Inbreeding,fluctuating asymmetry,and ejaculate quality in an endangered ungulate.

An ever-increasing number of species are suffering marked reductions in population size as a consequence of human activities. To understand the impact of these changes it is essential to assess how small population size affects individual fitness and the viability of populations. This issue acquires special relevance among endangered species in which numbers have decreased to such an extent that captive breeding must be established with a few founders. A major risk associated with small population size is inbreeding depression. The effects of inbreeding upon male reproductive traits are the subject of an ongoing controversy, since the evidence linking lack of genetic variability and poor ejaculate quality at the population level has been criticized recently by several authors. We report that among Gazella cuvieri males, inbreeding coefficient shows a strong inverse relationship with ejaculate quality. Furthermore, the degree of fluctuating asymmetry is positively related to the coefficient of inbreeding and negatively related to the proportion of normal sperm, suggesting that it is a reliable indicator of genetic stress and of ejaculate quality.     

Pedigrees and microsatellites among endangered ungulates: what do they tell us?

Relationships between pedigree coefficients of inbreeding and molecular metrics are generally weak, suggesting that measures of heterozygosity estimated using microsatellites may be poor surrogates of genome-wide inbreeding. We compare three endangered species of gazelles ( Gazella ) with different degrees of threat in their natural habitats, for which captive breeding programmes exist. For G. dorcas, the species with the largest founding population, the highest and most recent number of founding events, the correlation between pedigree coefficient of inbreeding and molecular metrics was higher than for outbred populations of mammals, probably because it has both higher mean f and variance. For the two species with smaller founding populations, conventional assumptions about founders, i.e. outbred and unrelated, are unrealistic. When realistic assumptions about the founders were made, clear relationships between pedigree coefficients of inbreeding and molecular metrics were revealed for G. cuvieri. This population had a small founding population, but it did experience admixture years later; thus, the relationship between inbreeding and molecular metrics in G. cuvieri is very similar to the expected values but lower than in G. dorcas . In contrast, no relationship was found for G. dama mhorr which had a much smaller founding population than had been previously assumed, which probably had high levels of inbreeding and low levels of genetic variability, and no admixture. In conclusion, the strength of the association between pedigree coefficient of inbreeding and molecular metrics among endangered species depends on the level of inbreeding and genetic variability present in the founding population, its size and its history.     

Sperm cryopreservation in three species of endangered gazelles (Gazella cuvieri,G. dama mhorr,and G. dorcas neglecta)

Long-term storage of semen by cryopreservation, with high recovery rates on thawing, is essential for the establishment of genetic resource banks of endangered species. The purpose of the present study was to evaluate various diluents for the cryopreservation of spermatozoa from three species of gazelles (genus Gazella) in a captive breeding program. The diluents compared were Tes (N-tris(hydroxymethyl)methyl-2 aminoethane sulfonic acid)-Tris with 5% egg yolk and 6% glycerol (TEST) and Triladyl, yolk-citrate, Tris-trehalose, and Tris-lactose-all of them with 20% egg yolk and 6% (Triladyl) or 8% glycerol. Semen was obtained by electroejaculation from 12 G. cuvieri, 12 G. dama, and 13 G. dorcas males. Samples with less than 50% motile sperm, positive endosmosis, or acrosome integrity were not used. Diluted samples were loaded into 0.25-ml straws, cooled slowly to 5 degrees C over 1.5 h (-0.16 degrees C/min), equilibrated at that temperature for 2 h, frozen in nitrogen vapors for 10 min, and plunged into liquid nitrogen. Subsamples were assessed fresh, after refrigeration-equilibration, after freezing and thawing, and 2 h after thawing. Differences were seen between diluents, with best overall recovery rates after freezing and thawing found with Triladyl, TEST, and Tris-trehalose in G. cuvieri, TEST in G. dama, and Triladyl and TEST in G. dorcas. Differences were observed between species in the ability to withstand freezing and thawing, with best results seen in G. dorcas, intermediate results in G. dama, and worst results in G. cuvieri. These differences were inversely related to the average values of inbreeding of these populations. The underlying mechanism responsible for these differences may be a differential resistance to osmotic shock.     

Hematologic and biochemical observations in Gazella dama, G. dorcas and G. cuvieri.

1. Blood samples obtained from 114 animals of three species of the genus Gazella (Gazella dama, Gazella dorcas and Gazella cuvieri) were analyzed from hematology (osmotic fragility, red blood cells morphology and hemoglobin electrophoresis) and biochemical values (glucose-6-phosphate dehydrogenase, pyruvate kinase and glutathione reductase deficiencies and superoxide dismutase activity). 2. Standard methods were used. Hemoglobin polymorphism was found. 3. There was no abnormality in the osmotic fragility and red blood cells morphology. 4. The biochemical results are compared with information from the literature and with the normal human range.     

Abomasal and small intestinal nematodes from captive gazelles in Spain

The abomasal and small intestinal helminth fauna of three species of captive gazelles (Gazella dama mhorr, G. cuvieri and G. dorcas neglecta) kept in captivity in Almería (southeast Spain) have been studied, and the following species were identified: Nematodirus spathiger, N. filicollis, N. helvetianus, Camelostrongylus mentulatus, Trichostrongylus vitrinus, T. probolurus, T. colubriformis, Ostertagia ostertagi, O. harrisi, Teladorsagia (Ostertagia) circumcincta, and T. (Ostertagia) davtiani. Camelostrongylus mentulatus and N. spathiger were the most prevalent and abundant parasites. Ostertagia ostertagi, O. harrisi, N. helvetianus, and T. (Ostertagia) davtiani were identified for the first time in the genus Gazella. In addition, O. harrisi and Trichostrongylus probolurus are new records for Spain.     

Does stress mess with rodents’ heads? Influence of habitat amount and genetic factors in mandible fluctuating asymmetry in South American water rats (Nectomys squamipes,Sigmodontinae) from Brazilian Atlantic rainforest remnants

Loss of developmental stability can lead to deviations from bilateral symmetry (i.e. Fluctuating Asymmetry ‐ FA), and is thought to be caused by environmental and genetic factors associated with habitat loss and stress. Therefore, levels of FA might be a valuable tool to monitor wild populations if FA serves as an indicator of exposure to stress due to impacts of habitat loss and fragmentation. In studies examining FA and habitat fragmentation, FA levels are often explained by loss of genetic variation, though few studies have addressed FA’s use as indicator of environmental impact. Here, we investigated whether habitat loss, genetic variation, and/or inbreeding affect the developmental instability in Brazilian Atlantic forest populations of a Neotropical water rat (Nectomys squamipes). We sampled individuals from eight sites within Atlantic forest remnants with different amounts of available forest habitat and assessed FA levels with geometric morphometric techniques using adult mandibles. We used observed heterozygosity (H_o) and inbreeding coefficient (F_is), from seven microsatellite markers, as a proxy of genetic variation at individual and population levels. Populations were not significantly different for shape or size FA levels. Furthermore, interindividual variation in both shape and size FA levels and interpopulational differences in size FA levels were best explained by chance. However, habitat amount was negatively associated with both interpopulational variance and average shape FA levels. This association was stronger in populations living in areas with <28% of forest cover, which presented higher variance and higher average FA, suggesting that Nectomys squamipes might have a tolerance threshold to small availability of habitat. Our work is one of the first to use FA to address environmental stress caused by habitat loss in small mammal populations from a Neotropical biome. We suggest that shape FA might serve as a conservation tool to monitor human impact on natural animal populations.     

Heat stress but not inbreeding affects offensive sperm competitiveness in Callosobruchus maculatus

Environmental and genetic stress have well‐known detrimental effects on ejaculate quality, but their concomitant effect on male fitness remains poorly understood. We used competitive fertilization assays to expose the effects of stress on offensive sperm competitive ability in the beetle Callosobruchus maculatus, a species where ejaculates make up more than 5% of male body mass. To examine the effects of environmental and genetic stress, males derived from outcrosses or sib matings were heat shocked at 50°C for 50 min during the pupal stage, while their siblings were maintained at a standard rearing temperature of 28°C. Heat‐shocked males achieved only half the offensive paternity success of their siblings. While this population exhibited inbreeding depression in body size, sperm competitiveness was unaffected by inbreeding, nor did the effect of heat shock stress on sperm competitiveness depend on inbreeding status. In contrast, pupal emergence success was increased by 34% among heat‐stressed individuals, regardless of their inbreeding status. Heat‐shocked males' ejaculate size was 19% reduced, but they exhibited 25% increased mating duration in single mating trials. Our results highlight both the importance of stress in postcopulatory sexual selection, and the variability among stressors in affecting male fitness.     

Is phenotypic canalization involved in the decline of the endemic Aquilegia thalictrifolia? Rethinking relationships between fluctuating asymmetry and species conservation status

Fluctuating asymmetry (FA), the deviation from the normal symmetrical condition of a morphological trait having specific morphological symmetry, increases in response to environmental and genetic stress, is related to phenotypic plasticity and is considered a tool for monitoring a species conservation status. However, FA–stress relations are dependent on measured traits or species‐specific characteristics such as mating system and habitat. This study investigates the relationships between FA, genetic diversity, population size, density and individual fitness traits (plant height, fruit and seed set), in the endemic Aquilegia thalictrifolia, a mixed breeder that is declining, but maintaining high levels of heterozygosity. Leaf and flower FA and other traits were investigated in 10 populations of A. thalictrifolia, the whole species range. As a result, we found similar patterns of FA in leaves and flowers between populations, indicating a homogenous level of stress between populations that differed for other traits. FA and the other traits were not related, including heterozygosity. Heterozygosity was not related to individual fitness traits with the exception of plant height. In accordance with other studies, we found that the role of FA as a tool for assessing the conservation status of a species or population should be reconsidered. However, we conclude that a low level of FA should not automatically be considered an indicator of good conservation status or low level of stress, because in species that evolved in highly stable environments it may indicate a scarce ability to plastically respond to environmental changes, as a consequence of environmental and genetic canalization. Further investigation of this point is needed.     

Effect of directional selection for body size on fluctuating asymmetry in certain morphological traits in <Emphasis Type="Italic">Drosophila ananassae</Emphasis>

Variation in the subtle differences between the right and left sides of bilateral characters or fluctuating asymmetry (FA) has been considered as an indicator of an organism’s ability to cope with genetic and environmental stresses during development. However, due to inconsistency in the results of empirical studies, the relationship between FA and stress has been the subject of intense debate. In this study, we investigated whether stress caused by artificial bidirectional selection for body size has any effect on the levels of FA of different morphological traits in Drosophila ananassae. The realised heritability (h²) was higher in low-line females and high-line males, which suggests an asymmetrical response to selection for body size. Further, the levels of FA were compared across 10 generations of selection in different selection lines in both sexes for sternopleural bristle number, wing length, wing-to-thorax ratio, sex comb-tooth number and ovariole number. The levels of FA differed significantly among generations and selection lines but did not change markedly with directional selection. However, the levels of FA were higher in the G10 generation (at the end of selection) than G0 (at the start of selection) but lower than the G5 generation in different selection lines, suggesting that the levels of FA are not affected by the inbreeding generated during the course of selection. Also, the levels of FA in the hybrids of high and low lines were significantly lower than the parental selection lines, suggesting that FA is influenced by hybridisation. These results are discussed in the framework of the literature available on FA and its relationship with stress.     

Fluctuating asymmetry of leaves is a poor indicator of environmental stress and genetic stress by inbreeding in Silene vulgaris

Fluctuating asymmetry (FA) is often used as a measure of developmental instability and has been proposed as an indicator of both environmental and genetic stress. However, the empirical evidence for the effects of stress on FA in plants is inconsistent, and there are few controlled experimental studies. We analyzed different distance- and shape-based measures of the fluctuating asymmetry of the leaves of clonally replicated self- and cross-pollinated lineages of Silene vulgaris (Moench) Garcke (Caryophyllaceae) grown under a control and seven different stress treatments (drought, copper, simulated herbivory, and two levels of nutrient deficiency and of shade). Overall, FA differed among stress treatments, but was not generally higher under stress and even reduced in some treatments. Different measures of FA were only weakly correlated. Inbreeding increased only one measure of leaf FA, and only under high stress intensities. Our findings suggest that in S. vulgaris leaf fluctuating asymmetry does not serve as an indicator of environmental stress or of genetic stress by inbreeding.     

Relationship of inbreeding with sperm quality and reproductive success in Mexican gray wolves

The ultimate goal of the Mexican gray wolf Canis lupus baileyi captive management program is reintroduction of healthy individuals into wild habitats. To this end, zoo population managers work to provide not only for the physical well-being but also for the genetic health of these animals. However, the very limited genetic founder base, exacerbated by breeding within three distinct lineages, resulted in very high coefficients of inbreeding. Because support for measurable levels of inbreeding depression in the captive wolf population, as defined by reductions in common fitness measures such as juvenile survival or reproductive success, has been weak, we investigated the potential effects on male reproductive capacity. We analyzed semen samples from wolves from all three lineages and compared them with samples from subsequent lineage crosses and from generic gray wolves. We not only found a significant effect of inbreeding on sperm quality but we related both inbreeding and sperm quality to reproductive success. Samples from male offspring of lineage crosses, with inbreeding coefficients of zero were similar in quality to those from generic gray wolves. However, samples from a limited number of offspring from back-crosses were of extremely poor quality. Although it is reassuring that sperm quality was so much improved in male offspring of lineage crosses, the concomitant reduction in inbreeding coefficient does not eliminate the potentially deleterious alleles. Our results demonstrate that sperm quality is an important indicator of fertility and reproductive success in Mexican wolves. In addition, our data lend further support to the presence of inbreeding depression in this taxon.     

Fluctuating feather asymmetry in relation to corticosterone levels is sex-dependent in Eurasian treecreeper (Certhia familiaris) nestlings

Fluctuating asymmetry (FA) has been widely used as a stress-related phenotypic marker of developmental instability. However, previous studies relating FA to various stressful conditions have produced inconsistent results and we still lack quantitative individual-level evidence that high FA is related to stress in wild vertebrate species. We studied how baseline plasma levels of corticosterone predicted FA of wing and tail feathers in free-living Eurasian treecreeper (Certhia familiaris) nestlings. We found a sex-specific association between corticosterone levels and FA: high corticosterone levels were related to an increased FA in male but not in female nestlings. These results suggest that in treecreepers, FA may correlate with individual stress hormone levels, male developmental trajectory being potentially more sensitive to stress than that of the female.     

No evidence for postcopulatory inbreeding avoidance in Drosophila melanogaster

Selection to avoid inbreeding is predicted to vary across species due to differences in population structure and reproductive biology. Over the past decade, there have been numerous investigations of postcopulatory inbreeding avoidance, a phenomenon that first requires discrimination of mate (or sperm) relatedness and then requires mechanisms of male ejaculate tailoring and/or cryptic female choice to avoid kin. The number of studies that have found a negative association between male-female genetic relatedness and competitive fertilization success is roughly equal to the number of studies that have not found such a relationship. In the former case, the underlying mechanisms are largely unknown. The present study was undertaken to verify and expand upon a previous report of postcopulatory inbreeding avoidance in D. melanogaster, as well as to resolve underlying mechanisms of inbreeding avoidance using transgenic flies that express a sperm head-specific fluorescent tag. However, siblings did not have a lower fertilization success as compared to unrelated males in either the first (P(1) ) or second (P(2) ) mate role in sperm competition with a standard unrelated competitor male in our study population of D. melanogaster. Analyses of mating latency, copulation duration, egg production rate, and remating interval further revealed no evidence for inbreeding avoidance.     

Fluctuating asymmetry in a secondary sexual trait: no associations with individual fitness, environmental stress or inbreeding, and no heritability

It has been suggested that fluctuating asymmetry (FA) in secondary sexual traits may be a useful indicator of either individual quality or environmental stress. We tested this concept using a series of analyses of FA in male antler size in a wild red deer (Cervus elaphus) population, using four measures of size repeated across successive years on the same individuals. We found no consistent evidence of correlations between traits in levels of FA, nor of any associations between known environmental or developmental conditions. None of the four measures of FA showed a significant heritability (average h2 = 0.041), nor was there any evidence of inbreeding depression. For three of the four traits, fluctuating asymmetry did not predict either annual or lifetime breeding success. However there were significant associations between breeding success and FA in antler length. Given the series of null results in our other tests, it seems likely that this was a direct mechanistic effect rather than because measures of FA were indicative of individual quality or condition.     

Olfactory bulbectomy-induced changes in the reproductive behaviour of the south Indian gerbil, Tatera indica cuvieri

Male South Indian gerbils (T. indica cuvieri), both adults and weanlings, were olfactory bulbectomized (OBX) and changes in male reproductive behaviour were assessed. Consequent to OBX, majority of the adult males failed to ejaculate, and courtship behaviour has also been considerably reduced. All OBX weanlings were rendered incapable of ejaculation. However, maturational parameters, and organ weights (testes, epididymis and seminal vesicle) remained unchanged in OBX males.     

Fluctuating asymmetry in leaves and flowers of sympatric species in a tropical montane environment

Fluctuating asymmetry (FA) represents small, random variations in traits, presumably with bilateral symmetry, and is widely used as a tool to measure developmental instability in plants and animals. Because FA is a quick, simple and reliable measure, it has been frequently used for monitoring levels of environmental stress. This study investigated whether FA can be used as a predictor of individual developmental instability for four sympatric Melastomataceae species. To achieve that aim, 20 individuals of Trembleya laniflora, T. parviflora, Lavoisiera campos‐portoana and Tibouchina heteromalla were marked in southeastern Brazil and monitored before and during the flowering season. The FA index was calculated as the mean of the difference in the width or length between the left and right sides measured for each leaf or flower. All studied species exhibited asymmetry in the leaves and flowers, but the leaves of L. campos‐portoana and the petal width of T. heteromalla exhibited directional asymmetry, also an indicator of developmental instability. The highest level of leaf asymmetry was found in T. heteromalla and on flowers of L. campos‐portoana. None of the studied species exhibited a significant relationship between the FA level of the leaves and flowers on an individual basis, indicating that environmental and/or genetic sources of stress might act differently on different plant traits. For the studied species, measurements of FA can be suggested as useful tools to biomonitor levels of stress experienced by both leaves and flowers within the Melastomataceae family.     

Study of closely related species within the Physalaemus cuvieri group (Anura): contribution of microsatellite markers

Various species of the Physalaemus cuvieri group of frogs are difficult to distinguish morphologically, making molecular analysis an attractive alternative for indentifying members of this group, which is considered to be at risk because of loss of habitat. The genetic structure of natural populations of P. ephippifer and P. albonotatus species was investigated and analyzed, together with that of five previously studied populations of P. cuvieri. Nine microsatellite loci were used in the analyses. The overall G(ST) value (0.46) revealed high genetic variation among the populations, as expected for different species. Bayesian analysis implemented by the STRUCTURE software clustered the seven populations into seven groups (K = 7). All the P. albonotatus and P. ephippifer specimens were grouped into a single cluster, both species showing clear differentiation from P. cuvieri. The different grouping based on these microsatellites of some P. cuvieri individuals from Porto Nacional and from Passo Fundo suggests that they could be a new species, indicating a necessity for taxonomic reevaluation. Despite the intrinsic difficulties in analyzing closely related species, the nine microsatellite loci were found to be adequate for distinguishing these three species of the P. cuvieri group and their populations.     

No evidence of inbreeding avoidance despite demonstrated survival costs in a polygynous rodent

Individuals are generally predicted to avoid inbreeding because of detrimental fitness effects. However, several recent studies have shown that limited inbreeding is tolerated by some vertebrate species. Here, we examine the costs and benefits of inbreeding in a largely polygynous rodent, the yellow-bellied marmot (Marmota flaviventris). We use a pedigree constructed from 8 years of genetic data to determine the relatedness of all marmots in our study population and examine offspring survival, annual male reproductive success, relatedness between breeding pairs and the effects of group composition on likelihood of male reproduction to assess inbreeding in this species. We found decreased survival in inbred offspring, but equal net reproductive success among males that inbred and those that avoided it. Relatedness between breeding pairs was greater than that expected by chance, indicating that marmots do not appear to avoid breeding with relatives. Further, male marmots do not avoid inbreeding: males mate with equal frequency in groups composed of both related and unrelated females and in groups composed of only female relatives. Our results demonstrate that inbreeding can be tolerated in a polygynous species if the reproductive costs of inbreeding are low and individuals that mate indiscriminately do not suffer decreased reproductive success.     

Effects of inbreeding on male function and self-fertility in the partially self-incompatible herb Campanula rapunculoides (Campanulaceae)

We examined the effect of inbreeding on fitness (through both male and female functions) and changes in self-fertility in the partially self-incompatible species Campanula rapunculoides. Individuals in natural populations of C. rapunculoides varied extensively in their strength of self-incompatibility (SI). We crossed 11 individuals that differed in their strength of SI to generate families with four levels of inbreeding (f = 0.0, 0.25, 0.5, and 0.75). Progeny were scored for three traits related to male fitness and for outcrossed and selfed seed production. Analyses of variance revealed significant inbreeding depression for the three male traits and seed set. Families with strong or weak SI differed in their response to inbreeding. Families with weak SI had lower levels of inbreeding depression for most traits than families with strong SI, but strong SI families had a greater increase in selfed seed set, but not self-fertility, with inbreeding. Finally, we found evidence of a significant linear response to inbreeding for all three male reproductive traits and outcrossed seed, indicating that inbreeding depression was primarily caused by partially or fully recessive deleterious alleles. Variation in genetic load was associated with variation in self-fertility, a finding that suggests an evolutionary role for partial self-fertility in natural populations of C. rapunculoides.     

Sex differences in the drivers of reproductive skew in a cooperative breeder

Many cooperatively breeding societies are characterized by high reproductive skew, such that some socially dominant individuals breed, while socially subordinate individuals provide help. Inbreeding avoidance serves as a source of reproductive skew in many high‐skew societies, but few empirical studies have examined sources of skew operating alongside inbreeding avoidance or compared individual attempts to reproduce (reproductive competition) with individual reproductive success. Here, we use long‐term genetic and observational data to examine factors affecting reproductive skew in the high‐skew cooperatively breeding southern pied babbler (Turdoides bicolor). When subordinates can breed, skew remains high, suggesting factors additional to inbreeding avoidance drive skew. Subordinate females are more likely to compete to breed when older or when ecological constraints on dispersal are high, but heavy subordinate females are more likely to successfully breed. Subordinate males are more likely to compete when they are older, during high ecological constraints, or when they are related to the dominant male, but only the presence of within‐group unrelated subordinate females predicts subordinate male breeding success. Reproductive skew is not driven by reproductive effort, but by forces such as intrinsic physical limitations and intrasexual conflict (for females) or female mate choice, male mate‐guarding and potentially reproductive restraint (for males). Ecological conditions or “outside options” affect the occurrence of reproductive conflict, supporting predictions of recent synthetic skew models. Inbreeding avoidance together with competition for access to reproduction may generate high skew in animal societies, and disparate processes may be operating to maintain male vs. female reproductive skew in the same species.