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Polyploids are cells or organisms with a genome consisting of more than two sets of homologous chromosomes. Polyploid plants have important traits that facilitate speciation and are thus often model systems for evolutionary, molecular ecology and agricultural studies. However, due to their unusual mode of inheritance and double-reduction, diploid models of population genetic analysis cannot properly be applied to autopolyploids. To overcome this problem, we developed a software package entitled vcfpop to perform a variety of population genetic analyses for autopolyploids, such as parentage analysis, analysis of molecular variance, principal coordinates analysis, hierarchical clustering analysis and Bayesian clustering. We use three datasets to evaluate the capability of vcfpop to analyze large datasets on a desktop computer. This software is freely available at http://github.com/huangkang1987/vcfpop.

International audience; Many insects metamorphose from antagonistic larvae into mutualistic adult pollinators, with reciprocal adaptation leading to specialized insect-plant associations. It remains unknown how such interactions are established at molecular level. Here we assemble high-quality genomes of a fig species, Ficus pumila var. pumila, and its specific pollinating wasp, Wiebesia pumilae. We combine multi-omics with validation experiments to reveal molecular mechanisms underlying this specialized interaction. In the plant, we identify the specific compound attracting pollinators and validate the function of several key genes regulating its biosynthesis. In the pollinator, we find a highly reduced number of odorant-binding protein genes and an odorant-binding protein mainly binding the attractant. During antagonistic interaction, we find similar chemical profiles and turnovers throughout the development of galled ovules and seeds, and a significant contraction of detoxification-related gene families in the pollinator. Our study identifies some key genes bridging coevolved mutualists, establishing expectations for more diffuse insect-pollinator systems.

Fig tree–fig wasp mutualisms are diverse and underpin much biodiversity. The wasps (Agaonidae) are the sole pollinators of the trees (Ficus). Figs are enclosed inflorescences, each of which contains many small flowers. Female wasps (foundresses) enter receptive figs to spread pollen and to lay their eggs individually into fig flowers. As they oviposit, wasps also inject chemicals that transform individual flower ovaries into galls that will feed and house wasp offspring. For fig tree–fig wasp mutualisms to persist, the trees must set seed; therefore, the wasps have both to pollinate and to fail to gall all flower ovaries. However, wasps that avoid pollination costs and/or gall all flowers are predicted to outcompete more cooperative conspecifics, resulting in destabilisation of the mutualism. Here, I review the literature on why wasps pollinate by focusing on how trees reduce investment to unpollinated figs, resulting in ‘sanctions’ to wasps that fail to pollinate via reduced production of offspring. I also review the mechanisms that prevent wasps from galling all flowers, mainly those in monoecious Ficus, that also result in wasps predominantly galling longer flowers whilst leaving shorter flowers to become seeds. I make suggestions for future work and conclude by reaffirming why multiple processes promote stability in fig tree–fig wasp mutualisms.

Existing F-statistic estimators fail to account for any genetic correlations among individuals or subpopulations and assume that all samples are independent. This may result in inaccurate F-statistic estimations for natural populations. Here, we derive the expectations of previous F-statistics estimates using extended kinship coefficients. On this basis, we developed a new method for F-statistic estimation that accounts for non-independence of samples, finite sample sizes, and autopolyploidy. As proof of principle, using the same simulated datasets we compared the accuracy of several established F-statistic estimators with our new estimator. We found that our new method outperformed all of the other methods we used and showed almost no bias. Our new method has been added as a new function to our existing software package polygene V1.4, which is freely available at http://github.com/huangkang1987/polygene.

Linkage disequilibrium (LD) is the non-random association of alleles at different loci. Squared LD coefficients r(2) (for phased genotypes) and [Formula: see text] (for unphased genotypes) will converge to constants that are determined by the sample size, the recombination frequency, the effective population size and the mating system. LD can therefore be used for gene mapping and the estimation of effective population size. However, current methods work only with diploids. To resolve this problem, we here extend the linkage disequilibrium measures to include polysomic inheritance. We derive the values of r(2) and [Formula: see text] at equilibrium state for various mating systems and different ploidy levels. For unlinked loci, [Formula: see text] for monoecious and dioecious (with random pairing) mating systems or [Formula: see text] for dioecious mating systems (with lifetime pairing), where f is the number of females in a half-sib family and η is a constant related to the ploidy level. We simulate the application of estimating N(e) using unphased genotypes. We find that estimating N(e) in polyploids requires similar sample sizes and numbers of loci as in diploids, with the main source of bias due to using 0.5 as the recombination frequency.

In mammal herbivores, fiber digestion usually occurs predominantly in either the foregut or the hindgut. Reports of mechanisms showing synergistic function in both gut regions for the digestion of fiber and other nutrients in wild mammals are rare because it requires integrative study of anatomy, physiology, and gut microbiome. Colobine monkeys (Colobinae) are folivorous, with high-fiber foods fermented primarily in their foreguts. A few colobine species live in temperate regions, so obtaining energy from fiber during the winter is essential. However, the mechanisms enabling this remain largely unknown. We hypothesized that such species possess specialized mechanisms to enhance fiber digestion in the hindgut and studied microbial and morphological digestive adaptations of golden snub-nosed monkeys (GSMs), Rhinopithecus roxellana. which is a temperate forest colobine from central China that experiences high-thermal-energy demands while restricted to a fibrous, low-energy winter diet. We tested for synergistic foregut and hindgut fiber digestion using comparisons of morphology, microbiome composition and function, and digestive efficiency. We found that the GSM colon has a significantly greater volume than that of other foregut-fermenting colobines. The microbiomes of the foregut and hindgut differed significantly in composition and abundance. However, while digestive efficiency and the expression of microbial gene functions for fiber digestion were higher in the foregut than in the hindgut, both gut regions were dominated by microbial taxa producing enzymes to enable active digestion of complex carbohydrates. Our data suggest that both the GSM foregut and hindgut facilitate fiber digestion and that an enlarged colon is likely an adaptation to accommodate high throughput of fiber-rich food during winter.

Duplicated loci, for example those associated with major histocompatibility complex (MHC) genes, often have similar DNA sequences that can be coamplified with a pair of primers. This results in genotyping difficulties and inaccurate analyses. Here, we present a method to assign alleles to different loci in amplifications of duplicated loci. This method simultaneously considers several factors that may each affect correct allele assignment. These are the sharing of identical alleles among loci, null alleles, copy number variation, negative amplification, heterozygote excess or heterozygote deficiency, and linkage disequilibrium. The possible multilocus genotypes are extracted from the alleles for each individual and weighted to estimate the allele frequencies. The likelihood of an allele configuration is calculated and is optimized with a heuristic algorithm. Monte-Carlo simulations and three empirical MHC data sets are used as examples to evaluate the efficacy of our method under different conditions. Our new software, mhc-typer V1.1, is freely available at https://github.com/huangkang1987/mhc-typer.

Natal dispersals are male-biased in most Old World monkey species, especially those that are polygynous. We examined patterns of male dispersal in golden snub-nosed monkeys, in which male offspring mainly disperse as juveniles from their natal one-male units (OMUs) to a bachelor group. However, out of a total of 112 male dispersals from 2001–2016, we documented six cases in which male offspring remained in their natal OMU until sub-adulthood. Based on monitoring of dispersal dynamics, we found that male offspring were more likely to delay dispersal if the tenure of leader males was lengthy. Social network analysis showed male offspring who delayed dispersal exhibited strong social relationships with each other and their OMU leader male but not with the females of their OMU. Our results suggest that the length of leader male tenure may affect natal male dispersal due to benefits to leader males accrued via intrasexual competition and kin selection.

Background: In mammal herbivores, the digestion of fiber usually occurs predominantly in either the foregut or in the hindgut. However, how both gut regions function synergistically in the digestion of fiber and other nutrients has rarely been reported in wild mammals. This requires an integrative study of host anatomy, physiology and gut microbiome. Colobine monkeys (Colobinae) are folivorous, with fiber fermentation primarily occurring in the foregut, with residual fermentation in the hindgut. For the few colobine species that live in temperate regions obtaining energy from fiber during winter is critical but the mechanisms enabling this remain unclear. Results: We studied microbial and morphological digestive adaptations of golden snub-nosed monkeys (GSMs), Rhinopithecus roxellana, a temperate forest colobine from central China. We tested for synergistic foregut and hindgut fiber digestion in a species that experiences high thermal energy demands while restricted to a fibrous, low-energy winter diet. We found that the GSM’s colon has a significantly greater volume than that of other foregut fermenting colobines, and both gut regions of GSMs are dominated by microbial taxa producing enzymes to enable active digestion of complex carbohydrates. The microbiomes of the fore- and hindgut differed significantly in composition and abundance. Although the expression of microbial gene functions for fiber digestion were higher in the foregut than in the hindgut, our microbiome analysis in conjunction with that for morphology, enzyme activity and fiber-protein digestion, suggests complementary fiber and protein metabolism in both gut regions. Conclusions: Our results support that both the GSM fore- and hindgut facilitate fiber digestion, with an enlarged colon consistent as an adaptation to accommodate high throughput of fiber-rich food during winter.

The influence of genetic drift on linkage disequilibrium in finite populations has been extensively studied in diploids. However, to date the effects of ploidy on LD has not been extensively studied. We here extend the linkage disequilibrium measureDand Burrow’s Δ statistic to include polysomic inheritance, as well as their corresponding squared correlation coefficientsr2and, where the former is for phased genotypes and the latter for unphased genotypes. Weir & Hill’s double non-identity framework is also extended to include polysomic inheritance, and the expressions of double non-identity coefficients are derived under five mating systems. On this basis, the approximated expectations of estimatedr2andat equilibrium state,d2andδ2, are derived under five mating systems. We assess the behaviors of the estimatedr2andand the influence of the recombination rate ond2orδ2, simulate the application of estimating effective population size, and evaluate the statistical performance of the method of estimating.

Polyploidy poses several problems for parentage analysis. We present a new polysomic inheritance model for parentage analysis based on genotypes or allelic phenotypes to solve these problems. The effects of five factors are simultaneously accommodated in this model: (i) double-reduction, (ii) null alleles, (iii) negative amplification, (iv) genotyping errors and (v) self-fertilization. To solve genotyping ambiguity (unknown allele dosage), we developed a new method to establish the likelihood formulas for allelic phenotype data and to simultaneously include the effects of our five chosen factors. We then evaluated and compared the performance of our new method with three established methods by using both simulated data and empirical data from the cultivated blueberry (Vaccinium corymbosum). We also developed and compared the performance of two additional estimators to estimate the geno-typing error rate and the sample rate. We make our new methods freely available in the software packagepolygene, athttp://github.com/huangkang1987/polygene.

The analysis of molecular variance (AMOVA) is a widely used statistical method in population genetics and molecular ecology. The classic framework of AMOVA only supports haploid and diploid data, in which the number of hierarchies ranges from two to four. In practice, natural populations can be classified into more hierarchies, and polyploidy is frequently observed in extant species. The ploidy level may even vary within the same species, and/or within the same individual. We generalized the framework of AMOVA such that it can be used for any number of hierarchies and any level of ploidy. Based on this framework, we present four methods to account for data that are multilocus genotypic and allelic phenotypic (with unknown allele dosage). We use simulated datasets and an empirical dataset to evaluate the performance of our framework. We make freely available our methods in a new software package, polygene, which is freely available at https://github.com/huangkang1987/polygene.

Parentage analysis is an important method that is used widely in zoological and ecological studies. Current mathematical models of parentage analyses usually assume that a population has a uniform genetic structure and that mating is panmictic. In a natural population, the geographic or social structure of a population, and/or nonrandom mating, usually leads to a genetic structure and results in genotypic frequencies deviating from those expected under the Hardy-Weinberg equilibrium (HWE). In addition, in the presence of null alleles, an observed genotype represents one of several possible true genotypes. The true father of a given offspring may thus be erroneously excluded in parentage analyses, or may have a low or negative LOD score. Here, we present a new mathematical model to estimate parentage that includes simultaneously the effects of inbreeding, null alleles, and negative amplification. The influences of these three factors on previous model are evaluated by Monte-Carlo simulations and empirical data, and the performance of our new model is compared under controlled conditions. We found that, for both simulated and empirical data, our new model outperformed other methods in many situations. We make available our methods in a new, free software package entitled parentage. This can be downloaded via http://github.com/huangkang1987/parentage.

Sexually selected ornaments in animals are costly, with parasitism often affecting the degree to which they are expressed. Male Calopteryx splendens damselflies exhibit melanised ‘wing spots’. Those possessing large spots are favoured by females but also have an increased likelihood of being attacked by Calopteryx virgo, a common sympatric competitor. Melanin is used to produce the wing spots, but it is also used in immune defence against parasites that commonly infect damselflies. A total of 261 C. splendens males were collected from 26 Finnish and Latvian populations, of which half were found to be sympatric with C. virgo. It was found that males which originated from populations in which eugregarine parasites were present had smaller wing spots than individuals from parasite-free populations. Contrary to previous studies, the wing spots of C. splendens males in populations sympatric with C. virgo were not found to be smaller than those in allopatric populations. Parasite presence in C. splendens was fo...

Objectives Social animals often have dominance hierarchies, with high rank conferring preferential access to resources. In primates, competition among males is often assumed to occur predominantly over reproductive opportunities. However, competition for food may occur during food shortages, such as in temperate species during winter. Higher-ranked males may thus gain preferential access to high-profitability food, which would enable them to spend longer engaged in activities other than feeding. Materials and methods We performed a field experiment with a breeding band of golden snub-nosed monkeys, a species that lives in a multi-level society in high-altitude forests in central China. We provisioned monkey's high-profitability food during winter when natural foods are limited, and then recorded the times individual adult males spent engaged in different behaviors. Results Higher-ranking males spent less time feeding overall and fed on provisioned foods at a higher rate than lower-ranking males. Higher-ranking males therefore had more time to spend on alternative behaviors. We found no significant difference according to rank in times spent moving or resting. However, high-ranking males spend significantly longer on affiliative behaviors with other members of their social sub-units, especially grooming and being groomed, behaviors known to promote social cohesion in primates. Discussion We show that preferential access to high-profitability foods likely relaxes time-budget constraints to higher-ranking males. High-ranking males thus spend more time on non-feeding activities, especially grooming, which may enhance social cohesion within their social sub-unit. We discuss the potential direct and indirect benefits to high-ranking males associated with preferential access to high-value food during winter.

Fig-pollinating wasps (Agaonidae) only reproduce within fig tree inflorescences (figs). Agaonid offspring sex ratios are usually female-biased and often concur with local mate competition theory (LMC). LMC predicts less female-bias when several foundresses reproduce in a fig due to reduced relatedness among intra-sexually competing male offspring. Clutch size, the offspring produced by each foundress, is a strong predictor of agaonid sex ratios and correlates negatively with foundress number. However, clutch size variation can result from several processes including egg load (eggs within a foundress), competition among foundresses and oviposition site limitation, each of which can be used as a sex allocation cue. We introduced into individual Ficus racemosa figs single Ceratosolen fusciceps foundresses and allowed each to oviposit from zero to five hours thus variably reducing their eggs-loads and then introduced each wasp individually into a second fig. Offspring sex ratio (proportion males) in second figs correlated negatively with clutch size, with males produced even in very small clutches. Ceratosolen fusciceps lay mainly male eggs first and then female eggs. Our results demonstrate that foundresses do not generally lay or attempt to lay a 'fixed' number of males, but do 'reset to zero' their sex allocation strategy on entering a second fig. With decreasing clutch size, gall failure increased, probably due to reduced pollen. We conclude that C. fusciceps foundresses can use their own egg loads as a cue to facultatively adjust their offspring sex ratios and that foundresses may also produce more 'insurance' males when they can predict increasing rates of offspring mortality.

Premise Rosebay willowherb, or fireweed (Chamerion angustifolium: Onagraceae), has diploid, tetraploid, and hexaploid cytotypes. There are known physiological and ecological differences among the three cytotypes, but genetic differences remain undetermined. We developed simple sequence repeat (SSR) markers for this species. Methods and Results Leaf samples were collected from three hexaploid C. angustifolium populations. We successfully amplified 16 SSR loci, which were found to be highly polymorphic. The number of alleles, the observed heterozygosity levels, and the expected heterozygosity levels ranged from four to 13, 0.286-0.899, and 0.372-0.871, respectively. Most primers could also be amplified successfully in C. conspersum and the closely related species Epilobium palustre. Conclusions The 16 polymorphic SSR markers developed here will be useful for genetic studies in C. angustifolium and related species.

The analysis of molecular variance (AMOVA) is a widely used statistical model in the studies of population genetics and molecular ecology. The classical framework of AMOVA only supports haploid and diploid data, in which the number of hierarchies ranges from two to four. In practice, natural populations can be classified into more hierarchies, and polyploidy is frequently observed in contemporary species. The ploidy level may even vary within the same species, even within the same individual. We generalized the framework of AMOVA such that it can be used for any number of hierarchies and any level of ploidy. Based on this framework, we present four methods to account for the multilocus genotypic and allelic phenotypic data. We use simulated datasets and an empirical dataset to evaluate the performance of our framework. We make freely available our methods in a software, POLYGENE, which is freely available athttps://github.com/huangkang1987/.

The collapse of mutualisms owing to anthropogenic changes is contributing to losses of biodiversity. Top predators can regulate biotic interactions between species at lower trophic levels and may contribute to the stability of such mutualisms, but they are particularly likely to be lost after disturbance of communities. We focused on the mutualism between the fig tree Ficus microcarpa and its host-specific pollinator fig wasp and compared the benefits accrued by the mutualists in natural and translocated areas of distribution. Parasitoids of the pollinator were rare or absent outside the natural range of the mutualists, where the relative benefits the mutualists gained from their interaction were changed significantly away from the plant's natural range owing to reduced seed production rather than increased numbers of pollinator offspring. Furthermore, in the absence of the negative effects of its parasitoids, we detected an oviposition range expansion by the pollinator, with the use of a wider range of ovules that could otherwise have generated seeds. Loss of top-down control has therefore resulted in a change in the balance of reciprocal benefits that underpins this obligate mutualism, emphasizing the value of maintaining food web complexity in the Anthropocene.

Polyploids are organisms whose genomes consist of more than two complete sets of chromosomes. Both autopolyploids and allopolyploids may display polysomic inheritance. A peculiarity of polysomic inheritance is multivalent formation during meiosis resulting in double-reduction, which occurs when sister chromatid fragments are segregated into the same gamete. Double-reduction can result in gametes carrying identical-by-descent alleles and slightly increasing homozygosity. This will cause the genotypic frequencies to deviate from expected values and will thus bias the results of standard population genetic analytical methods used in molecular ecology and selective breeding. In this study, we extend existing double-reduction models to account for any even level of ploidy, and derive the symbolic expressions for genotypic frequencies via two methods. Inbreeding coefficients and heterozygosity under double-reduction and inbreeding are also calculated. Numerical solutions obtained by computer simulations are compared with analytical solutions predicted by the model to validate the model.

Premise Rosebay willowherb, or fireweed (Chamerion angustifolium: Onagraceae), has diploid, tetraploid, and hexaploid cytotypes. There are known physiological and ecological differences among the three cytotypes, but genetic differences remain undetermined. We developed simple sequence repeat (SSR) markers for this species. Methods and Results Leaf samples were collected from three hexaploid C. angustifolium populations. We successfully amplified 16 SSR loci, which were found to be highly polymorphic. The number of alleles, the observed heterozygosity levels, and the expected heterozygosity levels ranged from four to 13, 0.286–0.899, and 0.372–0.871, respectively. Most primers could also be amplified successfully in C. conspersum and the closely related species Epilobium palustre. Conclusions The 16 polymorphic SSR markers developed here will be useful for genetic studies in C. angustifolium and related species.

Habitat loss via human activity has fragmented populations of the golden snub-nosed monkey (Rhinopithecus roxellana), and thus affected patterns of gene flow. We investigated in-depth a single troop in the Qinling Mountains, central China, two major histocompatibility complex (MHC) class II loci, DQA1 and DQB1, and compared the resulting data with data from troops from the wider Qinling Mountains region. We found that a novel DQB1 allele was only present in the study troop and relatively few divergent alleles at the DQA1 and DQB1 loci were present compared with the wider population. The inbreeding coefficient (Fis) at the MHC region was lower than previous measurements, which may have reflected different sampling strategies. However, R. roxellana has relatively high diversity in MHC genes, even though it has probably experienced serious past population bottlenecks and reduced gene flow between populations. We also found that some alleles present in the wider population had been lost in the study troop, and suggest that conservation management strategies be implemented to increase gene flow between troops in order to increase genetic variation.

A significant portion of plant species are polyploids, with ploidy levels sometimes varying among individuals and/or populations. Current techniques to determine the individual ploidy, e.g., flow cytometry, chromosome counting or genotyping-by-sequencing, are often cumbersome. Based on the genotypic probabilities for polysomic inheritance under double-reduction, we developed a model to estimate allele frequency and infer the ploidy status of individuals from the allelic phenotypes of codominant genetic markers. The allele frequencies are estimated by an expectation-maximization algorithm in the presence of null alleles, false alleles, negative amplifications and self-fertilization, and the posterior probabilities are used to assign individuals into different levels of ploidy. The accuracy of this method under different conditions is evaluated. Our methods are freely available in a new software package, ploidyinfer, for use by other researchers which can be downloaded from http://github.com/huangkang1987/ploidyinfer.

In some insect nursery pollination mutualisms, plant hosts impose net costs to uncooperative "cheater" symbionts. These "sanctions" promote mutualism stability but their precise adaptive nature remains unclear. In fig-wasp mutualisms host trees (Ficus spp.) are only pollinated by female agaonid wasps whose larvae only use galled fig flowers as food. In actively pollinated systems, if wasps fail to pollinate, sanctions can result via fig abortion, killing all wasp offspring, or by increased offspring mortality within un-aborted figs. These sanctions result from selective investment to pollinated inflorescences, a mechanism present in almost all angiosperms. To more fully understand how selective investment functions as sanctions requires the measurement of variation in their costs and benefits to both hosts and symbionts. Gynodioecious fig-tree-fig-wasp mutualisms are particularly suitable for this because pollen and wasps are produced only in the figs of "male" trees and seeds only in the figs of "female" trees. Male and female trees thus incur different net costs of pollen absence, and costs of sanctions to pollen-free "cheater" wasps only occur in male trees. We used the actively pollinated host tree Ficus hispida and introduced into male and female figs either 1, 3, 5, 7, or 9 all pollen-laden "cooperative" (P+) or all pollen-free "cheater" (P-) wasps. Abortion in both male and female trees was highest in P- figs, with P- fig abortion higher in females (~90%) than in males (~40%). Fig abortion was negatively associated with foundress number mainly in P+ figs; in P- figs abortion was only weakly associated with the number of "cheater" wasps, especially in female figs. In un-aborted male figs, wasp offspring mortality was higher in P- figs than in P+ figs, and in P- figs correlated positively with foundress (cheater) number. Increased offspring mortality was biased against female wasp offspring and likely resulted from reduced larval nutrition in unpollinated flowers. Variation in selective investment to P- figs thus reflects costs and benefits of pollen absence/presence to hosts, variation that translates directly to net costs to cheater wasps.

Different types of mutualisms may interact, co-evolve and form complex networks of interdependences, but how species interact in networks of a mutualistic community and maintain its stability remains unclear. In a mutualistic network between treehoppers-weaver ants and fig-pollinating wasps, we found that the cuticular hydrocarbons of the treehoppers are more similar to the surface chemical profiles of fig inflorescence branches (FIB) than the cuticular hydrocarbons of the fig wasps. Behavioral assays showed that the cuticular hydrocarbons from both treehoppers and FIBs reduce the propensity of weaver ants to attack treehoppers even in the absence of honeydew rewards, suggesting that chemical camouflage helps enforce the mutualism between weaver ants and treehoppers. High levels of weaver ant and treehopper abundances help maintain the dominance of pollinating fig wasps in the fig wasp community and also increase fig seed production, as a result of discriminative predation and disturbance by weaver ants of ovipositing non-pollinating fig wasps (NPFWs). Ants therefore help preserve this fig-pollinating wasp mutualism from over exploitation by NPFWs. Our results imply that in this mutualistic network chemical camouflage plays a decisive role in regulating the behavior of a key species and indirectly shaping the architecture of complex arthropod-plant interactions.

Aggressive contests between animals are common but rarely result in death because the benefits of winning a fight rarely exceed the cost of losing. Lethal combat can evolve, however, when the contested resource translates to much of the future reproductive success of each combatant. Female agaonid fig wasps pollinate and lay their eggs in the flowers within the enclosed inflorescences (“figs”) of fig trees (Ficus spp.). Wasps rarely leave the first fig entered so the reproductive success of each “foundress” usually depends on the availability of flowers within a single fig. We report for the first time lethal combat between female agaonids, in the undescribed Pegoscapus sp. that pollinates Ficus citrifolia in southeastern Brazil. In staged dyadic contests, wasps showed no aggression outside or inside the fig until one foundress oviposited. The first wasp to oviposit then became aggressive, which usually resulted in the death of its competitor. Examination of dead foundresses in naturally occurring figs showed that injuring competitors, particularly through decapitation, was effective at reducing their oviposition rates. In a Panamanian F. citrifolia population, pollinated by another wasp species, Pegoscapus tonduzi , there was little aggression between foundresses in similar contests. We suggest that reduced aggression in P. tonduzi reflects less competition for resources essential for successful reproduction due to on average fewer foundresses per flower in Panama.

Estimates of relatedness coefficients, based on genetic marker data, are often necessary for studies of genetics and ecology. Whilst many estimates based on method-of-moment or maximum-likelihood methods exist for diploid organisms, no such estimators exist for organisms with multiple ploidy levels, which occur in some insect and plant species. Here, we extend five estimators to account for different levels of ploidy: one relatedness coefficient estimator, three coefficients of coancestry estimators and one maximum-likelihood estimator. We use arrhenotoky (when unfertilized eggs develop into haploid males) as an example in evaluations of estimator performance by Monte Carlo simulation. Also, three virtual sex-determination systems are simulated to evaluate their performances for higher levels of ploidy. Additionally, we used two real data sets to test the robustness of these estimators under actual conditions. We make available a software package, PolyRelatedness, for other researchers to apply to organisms that have various levels of ploidy.

A small number of primate species including snub-nosed monkeys (colobines), geladas (papionins) and humans live in multilevel societies (MLSs), in which multiple one-male polygamous units (OMUs) coexist to form a band, and non-breeding males associate in bachelor groups. Phylogenetic reconstructions indicate that the papionin MLS appears to have evolved through internal fissioning of large mixed-sex groups, whereas the colobine MLS evolved through the aggregation of small, isolated OMUs. However, how agonistic males maintain tolerance under intensive competition over limited breeding opportunities remains unclear. Using a combination of behavioural analysis, satellite telemetry and genetic data, we quantified the social network of males in a bachelor group of golden snub-nosed monkeys. The results show a strong effect of kinship on social bonds among bachelors. Their interactions ranged from cooperation to agonism, and were regulated by access to mating partners. We suggest that an ‘arms race’ between breeding males' collective defence against usurpation attempts by bachelor males and bachelor males' aggregative offence to obtain reproductive opportunities has selected for larger group size on both sides. The results provide insight into the role that kin selection plays in shaping inter-male cohesion which facilities the evolution of multilevel societies. These findings have implications for understanding human social evolution, as male–male bonds are a hallmark of small- and large-scale human societies.

Endophytic insects and their parasitoids provide valuable models for community ecology. The wasp communities in inflorescences of fig trees have great potential for comparative studies, but we must first describe individual communities. Here, we add to the few detailed studies of such communities by describing the one associated with Ficus rubiginosa in Australia. First, we describe community composition, using two different sampling procedures. Overall, we identified 14 species of non-pollinating fig wasp (NPFW) that fall into two size classes. Small wasps, including pollinators, gallers and their parasitoids, were more abundant than large wasps (both galler and parasitoid species). We show that in figs where wasps emerge naturally, the presence of large wasps may partly explain the low emergence of small wasps. During fig development, large gallers oviposit first, before and around the time of pollination, while parasitoids lay eggs after pollination. We further show that parasitoids in the subfamily Sycoryctinae, which comprise the majority of all individual NPFWs, segregate temporally by laying eggs at different stages of fig development. We discuss our results in terms of species co-existence and community structure and compare our findings to those from fig wasp communities on other continents.

In some mutualisms, cooperation in symbionts is promoted by hosts sanctioning "cheats," who obtain benefits but fail to reciprocate. In fig-wasp mutualisms, agaonid wasps pollinate the trees (Ficus spp.), but are also exploitative by using some flowers as larval food. Ficus can sanction cheats that fail to pollinate by aborting some un-pollinated figs. However, in those un-pollinated figs retained by trees, cheats successfully reproduce. When this occurs, wasp broods are reduced, suggesting sanctions increase offspring mortality within un-pollinated figs. We investigated sanction mechanisms of abortion and larval mortality against wasp cheats in the monoecious Ficus racemosa by introducing into figs 1, 3, 5, 7, or 9 female wasps (foundresses) that were either all pollen-laden (P+) or all pollen-free (P-). The abortion rates of P- figs were highest (-60%) when single foundresses were present. Abortion declined with increased foundresses and ceased with seven or more wasps present, irrespective of pollination. In un-aborted figs, wasp fitness (mean offspring per foundress) declined as foundress number increased, especially in P- figs. Reduced broods in P- figs resulted from increased larval mortality of female offspring as foundress number increased, resulting in more male-biased sex ratios. Overall sanctions estimated from both abortion rates and reduced offspring production strengthened as the number of cheats increased. In a second experiment, we decoupled pollination from wasp oviposition by introducing one pollen-free foundress, followed 24 h later by seven pollen-laden ovipositor-excised wasps. Compared with P+ and P- single-foundress figs, delayed pollination resulted in intermediate larval mortality and wasp fitness, which concurred with patterns of female offspring production. We conclude that fig abortion reflects both pollinator numbers and pollen presence. Sanctions within P- figs initiate soon after oviposition and discriminate against female offspring, thus reducing the benefits to cheats from adaptively biasing their offspring sex ratios. We suggest that costs to cheats via these discriminative sanctions are likely to promote stability in this mutualism.

The mutualism between fig trees and their wasp pollinators is a model system for many ecological and evolutionary studies. However, the immature stages of pollinating fig wasps have rarely been studied. We monitored developing fig wasps of known ages and performed a series of dissections at 24 h intervals to identify key developmental traits of Ceratosolen solmsi marchali Mayr (Hymenoptera: Agaonidae), a pollinator of Ficus hispida L. (Moraceae). We identified where in the Ficus ovary eggs were deposited and time to hatch. We were also able to identify the timing and key underlying characters of five larval instars, three sub-pupal stages, and a single prepupal stage. We provide detailed morphological descriptions for the key stages and report some behavioral observations of the wasps in the several developmental stages we recorded. Scanning electron microscope images were taken.

Environmental barriers and habitat fragmentation can restrict gene flow, leading to genetic divergence among animal populations. The golden snub-nosed monkey, Rhinopithecus roxellana, is endemic to China, and ranges across 4 provinces. However, over the past 40 years its populations have become fragmented. We investigated the genetic diversity, demographic history and population structure of R. roxellana in 5 reserves in one of its strongholds, the Qinling Mountain forests of Shaanxi. We collected genetic material from 11 monkey bands (a group of individuals containing multiple 1-male units) with a total of 428 samples genotyped at 20 microsatellite loci. Allelic richness and heterozygosity suggested a relatively high level of intra-band genetic diversity. We found no evidence of any genetic bottleneck in these R. roxellana populations. AMOVA and Bayesian cluster analysis revealed that R. roxellana in the 5 reserves are highly structured and form at least 3 distinct subpopulations. These subpopulations concur with major topographical features in the study area, such as mountain ridges, suggesting that dispersal of R. roxellana may be restricted by geographical barriers.

We report evidence that helps resolve two competing explanations for stability in the mutualism between Ficus racemosa fig trees and the Ceratosolen fusciceps wasps that pollinate them. The wasps lay eggs in the tree's ovules, with each wasp larva developing at the expense of a fig seed. Upon maturity, the female wasps collect pollen and disperse to a new tree, continuing the cycle. Fig fitness is increased by producing both seeds and female wasps, whereas short-term wasp fitness increases only with more wasps, thereby resulting in a conflict of interests. We show experimentally that wasps exploit the inner layers of ovules first (the biased oviposition explanation), which is consistent with optimal-foraging theory. As oviposition increases, seeds in the middle layer are replaced on a one-to-one basis by pollinator offspring, which is also consistent with biased oviposition. Finally, in the outer layer of ovules, seeds disappear but are only partially replaced by pollinator offspring, which suggests high wasp mortality (the biased survival or ‘unbeatable seeds’ explanation). Our results therefore suggest that both biased oviposition and biased survival ensure seed production, thereby stabilizing the mutualism. We further argue that biased oviposition can maintain biased survival by selecting against wasp traits to overcome fig defenses. Finally, we report evidence suggesting that F. racemosa balances seed and wasp production at the level of the tree. Because figs are probably selected to allocate equally to male and female function, a 1:1 seed:wasp ratio suggests that fig trees are in control of the mutualism.

Fig trees (Ficus spp.) are pollinated by tiny wasps that enter their enclosed inflorescences (syconia). The wasp larvae also consume some fig ovules, which negatively affects seed production. Within syconia, pollinator larvae mature mostly in the inner ovules whereas seeds develop mostly in outer ovules—a stratification pattern that enables mutualism persistence. Pollinators may prefer inner ovules because they provide enemy-free space from externally ovipositing parasitic wasps. In some Australasian Ficus, this results in spatial segregation of pollinator and parasite offspring within syconia, with parasites occurring in shorter ovules than pollinators. Australian figs lack non-pollinating fig wasps (NPFW) that enter syconia to oviposit, but these occur in Africa and Asia, and may affect mutualist reproduction via parasitism or seed predation. We studied the African fig, F. burkei, and found a similar general spatial pattern of pollinators and NPFWs within syconia as in Australasian figs. However, larvae of the NPFW Philocaenus barbarus, which enters syconia, occurred in inner ovules. Philocaenus barbarus reduced pollinator abundance but not seed production, because its larvae replaced pollinators in their favoured inner ovules. Our data support a widespread role for NPFWs in contributing to factors preventing host overexploitation in fig-pollinator mutualisms.

Lifetime reproductive success in female insects is often egg- or time-limited. For instance in pro-ovigenic species, when oviposition sites are abundant, females may quickly become devoid of eggs. Conversely, in the absence of suitable oviposition sites, females may die before laying all of their eggs. In pollinating fig wasps (Hymenoptera: Agaonidae), each species has an obligate mutualism with its host fig tree species [Ficus spp. (Moraceae)]. These pro-ovigenic wasps oviposit in individual ovaries within the inflorescences of monoecious Ficus (syconia, or ‘figs’), which contain many flowers. Each female flower can thus become a seed or be converted into a wasp gall. The mystery is that the wasps never oviposit in all fig ovaries, even when a fig contains enough wasp females with enough eggs to do so. The failure of all wasps to translate all of their eggs into offspring clearly contributes to mutualism persistence, but the underlying causal mechanisms are unclear. We found in an undescribed Brazilian Pegoscapus wasp population that the lifetime reproductive success of lone foundresses was relatively unaffected by constraints on oviposition. The number of offspring produced by lone foundresses experimentally introduced into receptive figs was generally lower than the numbers of eggs carried, despite the fact that the wasps were able to lay all or most of their eggs. Because we excluded any effects of intraspecific competitors and parasitic non-pollinating wasps, our data suggest that some pollinators produce few offspring because some of their eggs or larvae are unviable or are victims of plant defences.

Character displacement is a process by which interactions between two species that exhibit similar traits, results in geographical patterns of trait divergence in one or both species. These traits evolve to reduce costs of interspecific interactions in sympatry and thus differ from their condition in allopatry. In male damselflies Calopteryx splendens, large wing spots are sexually selected. However, in sympatric populations with Calopteryx virgo, wing spot size decreases as C. virgo abundance increases. The stability of this pattern is unclear, because previous studies have focused on sympatric populations with potentially fluctuating relative abundances. We studied the wing spot sizes of C. splendens in both sympatric and allopatric populations. Our data show that male C. splendens’ wing spots are larger in allopatry than in sympatry with C. virgo. We suggest that both interspecific aggression and avoidance of interspecific reproductive interactions may result in this pattern, although their relative importance remains unclear.

Understanding the factors that enable mutualisms to evolve and to subsequently remain stable over time, is essential to fully understand patterns of global biodiversity and for evidence based conservation policy. Theoretically, spatial heterogeneity of mutualists, through increased likelihood of fidelity between cooperative partners in structured populations and ‘self-restraint’ of symbionts, due to selection against high levels of virulence leading to short-term host overexploitation, will result in either a positive correlation between the reproductive success of both mutualists prior to the total exploitation of any host resource or no correlation after any host resource has been fully exploited. A quantitative review by meta-analysis on the results of 96 studies from 35 papers, showed no evidence of a significant fitness correlation between mutualists across a range of systems that captured much taxonomic diversity. However, when the data were split according to four categories of host: 1) cnidarian corals, 2) woody plants, 3) herbaceous plants and 4) insects, a significantly positive effect in corals was revealed. The trends for the remaining three categories did not significantly differ to zero. Our results suggest that stability in mutualisms requires alternative processes, or mechanisms in addition to, spatial heterogeneity of hosts and/or ‘self-restraint’ of symbionts.

Fig wasp is one of the most well known model systems in examining whether or not the parents could adjust their offspring sex ratio to maximize their gene frequency transmission in next generations. Our manipulative experiments showed that, in all of the five pollinator wasps of figs (Agaonidae) that have different averages of foundress numbers per syconium, almost the same proportions of male offspring are produced in the experiment that foundresses deposit one hour then are killed with ether (66.1%–70.1%) and over the lifespan of each foundress (14.0%–21.0%). The foundresses tend to deposit their male eggs prior to female eggs. The observed increase in the proportion of male offspring as a function of foundress number results from density-dependent interference competition among the foundresses. These results showed that the selection of gene frequency transmission through the behavioral adjustment in the evolution of sex ratio does not exist in these five fig wasps. The results here implied that genetic adjustment mechanisms of the sex ratio of fig wasps can only be triggered to be on or off and that the foundresses can not quantitatively adjust their sex ratio according to increased environmental selection pressure.