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Long life requires individuals to defend themselves against pathogens over prolonged periods of time whilst minimising damage to themselves. In vertebrates, pathogen defence is provided by two integrated systems, innate and adaptive immunity. Innate immunity is relatively nonspecific, resulting in collateral damage to hosts, and does not involve canonical immunological memory. In contrast, adaptive immunity is highly specific and confers long-lasting memory, which are features that are predicted to facilitate long life. However, there is long-standing debate over the general importance of adaptive immunity for the evolution of extended lifespans, partly because this is difficult to test. We highlight how recent improvements in whole genome assemblies open the door to immunogenomic comparative analyses that enable the coevolution of longevity and specific immune traits to be disentangled.

For over 300 million years, insects have relied on symbiotic microbes for nutrition and defence. However, it is unclear whether specific ecological conditions have repeatedly favoured the evolution of symbioses, and how this has influenced insect diversification. Here, using data on 1,850 microbe–insect symbioses across 402 insect families, we found that symbionts have allowed insects to specialize on a range of nutrient-imbalanced diets, including phloem, blood and wood. Across diets, the only limiting nutrient consistently associated with the evolution of obligate symbiosis was B vitamins. The shift to new diets, facilitated by symbionts, had mixed consequences for insect diversification. In some cases, such as herbivory, it resulted in spectacular species proliferation. In other niches, such as strict blood feeding, diversification has been severely constrained. Symbioses therefore appear to solve widespread nutrient deficiencies for insects, but the consequences for insect diversification depend on the feeding niche that is invaded.

The evolution of multicellular life has played a pivotal role in shaping biological diversity. However, we know surprisingly little about the natural environmental conditions that favour the formation of multicellular groups. Here we experimentally examine how key environmental factors (predation, nitrogen and water turbulence) combine to influence multicellular group formation in 35 wild unicellular green algae strains (19 Chlorophyta species). All environmental factors induced the formation of multicellular groups (more than four cells), but there was no evidence this was adaptive, as multicellularity (% cells in groups) was not related to population growth rate under any condition. Instead, population growth was related to extracellular matrix (ECM) around single cells and palmelloid formation, a unicellular life-cycle stage where two to four cells are retained within a mother-cell wall after mitosis. ECM production increased with nitrogen levels resulting in more cells being in palmelloids and higher rates of multicellular group formation. Examining the distribution of 332 algae species across 478 lakes monitored over 55 years, showed that ECM and nitrogen availability also predicted patterns of obligate multicellularity in nature. Our results highlight that adaptations of unicellular organisms to cope with environmental challenges may be key to understanding evolutionary routes to multicellular life.

For over 300 million years, insects have relied on symbiotic microbes for nutrition and defence1,2. However, it is unclear whether specific ecological conditions have repeatedly favoured the evolution of symbioses, and how this has influenced insect diversification1,3,4. Using data on 1849 microbe-insect symbioses across 402 insect families, we found that symbionts have allowed insects to specialize on a range of nutrient-imbalanced diets, including phloem, blood and wood. Across diets, the only limiting nutrient consistently associated with the evolution of obligate symbiosis was B vitamins. The shift to new diets, facilitated by symbionts, had mixed consequences for insect diversification. In some cases, such as herbivory, it resulted in spectacular species proliferation. In other niches, such as strict blood feeding, diversification has been severely constrained. Symbioses therefore appear to solve widespread nutrient deficiencies for insects, but the consequences for insect diversification depend on the feeding niche invaded.

Temperature has a crucial influence on the places where species can survive and reproduce. Past research has primarily focused on survival, making it unclear if temperature fluctuations constrain reproductive success, and if so whether populations harbour the potential to respond to climatic shifts. Here, using two decades of data from a large experimental breeding programme of the iconic ostrich (Struthio camelus) in South Africa, we show that the number of eggs females laid and the number of sperm males produced were highly sensitive to natural temperature extremes (ranging from −5 °C to 45 °C). This resulted in reductions in reproductive success of up to 44% with 5 °C deviations from their thermal optimum. In contrast, gamete quality was largely unaffected by temperature. Extreme temperatures also did not expose trade-offs between gametic traits. Instead, some females appeared to invest more in reproducing at high temperatures, which may facilitate responses to climate change. These results show that the robustness of fertility to temperature fluctuations, and not just temperature increases, is a critical aspect of species persistence in regions predicted to undergo the greatest change in climate volatility., Climate change may pose a challenge not only for survival of animals but also for their reproduction. Here, Schou et al. analyse how male and female ostrich fertility relates to fluctuating temperature across 20 years, finding reduced fertility away from the thermal optimum, but also individual variation in thermal tolerance.

Organisms inhabiting extreme thermal environments, such as desert birds, have evolved spectacular adaptations to thermoregulate during hot and cold conditions. However, our knowledge if and how selection acts on thermoregulation and the potential for evolutionary responses is limited, particularly for large organisms experiencing extreme temperature fluctuations. Here we use thermal imaging to quantify selection and genetic variation in thermoregulation in ostriches (Struthio camelus), the world’s largest bird species that is regularly exposed to increasingly volatile temperatures. We found that females that are better at regulating their head temperatures had higher egg-laying rates under hotter conditions. Thermoregulatory capacity was both heritable and showed signatures of local adaptation. Females originating from populations exposed to more unpredictable climates were better at regulating their head temperatures in response to temperature fluctuations. Together these results reveal that past and present evolutionary processes have shaped genetic variation in thermoregulatory capacity, which appears to protect critical organs, such as the brain, from extreme temperatures during reproduction.Impact SummaryLarge animals inhabiting extreme thermal environments, such as deserts, are predicted to be particularly vulnerable to the sustained heat waves expected in their future climate. However, previous research on the evolution of thermoregulation has focused on the effect of increasing temperatures on survival in ectotherms. Here we study the ostrich (Struthio camelus), the world’s largest bird, that inhabits some of the hottest and driest regions on Earth. We show that the ability of females to reproduce during hot conditions is associated with their thermoregulatory capacity. Furthermore, variation in thermoregulation is heritable and related to past climatic conditions: Females from populations originating from parts of Africa with more extreme temperature fluctuations were better able to thermoregulate, indicating local adaptation to different climatic conditions. Together, these results suggest that thermoregulation in this large desert bird has evolved in response to past climatic conditions, remains genetically variable and is currently under selection due to its effect on reproduction.

Cooperative breeding societies allow the costs of reproduction to be shared. However, as groups become larger, such benefits often decrease and competition increases. This is predicted to select for an optimal group size, yet variation in groups is a ubiquitous feature of cooperative breeding animals. Here we experimentally established groups (ngroups=96) of cooperative breeding ostriches, Struthio camelus, with different numbers of males and females and manipulated the potential for cooperation over incubation. There was a clear optimal group size for males. Their reproductive success was maximized in groups with four or more females and no other males, irrespective of cooperation over incubation. Conversely, female reproductive success was strongly dependent on the benefits of cooperating over incubation, being maximized in groups with either many males or many females. In intermediate sized groups, both male and female reproductive success was reduced by sexual conflict over the timing of mating and incubation. Our experiments show that sex differences in the opposing forces of cooperation and competition can explain why variation in cooperative groups is widespread.

In birds that breed cooperatively in family groups, adult offspring often delay dispersal to assist the breeding pair in raising their young. Kin selection is thought to play an important role in the evolution of this breeding system. However, evidence supporting the underlying assumption that helpers increase the reproductive success of breeders is inconsistent. In 10 out of 19 species where the effect of helpers on breeder reproductive success has been estimated while controlling for the effects of breeder and territory quality, no benefits of help were detected. Here, we use phylogenetic meta-analysis to show that the inconsistent evidence for helper benefits across species is explained by study design. After accounting for low sample sizes and the different study designs used to control for breeder and territory quality, we found that helpers consistently enhanced the reproductive success of breeders. Therefore, the assumption that helpers increase breeder reproductive success is supported by evidence across cooperatively breeding birds.

Group-living species show a diversity of social organization, from simple mated pairs to complex communities of interdependent individuals performing specialized tasks. The advantages of living in cooperative groups are well understood, but why some species breed in small aggregations while others evolve large, complex groups with clearly divided roles is unclear. We address this problem by reconstructing the evolutionary pathways to cooperative breeding across 4,730 bird species. We show that differences in the way groups form at the origin of cooperative breeding predicts the level of group complexity that emerges. Groups that originate through the retention of offspring have a clear reproductive divide with distinct breeder and helper roles. This is associated with reproductive specialization, where breeders invest more in fecundity and less in care. In contrast, groups formed through the aggregation of unrelated adults are smaller and lack specialization. These results help explain why some species have not transitioned beyond simple groups while others have taken the pathway to increased group complexity. Analysing data on group size and breeding systems of >4,700 species of birds, the authors show that complex sociality is more likely to arise in cooperative family groups than groups with unrelated individuals.

The evolutionary potential of species to cope with short-term temperature fluctuations during reproduction is critical to predicting responses to future climate change. Despite this, vertebrate research has focused on reproduction under high or low temperatures in relatively stable temperate climates. Here, we characterize the genetic basis of reproductive thermal tolerance to temperature fluctuations in the ostrich that lives in tropical and sub-tropical Africa. Both heat and cold tolerance are under selection and heritable, indicating that evolutionary responses to mean temperature change are possible. However, a negative, genetic correlation between heat and cold tolerance limits the potential for adaptation to fluctuating temperatures. Genetic constraints between heat and cold tolerance appears a crucial, yet underappreciated, factor influencing responses to climate change.One-sentence summaryReproductive success in fluctuating climates is constrained by a negative genetic correlation between heat and cold tolerance

The development of gut microbiota during ontogeny is emerging as an important process influencing physiology, immunity and fitness in vertebrates. However, knowledge of how bacteria colonize the juvenile gut, how this is influenced by changes in the diversity of gut bacteria and to what extent this influences host fitness, particularly in nonmodel organisms, is lacking. Here we used 16S rRNA gene sequencing to describe the successional development of the faecal microbiome in ostriches (Struthio camelus, n = 66, repeatedly sampled) over the first 3 months of life and its relationship to growth. We found a gradual increase in microbial diversity with age that involved multiple colonization and extinction events and a major taxonomic shift in bacteria that coincided with the cessation of yolk absorption. Comparisons with the microbiota of adults (n = 5) revealed that the chicks became more similar in their microbial diversity and composition to adults as they aged. There was a five-fold difference in juvenile growth during development, and growth during the first week of age was strongly positively correlated with the abundance of the genus Bacteroides and negatively correlated with Akkermansia. After the first week, the abundances of six phylogenetically diverse families (Peptococcaceae, S24-7, Verrucomicrobiae, Anaeroplasmataceae, Streptococcaceae, Methanobacteriaceae) were associated with subsequent reductions in chick growth in an age-specific and transient manner. These results have broad implications for our understanding of the development of gut microbiota and its associations with animal growth.

Background Imbalances in the gut microbial community (dysbiosis) of vertebrates have been associated with several gastrointestinal and autoimmune diseases. However, it is unclear which taxa are associated with gut dysbiosis, and if particular gut regions or specific time periods during ontogeny are more susceptible. We also know very little of this process in non-model organisms, despite an increasing realization of the general importance of gut microbiota for health. Methods Here, we examine the changes that occur in the microbiome during dysbiosis in different parts of the gastrointestinal tract in a long-lived bird with high juvenile mortality, the ostrich (Struthio camelus). We evaluated the 16S rRNA gene composition of the ileum, cecum, and colon of 68 individuals that died of suspected enterocolitis during the first 3 months of life (diseased individuals), and of 50 healthy individuals that were euthanized as age-matched controls. We combined these data with longitudinal environmental and fecal sampling to identify potential sources of pathogenic bacteria and to unravel at which stage of development dysbiosis-associated bacteria emerge. Results Diseased individuals had drastically lower microbial alpha diversity and differed substantially in their microbial beta diversity from control individuals in all three regions of the gastrointestinal tract. The clear relationship between low diversity and disease was consistent across all ages in the ileum, but decreased with age in the cecum and colon. Several taxa were associated with mortality (Enterobacteriaceae, Peptostreptococcaceae, Porphyromonadaceae, Clostridium), while others were associated with health (Lachnospiraceae, Ruminococcaceae, Erysipelotrichaceae, Turicibacter, Roseburia). Environmental samples showed no evidence of dysbiosis-associated bacteria being present in either the food, water, or soil substrate. Instead, the repeated fecal sampling showed that pathobionts were already present shortly after hatching and proliferated in individuals with low microbial diversity, resulting in high mortality several weeks later. Conclusions Identifying the origins of pathobionts in neonates and the factors that subsequently influence the establishment of diverse gut microbiota may be key to understanding dysbiosis and host development.

Pathogen communities can vary substantially between geographical regions due to different environmental conditions. However, little is known about how host immune systems respond to environmental variation across macro-ecological and evolutionary scales. Here, we select 37 species of songbird that inhabit diverse environments, including African and Palaearctic residents and Afro-Palaearctic migrants, to address how climate and habitat have influenced the evolution of key immune genes, the major histocompatibility complex class I (MHC-I). Resident species living in wetter regions, especially in Africa, had higher MHC-I diversity than species living in drier regions, irrespective of the habitats they occupy. By contrast, no relationship was found between MHC-I diversity and precipitation in migrants. Our results suggest that the immune system of birds has evolved greater pathogen recognition in wetter tropical regions. Furthermore, evolving transcontinental migration appears to have enabled species to escape wet, pathogen-rich areas at key periods of the year, relaxing selection for diversity in immune genes and potentially reducing immune system costs.

In some species individuals altruistically delay their chance of reproducing to help others raise their young. This is commonly referred to as cooperative breeding and is widespread across the animal kingdom, occurring in insects, crustaceans, fish, birds, and mammals, including humans (1, 2). This behavior has puzzled biologists for decades because, why should one individual help another at an expense to itself (3⇓–5)? Surely natural selection should favor individuals that maximize their own reproductive success, and not that of others (3, 4). In PNAS, Wang and Lu (6) address how altruistic and selfish strategies can stably coexist within populations, an issue that remains poorly understood in cooperative breeding animals. In the mid 1960s, the problem of how altruism can evolve was largely resolved by W. D. Hamilton, with his theory of inclusive fitness (3). Hamilton illustrated that although to the observer helping behavior appears altruistic, from a genetic perspective it is a case of a gene in one individual helping to replicate an identical copy of itself in another individual. This concept, coined Hamilton’s rule ( rb – c > 0), demonstrated that altruism evolves when the gene copies an individual passes on by helping a breeder produce extra offspring ( b : benefits), scaled by the probability that they share the gene for altruism ( r : relatedness), outweighs the gene copies it loses by not producing its own offspring ( c : costs) (3⇓–5). Hamilton’s rule has played a highly influential role in explaining why cooperative breeding occurs. Painstaking, long-term studies of marked individuals over multiple generations have allowed proxies of rb and c to be estimated in many cooperative breeding species (1, 2). This has revealed that altruistic behavior almost always arises in family groups where mature offspring from previous breeding … [↵][1]1Email: charlie.cornwallis{at}biol.lu.se. [1]: #xref-corresp-1-1

Dysbiosis in the vertebrate gut microbiome has been associated with several diseases. However, it is unclear whether particular gut regions or specific time periods during ontogeny are responsible for the development of dysbiosis, especially in non-model organisms. Here we examine the microbiome associated with dysbiosis in different parts of the gastrointestinal tract (ileum, caecum, colon) in a long-lived bird with high juvenile mortality, the ostrich. Individuals that died of gut disease (n=68) had substantially different microbial composition from age-matched controls (n=50) throughout the gut. Several taxa were associated with mortality (Enterobacteriaceae, Peptostreptococcaceae, Porphyromonadaceae,Clostridium) and some with survival (Lachnospiraceae, Ruminococcaceae, Erysipelotrichaceae,Turicibacter). Repeated faecal sampling showed that pathobionts were already present shortly after hatching and proliferated in individuals with low diversity, resulting in mortality weeks later. The factors influencing seeding of the gut microbiota may therefore be key to understanding dysbiosis and host development.

Sex differences in age-dependent mortality and reproductive success are predicted to drive the evolution of sexually dimorphic patterns of reproductive investment over life. However, this prediction has not been fully explored because it is difficult to measure primary and secondary sexual traits over the life spans of males and females. Here we studied a population of fowl, Gallus gallus, to gain longitudinal data on a sexual ornament (the comb), quantity of gametes produced, and gamete quality (sperm velocity and egg mass) of males and females. Our results reveal pronounced differences between the sexes in age-specific patterns of reproductive investment. In males, comb size decreased linearly with age, high sperm quality early in life was associated with reduced sperm quality late in life, and high sperm production was related to early death. In contrast, female comb size and egg mass were maximized at intermediate ages, and fecundity was independent of life span. Finally, the way traits were related in males did not change over life, whereas in females the association between fecundity and comb size changed from positive to negative over the lifetime of a female, indicating that aging may lead to trade-offs in investment between traits in females. These results show that males and females differ in reproductive investment with age, in terms of both the expression of individual traits and their phenotypic covariance.

In many species that raise young in cooperative groups, breeders live an exceptionally long time despite high investment in offspring production. How is this possible given the expected trade-off between survival and reproduction? One possibility is that breeders extend their lifespans by outsourcing parental care to non-reproductive group members. Having help lightens breeder workloads and the energy that is saved can be allocated to survival instead. We tested this hypothesis using phylogenetic meta-analysis across 23 cooperatively breeding bird species. We found that breeders with helpers had higher rates of annual survival than those without helpers (8% on average). Increased breeder survival was correlated with reduced investment in feeding offspring, which in turn depended on the proportion of feeding provided by helpers. Helpers had similar effects on female and male breeder survival. Our results indicate that one of the secrets to a long life is reduced investment in parental care. This appears to be a unique feature of cooperative societies with hard-working helpers. This article is part of the theme issue ‘Ageing and sociality: why, when and how does sociality change ageing patterns?’

The development of gut microbiota during ontogeny in vertebrates is emerging as an important process influencing physiology, immune system, health, and adult fitness. However, we have little knowledge of how the gut microbiome is colonised and develops in non-model organisms, and to what extent microbial diversity and specific taxa influence changes in fitness-related traits. Here, we used 16S rRNA gene sequencing to describe the successional development of the faecal microbiota in juvenile ostriches (Struthio camelus; n = 71) over their first three months of life, during which time a five-fold difference in weight was observed. We found a gradual increase in microbial diversity with age, an overall convergence in community composition among individuals, multiple colonisation and extinction events, and major taxonomic shifts coinciding with the cessation of yolk absorption. In addition, we discovered significant but complex associations between juvenile growth and microbial diversity, and identified distinct bacterial groups that had positive (Bacteroidaceae) and negative (Enterobacteriaceae, Enterococcaceae, Lactobacillaceae) correlations with the growth of individuals at specific ages. These results have broad implications for our understanding of the development of gut microbiota and its association with juvenile growth.

Although extensive research has demonstrated host-retrovirus microevolutionary dynamics, it has been difficult to gain a deeper understanding of the macroevolutionary patterns of host-retrovirus interactions. Here we use recent technological advances to infer broad patterns in retroviral diversity, evolution, and host-virus relationships by using a large-scale phylogenomic approach using endogenous retroviruses (ERVs). Retroviruses insert a proviral DNA copy into the host cell genome to produce new viruses. ERVs are provirus insertions in germline cells that are inherited down the host lineage and consequently present a record of past host-viral associations. By mining ERVs from 65 host genomes sampled across vertebrate diversity, we uncover a great diversity of ERVs, indicating that retroviral sequences are much more prevalent and widespread across vertebrates than previously appreciated. The majority of ERV clades that we recover do not contain known retroviruses, implying either that retroviral lineages are highly transient over evolutionary time or that a considerable number of retroviruses remain to be identified. By characterizing the distribution of ERVs, we show that no major vertebrate lineage has escaped retroviral activity and that retroviruses are extreme host generalists, having an unprecedented ability for rampant host switching among distantly related vertebrates. In addition, we examine whether the distribution of ERVs can be explained by host factors predicted to influence viral transmission and find that internal fertilization has a pronounced effect on retroviral colonization of host genomes. By capturing the mode and pattern of retroviral evolution and contrasting ERV diversity with known retroviral diversity, our study provides a cohesive framework to understand host-virus coevolution better.

The gut microbiome of animals is emerging as an important factor influencing ecological and evolutionary processes. A major bottleneck in obtaining microbiome data from large numbers of samples is the time-consuming laboratory procedures, specifically the isolation of DNA and generation of amplicon libraries. Recently, direct PCR kits have been developed that circumvent conventional DNA extraction steps, thereby streamlining the laboratory process by reducing preparation time and costs. However, the reliability and efficacy of the direct PCR method for measuring host microbiomes has not yet been investigated other than in humans with 454-sequencing. Here, we conduct a comprehensive evaluation of the microbial communities obtained with direct PCR and the widely used MoBio PowerSoil DNA extraction kit in five distinct gut sample types (ileum – caecum – colon – faeces – cloaca) from 20 juvenile ostriches, using 16S rRNA Illumina MiSeq sequencing. We found that direct PCR was highly comparable over a range of measures to the DNA extraction method in caecal, colon, and faecal samples. However, the two methods recovered significantly different microbiomes in cloacal, and especially ileal samples. We also sequenced 100 replicate sample pairs to evaluate repeatability during both extraction and PCR stages, and found that both methods were highly consistent for caecal, colon, and faecal samples (rs > 0.7), but had low repeatability for cloacal (rs = 0.39) and ileal (rs = −0.24) samples. This study indicates that direct PCR provides a fast, cheap, and reliable alternative to conventional DNA extraction methods for retrieving 16S data, which will aid future gut microbiome studies of animals.

Colonization and migration have a crucial effect on patterns of biodiversity, with disease predicted to play an important role in these processes. However, evidence of the effect of pathogens on broad patterns of colonization and migration is limited. Here, using phylogenetic analyses of 1,311 species of Afro-Palaearctic songbirds, we show that colonization events from regions of high (sub-Saharan Africa) to low (the Palaearctic) pathogen diversity were up to 20 times more frequent than the reverse, and that migration has evolved 3 times more frequently from African- as opposed to Palaearctic-resident species. We also found that resident species that colonized the Palaearctic from Africa, as well as African species that evolved long-distance migration to breed in the Palaearctic, have reduced diversity of key immune genes associated with pathogen recognition (major histocompatibility complex class I). These results suggest that changes in the pathogen community that occur during colonization and migration shape the evolution of the immune system, potentially by adjusting the trade-off between the benefits of extensive pathogen recognition and the costs of immunopathology that result from high major histocompatibility complex class I diversity.

The gut microbiomes of birds and other animals are increasingly being studied in ecological and evolutionary contexts. While methods for preserving samples and processing high-throughput sequence data to characterise bacterial communities have received considerable attention, there has been little evaluation of non-invasive sampling methods. Numerous studies on birds and reptiles have made inferences about gut microbiota using cloacal sampling, however, it is not known whether the bacterial community of the cloaca provides an accurate representation of the avian gut microbiome. We examined the accuracy with which cloacal swabs and faecal samples measure the microbiota in three different parts of the gastrointestinal tract (ileum, caecum, and colon) using a case study on juvenile ostriches,Struthio camelus, and high-throughput 16S rRNA sequencing. We found that faeces were significantly better than cloacal swabs in representing the bacterial community of the colon. Cloacal samples had a higher abundance of Gammaproteobacteria and fewer Clostridia relative to the gut and faecal samples. However, both faecal and cloacal samples were poor representatives of the microbial communities in the caecum and ileum. Furthermore, the accuracy of the sampling methods in measuring the abundance of different bacterial taxa was highly variable: Bacteroidetes was the most highly correlated phylum between all three gut sections and both methods, whereas colonic Actinobacteria correlated strongly only with faecal samples. This study demonstrates that sampling methods can have significant effects on the inferred gut microbiome in studies of birds. Based on our results, we recommend sampling faeces, whenever possible, as this provides the most accurate assessment of the colon microbiome. The fact that neither sampling technique portrayed the bacterial community of the ileum or the caecum illustrates the difficulty in non-invasively monitoring gut bacteria located further up in the gastrointestinal tract. These results have important implications for the interpretation of avian gut microbiome studies.

Animals living in harsh environments, where temperatures are hot and rainfall is unpredictable, are more likely to breed in cooperative groups. As a result, harsh environmental conditions have been accepted as a key factor explaining the evolution of cooperation. However, this is based on evidence that has not investigated the order of evolutionary events, so the inferred causality could be incorrect. We resolved this problem using phylogenetic analyses of 4,707 bird species and found that causation was in the opposite direction to that previously assumed. Rather than harsh environments favouring cooperation, cooperative breeding has facilitated the colonization of harsh environments. Cooperative breeding was, in fact, more likely to evolve from ancestors occupying relatively cool environmental niches with predictable rainfall, which had low levels of polyandry and hence high within-group relatedness. We also found that polyandry increased after cooperative breeders invaded harsh environments, suggesting that when helpers have limited options to breed independently, polyandry no longer destabilizes cooperation. This provides an explanation for the puzzling cases of polyandrous cooperative breeding birds. More generally, this illustrates how cooperation can play a key role in invading ecological niches, a pattern observed across all levels of biological organization from cells to animal societies.

Parasite diversity and abundance (parasite load) vary greatly among host species. However, the influence of host traits on variation in parasitism remains poorly understood. Comparative studies of parasite load have largely examined measures of parasite species richness and are predominantly based on records obtained from published data. Consequently, little is known about the relationships between host traits and other aspects of parasite load, such as parasite abundance, prevalence and aggregation. Meanwhile, understanding of parasite species richness may be clouded by limitations associated with data collation from multiple independent sources. We conducted a field study of Lake Tanganyika cichlid fishes and their helminth parasites. Using a Bayesian phylogenetic comparative framework, we tested evolutionary associations between five key host traits (body size, gut length, diet breadth, habitat complexity and number of sympatric hosts) predicted to influence parasitism, together with multiple measures of parasite load. We find that the number of host species that a particular host may encounter due to its habitat preferences emerges as a factor of general importance for parasite diversity, abundance and prevalence, but not parasite aggregation. In contrast, body size and gut size are positively related to aspects of parasite load within, but not between species. The influence of host phylogeny varies considerably among measures of parasite load, with the greatest influence exerted on parasite diversity. These results reveal that both host morphology and biotic interactions are key determinants of host-parasite associations and that consideration of multiple aspects of parasite load is required to fully understand patterns in parasitism.

Interactions between species can promote evolutionary divergence of ecological traits and social signals, a process widely assumed to generate species differences in adaptive radiation. However, an alternative view is that lineages typically interact when relatively old, by which time selection for divergence is weak and potentially exceeded by convergent selection acting on traits mediating interspecific competition. Few studies have tested these contrasting predictions across large radiations, or by controlling for evolutionary time. Thus the role of species interactions in driving broad-scale patterns of trait divergence is unclear. Here we use phylogenetic estimates of divergence times to show that increased trait differences among coexisting lineages of ovenbirds (Furnariidae) are explained by their greater evolutionary age in relation to non-interacting lineages, and that--when these temporal biases are accounted for--the only significant effect of coexistence is convergence in a social signal (song). Our results conflict with the conventional view that coexistence promotes trait divergence among co-occurring organisms at macroevolutionary scales, and instead provide evidence that species interactions can drive phenotypic convergence across entire radiations, a pattern generally concealed by biases in age.

The sterile worker castes found in the colonies of social insects are often cited as the archetypal examples of altruism in nature. The challenge to biologists has been to explain how losing the ability to mate has evolved as a superior strategy for transmitting genes into future generations. Recent theory and evidence have demonstrated that monogamy provides a unifying explanation for the evolution of altruism in family groups. However, sterile helpers have not evolved in all monogamous cooperative species. Why? Although monogamy is necessary, it is not sufficient for the evolution of sterility. Crucially, breeders also need to live longer than helpers, as this allows helpers to invest in raising full siblings for the duration of their lives. This eliminates any potential conflict over reproduction. When these two conditions, monogamy and completely overlapping generations, are met we consistently observe the evolution of sterile helpers. We explain the theory and evidence behind these ideas, and discuss the importance of ecology in predicting whether sterility will evolve using examples from social birds, mammals and insects. In doing so, we offer an explanation for the extraordinary lifespans of some cooperative species which hint at ways in which we can unlock the secrets of long life.

Malaria parasites ( Plasmodium spp.) include some of the world9s most widespread and virulent pathogens, infecting a wide array of vertebrates. Our knowledge of the molecular mechanisms these parasites use to invade and exploit hosts other than mice and primates is, however, extremely limited. How do Plasmodium adapt to individual hosts and to the immune response of hosts throughout an infection? To better understand parasite plasticity, and identify genes that are conserved across the phylogeny, it is imperative that we characterize transcriptome-wide gene expression from non-model malaria parasites in multiple host individuals. Here, we used high-throughput Illumina RNA-sequencing on blood from wild-caught Eurasian siskins experimentally infected with a clonal strain of the avian malaria parasite, Plasmodium ashfordi (lineage GRW2). By using a multi-step approach to filter out all host transcripts, we successfully assembled the blood-stage transcriptome of P. ashfordi . A total of 11 954 expressed parasite transcripts were identified, and 7 860 were annotated with protein information. We further quantified gene expression levels of all parasite transcripts across three hosts during two infection stages - peak and decreasing parasitemia. Interestingly, parasites from the same host during different infection stages displayed remarkably similar expression profiles, but show large differences across hosts. This indicates that P. ashfordi adjusts its gene expression to specific host individuals, but contrary to expectation does not markedly change expression across different stages of infection. Finally, we examined genome-wide sequence similarity between P. ashfordi and other apicomplexan species, and searched for candidate genes involved in red blood cell invasion. The majority of transcripts were most similar to the human parasite Plasmodium falciparum , and a large number of invasion genes were discovered, suggesting conserved red blood cell invasion strategies between mammalian and avian Plasmodium spp. The transcriptome of P. ashfordi and its host-specific gene expression over two infection stages advances our understanding of Plasmodium plasticity and will become a valuable resource as it allows for further studies analyzing gene evolution and comparisons of parasite gene expression.

Hamilton demonstrated that the evolution of cooperative behaviour is favoured by high relatedness, which can arise through kin discrimination or limited dispersal (population viscosity). These two processes are likely to operate with limited overlap: kin discrimination is beneficial when variation in relatedness is higher, whereas limited dispersal results in less variable and higher average relatedness, reducing selection for kin discrimination. However, most empirical work on eukaryotes has focused on kin discrimination. To address this bias, we analysed how kin discrimination and limited dispersal interact to shape helping behaviour across cooperatively breeding vertebrates. We show that kin discrimination is greater in species where the: (i) average relatedness in groups is lower and more variable; (ii) effect of helpers on breeders reproductive success is greater; and (iii) probability of helping was measured, rather than the amount of help provided. There was also an interaction between these effects with the correlation between the benefits of helping and kin discrimination being stronger in species with higher variance in relatedness. Overall, our results suggest that kin discrimination provides a route to indirect benefits when relatedness is too variable within groups to favour indiscriminate cooperation.

Arising from M. A. Nowak, C. E. Tarnita & E. O. Wilson 466, 1057-1062 (2010); Nowak et al. reply. The paper by Nowak et al. has the evolution of eusociality as its title, but it is mostly about something else. It argues against inclusive fitness theory and offers an alternative modelling approach that is claimed to be more fundamental and general, but which, we believe, has no practical biological meaning for the evolution of eusociality. Nowak et al. overlook the robust empirical observation that eusociality has only arisen in clades where mothers are associated with their full-sibling offspring; that is, in families where the average relatedness of offspring to siblings is as high as to their own offspring, independent of population structure or ploidy. We believe that this omission makes the paper largely irrelevant for understanding the evolution of eusociality.

When a female is sexually promiscuous, the ejaculates of different males compete for the fertilization of her eggs; the more sperm a male inseminates into a female, the more likely he is to fertilize her eggs. Because sperm production is limited and costly, theory predicts that males will strategically allocate sperm (1) according to female promiscuity, (2) saving some for copulations with new females, and (3) to females producing more and/or better offspring. Whether males allocate sperm in all of these ways is not known, particularly in birds where the collection of natural ejaculates only recently became possible. Here we demonstrate male sperm allocation of unprecedented sophistication in the fowl Gallus gallus. Males show status-dependent sperm investment in females according to the level of female promiscuity; they progressively reduce sperm investment in a particular female but, on encountering a new female, instantaneously increase their sperm investment; and they preferentially allocate sperm to females with large sexual ornaments signalling superior maternal investment. Our results indicate that female promiscuity leads to the evolution of sophisticated male sexual behaviour.\ud \ud

Inbreeding often depresses offspring fitness. Because females invest more than males in a reproductive event, inbreeding is expected to be more costly to mothers than fathers, creating a divergence between the reproductive interests of each sex and promoting sex-specific inbreeding strategies. Males and females may bias the probability of inbreeding by selecting copulation partners, and, in sexually promiscuous species, through male strategic sperm investment in different females and female selection of the sperm of different males. However, these processes are often difficult to study, and the way that different male and female strategies interact to determine inbreeding remains poorly understood. Here we demonstrate sex-specific, counteracting responses to inbreeding in the promiscuous red junglefowl, Gallus gallus. First, a male was just as likely to copulate with his full-sib sister as with an unrelated female. In addition, males displayed a tendency to: (i) initiate copulation faster when exposed to an unrelated female than when exposed to a sister, and (ii) inseminate more sperm into sisters than into unrelated females. Second, females retained fewer sperm following insemination by brothers, thus reducing the risk of inbreeding and counteracting male inbreeding strategies.

Quantifying the factors that predict parasite outbreak and persistence is a major challenge for both applied and fundamental biology. Key to understanding parasite prevalence and disease outbreaks is determining at what age individuals show signs of infection, and whether or not they recover. Age-dependent patterns of the infection of a host population by parasites can indicate among-individual heterogeneities in their susceptibility to, or rate of recovery from, parasite infections. Here, we present a cross-sectional study of avian malaria in a long-lived bird species, the mute swan Cygnus olor, examining age-related patterns of parasite prevalence and modelling patterns of infection and recovery. One-hundred and fifteen swans, ranging from one to nineteen years old, were screened for infection with Plasmodium, Haemoproteus and Leucocytozoon parasites. Infections with three cytochrome-b lineages of Haemoproteus were found (pooled prevalence 67%), namely WW1 (26%), which is common in passerine birds, and two new lineages closely related to WW1: MUTSW1 (25%) and MUTSW2 (16%). We found evidence for age-related infection in one lineage, MUTSW1. Catalytic models examining patterns of infection and recovery in the population suggested that infections in this population were not life-long - recovery of individuals was included in the best fitting models. These findings support the results of recent studies that suggest hosts can clear infections, although patterns of infection-related mortality in older birds remain to be studied in more detail. © 2013 The Author. Journal of Avian Biology © 2013 Nordic Society Oikos.

As the chemical environment of semen diluents can have a profound effect on sperm quality, we examined the effect of temperature and pH on the motility and viability of sperm in the ostrich. Semen was collected from four males, each male being replicated three times. Ejaculates were diluted and incubated for 10 min at 20°C and 40°C in four different buffers, temperature adjusted at pH 6, 7, 8 and 9 respectively. Average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), linearity (LIN), beat cross frequency (BCF) and amplitude of lateral displacement (ALH) were then recorded for each sample using CASA. The viability of sperm was assessed using nigrosin-eosin staining. Sperm incubated at 40°C had higher motility parameters, except for ALH. At 40°C, VAP, VSL and LIN increased with pH while VCL, BCF and ALH were higher for lower pHs. The viability of sperm was not affected by temperature but decreased at pH values>7. A pH in the neutral range appeared to yield higher quality sperm after in vitro storage at 20°C. However, the effect of different pH levels and temperatures on sperm longevity needs to be investigated further to develop viable ostrich specific diluents.

Two commonly used techniques for estimating the effect of genes on traits in wild populations are the candidate gene approach and quantitative genetic analyses. However, whether these two approaches measure the same underlying processes remains unresolved. Here, we use these two methods to test whether they are alternative or complementary approaches to understanding genetic variation in the timing of reproduction - a key trait involved in adaptation to climate change - in wild tit populations. Our analyses of the candidate gene Clock show weak correlates with timing variables in blue tits, but no association in great tits, confirming earlier results. Quantitative genetic analyses revealed very low levels of both direct (female) and indirect (male) additive genetic variation in timing traits for both species, in contrast to previous studies on these traits, and much lower than generally assumed. Hence, neither method suggests strong genetic effects on the timing of breeding in birds, and further work should seek to assess the generality of these conclusions. We discuss how differences in the genetic control of traits, species life-history and confounding environmental variables may determine how useful integrating these two techniques is to understand the phenotypic variation in wild populations.

The success of an artificial insemination program in ostriches is highly dependent on the yield of viable semen. We, therefore, tested how semen output is affected by three different collection frequencies: once every 2 d (48 h interval), daily (24 h interval), and twice a day (6 h interval). Ejaculates were collected from seven male ostriches (aged 2–4 years) for 10 consecutive days using the dummy female method. We assessed semen characteristics (sperm motility, volume, concentration, number of sperm per ejaculate and sperm viability) and male libido (the delay between the presentation of the dummy and ejaculation, and the willingness to mount the dummy). The total daily output of semen and the number of sperm were greater at the 6 h collection interval than at the 24 h or 48 h interval while sperm motility and viability were not affected. At the 6 h interval, the number of live normal sperm increased over the treatment period while the number of live abnormal sperm was reduced. Furthermore, the time that males took to mount the dummy and their willingness to copulate with the dummy were unaffected by collection frequency. Across males we observed great individual variation in both semen characteristics and libido suggesting there is the potential to increase the efficiency of semen collection by selecting superior males. These results indicate not only that two collections per day yield maximum semen output and may improve semen viability, but also that quantifying variation between males may help further increase semen collection efficiency.

The evolution of helping behaviour in species that breed cooperatively in family groups is typically attributed to kin selection alone. However, in many species, helpers go on to inherit breeding positions in their natal groups, but the extent to which this contributes to selection for helping is unclear as the future reproductive success of helpers is often unknown. To quantify the role of future reproduction in the evolution of helping, we compared the helping effort of female and male retained offspring across cooperative birds. The kin selected benefits of helping are equivalent between female and male helpers—they are equally related to the younger siblings they help raise—but the future reproductive benefits of helping differ because of sex differences in the likelihood of breeding in the natal group. We found that the sex which is more likely to breed in its natal group invests more in helping, suggesting that in addition to kin selection, helping in family groups is shaped by future reproduction.

Genes of the major histocompatibility complex (MHC) form a vital part of the vertebrate immune system and play a major role in pathogen resistance. The extremely high levels of polymorphism observed at the MHC are hypothesised to be driven by pathogen-mediated selection. Although the exact nature of selection remains unclear, three main hypotheses have been put forward; heterozygote advantage, negative frequency-dependence and fluctuating selection. Here, we report the effects of MHC genotype on survival in a cohort of semi-natural red junglefowl (Gallus gallus) that suffered severe mortality as a result of an outbreak of the disease coccidiosis. The cohort was followed from hatching until 250 days of age, approximately the age of sexual maturity in this species, during which time over 80% of the birds died. We show that on average birds with MHC heterozygote genotypes survived infection longer than homozygotes and that this effect was independent of genome-wide heterozygosity, estimated across microsatellite loci. This MHC effect appeared to be caused by a single susceptible haplotype (CD_c) the effect of which was masked in all heterozygote genotypes by other dominant haplotypes. The CD_c homozygous genotype had lower survival than all other genotypes, but CD_c heterozygous genotypes had survival probabilities equal to the most resistant homozygote genotype. Importantly, no heterozygotes conferred greater resistance than the most resistant homozygote genotype, indicating that the observed survival advantage of MHC heterozygotes was the product of dominant, rather than overdominant processes. This pattern and effect of MHC diversity in our population could reflect the processes ongoing in similarly small, fragmented natural populations.

Empirical studies of sexual traits continue to generate conflicting results, leading to a growing awareness that the current understanding of this topic is limited. Here we argue that this is because studies of sexual traits fail to encompass three important features of evolution. First, sexual traits evolve via natural selection of which sexual selection is just one part. Second, selection on sexual traits fluctuates in strength, direction and form due to spatial and temporal environmental heterogeneity. Third, phenotypic plasticity is ubiquitous and generates selection and responses to selection within and across generations. A move from purely gene-focused theories of sexual selection towards research that explicitly integrates development, ecology and evolution is necessary to break the stasis in research on sexual traits.

An important predictor of male fitness is the fertilizing efficiency of their ejaculates. Ejaculates are costly to produce and males are predicted to devote greater resources to copulations with reproductively superior females. It is well established that males allocate different numbers of sperm to ejaculates. However, less is known about how males adjust their sperm quality, which has important implications for our understanding of fertilization and the evolution of sexual strategies. Here we test in the fowl, Gallus gallus , whether males adjust their sperm velocity by differentially allocating seminal fluid to copulations with attractive and unattractive females. To disentangle the contributions of sperm and seminal fluid to sperm velocity, we separated and remixed sperm and seminal fluid from ejaculates allocated to females of different attractiveness. We show that dominant males increase the velocity of the sperm they invest in more attractive females by allocating larger ejaculates that contain seminal fluid that increases sperm velocity. Furthermore, we find weak evidence that males also allocate sperm with higher velocity, irrespective of seminal fluid, to more attractive females.

In promiscuous species, male reproductive success is determined by the interaction between the ability to access and choose females of the highest reproductive quality and, after copulation, the ability to outcompete the ejaculates of rival males. Disentangling the factors regulating the interplay between traits conferring a reproductive advantage before and after copulation is therefore crucial to understanding how sexual strategies evolve. Here we show in the fowl Gallus gallus, where social status determines copulation success, that dominant males produce more sperm than subordinates but that the quality of dominant males' sperm decreases over successive copulations, whereas that of subordinates remains constant. Experimentally manipulating male social status confirmed that ejaculate quality (the number and quality of sperm produced) was a response to the social environment rather than the result of intrinsic differences between dominant and subordinate males. We further show that dominant males responded to variation in female sexual ornamentation, which signals reproductive quality, by adjusting the number and quality of sperm they transferred, whereas subordinate males did not: they transferred ejaculates of similar quality to females with different ornament sizes. These results indicate that trade-offs between traits influencing reproductive success before and after copulation, combined with variation in social dynamics and female quality, may favor the evolution of phenotypically plastic alternative reproductive strategies.

When females copulate with multiple males, paternity is determined by the competitive ability of a male to access females and by the ability of its ejaculates to out-compete those of other males over fertilization. The relationship between the social competitiveness of a male and the fertilizing quality of its sperm has therefore crucial implications for the evolution of male reproductive strategies in response to sexual selection. Here, we present a longitudinal experimental study of the relationship between social status and sperm quality. We monitored sperm quality in socially naive male domestic fowl, Gallus gallus domesticus , before and after exposure to a social challenge which comprised two stages. In the first stage, social dominance was established in male pairs divergent in sperm quality, and in the second, social status was experimentally manipulated by re-shuffling males across pairs. We show that sperm quality fluctuates within males both before and after a social challenge. Importantly, such fluctuations followed consistently different patterns in males that displayed different levels of social competitiveness in the social challenge. In particular, following the social challenge, sperm quality dropped in males that won both contests while the sperm quality of males that lost both contests remained constant. Together, these results indicate that males of different social competitiveness are predisposed to specific patterns of fluctuations in sperm quality. These rapid within-male fluctuations may help explain the recent findings of trade-offs between male social and gametic competitive abilities and may help maintain phenotypic variability in these traits.