related males. We did not find evidence for inbreeding avoidance in males and outbred females, suggesting sex‐specific evolutionary trade‐offs may have driven the evolution of mating behaviour. Our results demonstrate inbreeding depression in a long‐lived species and that the evolution of inbreeding avoidance is shaped by multiple selective forces.     

Male inbreeding status affects female fitness in a seed-feeding beetle

Inbreeding generally reduces male mating activity such that inbred males are less successful in male-male competition. Inbred males can also have smaller accessory glands, transfer less sperm and produce sperm that are less motile, less viable or have a greater frequency of abnormalities, all of which can reduce the fertilization success and fitness of inbred males relative to outbred males. However, few studies have examined how male inbreeding status affects the fitness of females with whom they mate. In this study, we examine the effect of male inbreeding status (inbreeding coefficient f = 0.25 vs. f = 0) on the fecundity, adult longevity and the fate of eggs produced by outbred females in the seed-feeding beetle, Callosobruchus maculatus. Females mated to inbred males were less likely to lay eggs. Of those that laid eggs, females mated to inbred males laid 6-12% fewer eggs. Females mated to inbred males lived on average 5.4% longer than did females mated to outbred males, but this effect disappeared when lifetime fecundity was used as a covariate in the analysis. There was no effect of male inbreeding status on the proportion of a female's eggs that developed or hatched, and no evidence that inbred males produced smaller nuptial gifts. However, ejaculates of inbred males contained 17-33% fewer sperm, on average, than did ejaculates of outbred males. Our study demonstrates that mating with inbred males has significant direct consequences for the fitness of female C. maculatus, likely mediated by effects of inbreeding status on the number of sperm in male ejaculates. Direct effects of male inbreeding status on female fitness should be more widely considered in theoretical models and empirical studies of mate choice.     

Lifetime inbreeding depression in a leaf beetle

Ongoing habitat loss and fragmentation result in rapid population size reductions, which can increase the levels of inbreeding. Consequently, many species are threatened by inbreeding depression, a loss of individual fitness following the mating of close relatives. Here, we investigated inbreeding effects on fitness‐related traits throughout the lifetime of the mustard leaf beetle (Phaedon cochleariae) and mechanisms for the avoidance of inbreeding. Previously, we found that these beetles have family‐specific cuticular hydrocarbon profiles, which are likely not used as recognition cue for precopulatory inbreeding avoidance. Thus, we examined whether adult beetles show postcopulatory inbreeding avoidance instead. For this purpose, we determined the larval hatching rate of eggs laid by females mated sequentially with two nonsiblings, two siblings, a nonsibling, and a sibling or vice versa. The beetles suffered from inbreeding depression throughout their entire ontogeny, as evinced by a prolonged larval development, a decreased larval and adult survival and a decreased reproductive output of inbred compared to outbred individuals. The highest larval hatching rates were detected when females were mated with two nonsiblings or first with a sibling and second with a nonsibling. Significantly lower hatching rates were measured in the treatments with a sibling as second male. Thus, the results do not support the existence of postcopulatory inbreeding avoidance in P. cochleariae, but revealed evidence for second male sperm precedence. Consequently, an alternative strategy to avoid inbreeding costs might exist in this beetle, such as a polyandrous mating system, potentially coupled with a specific dispersal behavior.     

Differential effects of offspring and maternal inbreeding on egg laying and offspring performance in the burying beetle Nicrophorus vespilloides

We investigate the effect of offspring and maternal inbreeding on maternal and offspring traits associated with early offspring fitness in the burying beetle Nicrophorus vespilloides. We conducted two experiments. In the first experiment, we manipulated maternal inbreeding only (keeping offspring outbred) by generating mothers that were outbred, moderately inbred or highly inbred. Meanwhile, in the second experiment, we manipulated offspring inbreeding only (keeping females outbred) by generating offspring that were outbred, moderately inbred or highly inbred. In both experiments, we monitored subsequent effects on breeding success (number of larvae), maternal traits (clutch size, delay until laying, laying skew, laying spread and egg size) and offspring traits (hatching success, larval survival, duration of larval development and average larval mass). Maternal inbreeding reduced breeding success, and this effect was mediated through lower hatching success and greater larval mortality. Furthermore, inbred mothers produced clutches where egg laying was less skewed towards the early part of laying than outbred females. This reduction in the skew in egg laying is beneficial for larval survival, suggesting that inbred females adjusted their laying patterns facultatively, thereby partially compensating for the detrimental effects of maternal inbreeding on offspring. Finally, we found evidence of a nonlinear effect of offspring inbreeding coefficient on number of larvae dispersing. Offspring inbreeding affected larval survival and larval development time but also unexpectedly affected maternal traits (clutch size and delay until laying), suggesting that females adjust clutch size and the delay until laying in response to being related to their mate.     

Pattern of inbreeding depression,condition dependence,and additive genetic variance in Trinidadian guppy ejaculate traits

In polyandrous species, a male's reproductive success depends on his fertilization capability and traits enhancing competitive fertilization success will be under strong, directional selection. This leads to the prediction that these traits should show stronger condition dependence and larger genetic variance than other traits subject to weaker or stabilizing selection. While empirical evidence of condition dependence in postcopulatory traits is increasing, the comparison between sexually selected and ‘control’ traits is often based on untested assumption concerning the different strength of selection acting on these traits. Furthermore, information on selection in the past is essential, as both condition dependence and genetic variance of a trait are likely to be influenced by the pattern of selection acting historically on it. Using the guppy (Poecilia reticulata), a livebearing fish with high levels of multiple paternity, we performed three independent experiments on three ejaculate quality traits, sperm number, velocity, and size, which have been previously shown to be subject to strong, intermediate, and weak directional postcopulatory selection, respectively. First, we conducted an inbreeding experiment to determine the pattern of selection in the past. Second, we used a diet restriction experiment to estimate their level of condition dependence. Third, we used a half‐sib/full‐sib mating design to estimate the coefficients of additive genetic variance (CV_A) underlying these traits. Additionally, using a simulated predator evasion test, we showed that both inbreeding and diet restriction significantly reduced condition. According to predictions, sperm number showed higher inbreeding depression, stronger condition dependence, and larger CV_A than sperm velocity and sperm size. The lack of significant genetic correlation between sperm number and velocity suggests that the former may respond to selection independently one from other ejaculate quality traits. Finally, the association between sperm number and condition suggests that this trait may mediate the genetic benefits of polyandry which have been shown in this species.     

Resource quality affects weapon and testis size and the ability of these traits to respond to selection in the leaf‐footed cactus bug,Narnia femorata

The size of weapons and testes can be central to male reproductive success. Yet, the expression of these traits is often extremely variable. Studies are needed that take a more complete organism perspective, investigating the sources of variation in both traits simultaneously and using developmental conditions that mimic those in nature. In this study, we investigated the components of variation in weapon and testis sizes using the leaf‐footed cactus bug, Narnia femorata (Hemiptera: Coreidae) on three natural developmental diets. We show that the developmental diet has profound effects on both weapon and testis expression and scaling. Intriguingly, males in the medium‐quality diet express large weapons but have relatively tiny testes, suggesting complex allocation decisions. We also find that heritability, evolvability, and additive genetic variation are highest in the high‐quality diet for testis and body mass. This result suggests that these traits may have an enhanced ability to respond to selection during a small window of time each year when this diet is available. Taken together, these results illustrate that normal, seasonal fluctuations in the nutritional environment may play a large role in the expression of sexually selected traits and the ability of these traits to respond to selection.     

Body size and sperm quality in queen‐ And worker‐produced ant males

Workers of many species of social Hymenoptera have functional ovaries and are capable of laying haploid, unfertilized eggs, at least in the absence of a queen. Except for honeybees, it remains largely unknown whether worker‐produced males have the same quality as queen‐produced males and whether workers benefit in direct fitness by producing their sons. Previous studies in the monogynous ant Temnothorax crassispinus revealed that a high proportion of males in natural and laboratory colonies are worker offspring. Here, we compare longevity, body size, sperm length and sperm viability between queen‐ and worker‐produced males. We either split queenright colonies into queenright and queenless halves or removed the queen from a fraction of the queenright colonies and then examined the newly produced males. Male quality traits varied considerably among colonies but differed only slightly between queen‐ and worker‐produced males. Worker‐produced males outnumbered queen‐produced males and also had a longer lifespan, but under certain rearing conditions sperm from queen‐produced males had a higher viability.     

Feed‐backs among inbreeding,inbreeding depression in sperm traits,and sperm competition can drive evolution of costly polyandry

Ongoing ambitions are to understand the evolution of costly polyandry and its consequences for species ecology and evolution. Emerging patterns could stem from feed‐back dynamics between the evolving mating system and its genetic environment, defined by interactions among kin including inbreeding. However, such feed‐backs are rarely considered in nonselfing systems. We use a genetically explicit model to demonstrate a mechanism by which inbreeding depression can select for polyandry to mitigate the negative consequences of mating with inbred males, rather than to avoid inbreeding, and to elucidate underlying feed‐backs. Specifically, given inbreeding depression in sperm traits, costly polyandry evolved to ensure female fertility, without requiring explicit inbreeding avoidance. Resulting sperm competition caused evolution of sperm traits and further mitigated the negative effect of inbreeding depression on female fertility. The evolving mating system fed back to decrease population‐wide homozygosity, and hence inbreeding. However, the net overall decrease was small due to compound effects on the variances in sex‐specific reproductive success and paternity skew. Purging of deleterious mutations did not eliminate inbreeding depression in sperm traits or hence selection for polyandry. Overall, our model illustrates that polyandry evolution, both directly and through sperm competition, might facilitate evolutionary rescue for populations experiencing sudden increases in inbreeding.     

Simultaneous inbreeding modifies inbreeding depression in a plant–herbivore interaction

Because inbreeding is common in natural populations of plants and their herbivores, herbivore‐induced selection on plants, and vice versa, may be significantly modified by inbreeding and inbreeding depression. In a feeding assay with inbred and outbred lines of both the perennial herb, Vincetoxicum hirundinaria, and its specialist herbivore, Abrostola asclepiadis, we discovered that plant inbreeding increased inbreeding depression in herbivore performance in some populations. The effect of inbreeding on plant resistance varied among plant and herbivore populations. The among‐population variation is likely to be driven by variation in plant secondary compounds across populations. In addition, inbreeding depression in plant resistance was substantial when herbivores were outbred, but diminished when herbivores were inbred. These findings demonstrate that in plant–herbivore interactions expression of inbreeding depression can depend on the level of inbreeding of the interacting species. Furthermore, our results suggest that when herbivores are inbred, herbivore‐induced selection against self‐fertilisation in plants may diminish.