Systematic approaches to assessing high-temperature limits to fertility in animals.

Critical thermal limits (CTLs) gauge the physiological impact of temperature on survival or critical biological function, aiding predictions of species range shifts and climatic resilience. Two recent Drosophila species studies, using similar approaches to determine temperatures that induce sterility (thermal fertility limits [TFLs]), reveal that TFLs are often lower than CTLs and that TFLs better predict both current species distributions and extinction probability. Moreover, many studies show fertility is more sensitive at less extreme temperatures than survival (thermal sensitivity of fertility [TSF]). These results present a more pessimistic outlook on the consequences of climate change. However, unlike CTLs, TFL data are limited to Drosophila, and variability in TSF methods poses challenges in predicting species responses to increasing temperature. To address these data and methodological gaps, we propose 3 standardized approaches for assessing thermal impacts on fertility. We focus on adult obligate sexual terrestrial invertebrates but also provide modifications for other animal groups and life-history stages. We first outline a "gold-standard" protocol for determining TFLs, focussing on the effects of short-term heat shocks and simulating more frequent extreme heat events predicted by climate models. As this approach may be difficult to apply to some organisms, we then provide a standardized TSF protocol. Finally, we provide a framework to quantify fertility loss in response to extreme heat events in nature, given the limitations in laboratory approaches. Applying these standardized approaches across many taxa, similar to CTLs, will allow robust tests of the impact of fertility loss on species responses to increasing temperatures.

Longitudinal covariation of testosterone and sperm quality across reproductive stages in the zebra finch.

Birds that breed opportunistically maintain partial activation of reproductive systems to rapidly exploit environmental conditions when they become suitable for breeding. Maintaining reproductive systems outside of a breeding context is costly. For males, these costs are thought to include continual exposure to testosterone. Males of seasonally breeding birds minimise these costs by downregulating testosterone production outside of a breeding context. Opportunistically breeding birds trade off the need to rapidly initiate reproduction with the costs of elevated testosterone production. One way opportunistically breeding males could minimise these costs is through fine scale changes in testosterone production across discrete reproductive stages which have a greater or lesser requirement for active sperm production. Although spermatogenesis broadly depends on testosterone production, whether changes in testosterone levels across the reproductive stages affect sperm quality and production is unknown. Here, we measured testosterone, sperm quality, and body condition in male zebra finches at discrete stages within reproductive bouts (egg laying, incubation, nestling provisioning, and fledging) and across two consecutive reproductive events in captive male zebra finches (Taeniopygia castanotis). We also examined associations between male testosterone, sperm quality/production, body condition, and nestling body condition. We found that testosterone levels varied across discrete reproductive stages with the lowest levels during incubation and the highest following chick fledging. Testosterone levels were positively associated with sperm velocity and the proportion of motile sperm but were not associated with male body condition. We found no associations between paternal body condition, testosterone levels, or sperm traits with nestling body condition (a proxy for the reproductive quality of a male and his partner). This study is the first to show that opportunistically breeding males vary testosterone synthesis and sperm traits at discrete stages within a reproductive event.

Experimentally testing mate preference in an avian system with unidirectional bill color introgression.

Mating behavior can play a key role in speciation by inhibiting or facilitating gene flow between closely related taxa. Hybrid zones facilitate a direct examination of mating behavior and the traits involved in establishing species barriers. The long-tailed finch (Poephila acuticauda) has two hybridizing subspecies that differ in bill color (red and yellow), and the yellow bill phenotype appears to have introgressed ~350 km eastward following secondary contact. To examine the role of mate choice on bill color introgression, we performed behavioral assays using natural and manipulated bill colors. We found an assortative female mating preference for males of their own subspecies when bill color was not manipulated. However, we did not find this assortative preference in trials based on artificially manipulated bill color. This could suggest that assortative preference is not fixed entirely on bill color and instead may be based on a different trait (e.g., song) or a combination of traits, or alternatively may be due to lower statistical power alongside the bill manipulations being unconvincing to the female choosers. Intriguingly, we find a bias in the inheritance of bill color in captive bred F1 hybrid females. Previous modeling suggests that assortative mate preference and this kind of partial dominance in the underlying genes may together contribute to introgression, making the genetic architecture of bill color in this system a priority for future research.

Extensive transgressive gene expression in testis but not ovary in the homoploid hybrid Italian sparrow.

Hybridization can result in novel allelic combinations which can impact the hybrid phenotype through changes in gene expression. While misexpression in F1 hybrids is well documented, how gene expression evolves in stabilized hybrid taxa remains an open question. As gene expression evolves in a stabilizing manner, break-up of co-evolved cis- and trans-regulatory elements could lead to transgressive patterns of gene expression in hybrids. Here, we address to what extent gonad gene expression has evolved in an established and stable homoploid hybrid, the Italian sparrow (Passer italiae). Through comparison of gene expression in gonads from individuals of the two parental species (i.e., house and Spanish sparrow) to that of Italian sparrows, we find evidence for strongly transgressive expression in male Italian sparrows-2530 genes (22% of testis genes tested for inheritance) exhibit expression patterns outside the range of both parent species. In contrast, Italian sparrow ovary expression was similar to that of one of the parent species, the house sparrow (Passer domesticus). Moreover, the Italian sparrow testis transcriptome is 26 times as diverged from those of the parent species as the parental transcriptomes are from each other, despite being genetically intermediate. This highlights the potential for regulation of gene expression to produce novel variation following hybridization. Genes involved in mitochondrial respiratory chain complexes and protein synthesis are enriched in the subset that is over-dominantly expressed in Italian sparrow testis, suggesting that selection on key functions has moulded the hybrid Italian sparrow transcriptome.

Sperm Numbers as a Paternity Guard in a Wild Bird.

Sperm competition is thought to impose strong selection on males to produce competitive ejaculates to outcompete rival males under competitive mating conditions. Our understanding of how different sperm traits influence fertilization success, however, remains limited, especially in wild populations. Recent literature highlights the importance of incorporating multiple ejaculate traits and pre-copulatory sexually selected traits in analyses aimed at understanding how selection acts on sperm traits. However, variation in a male's ability to gain fertilization success may also depend upon a range of social and ecological factors that determine the opportunity for mating events both within and outside of the social pair-bond. Here, we test for an effect of sperm quantity and sperm size on male reproductive success in the red-back fairy-wren (Malurus melanocephalus) while simultaneously accounting for pre-copulatory sexual selection and potential socio-ecological correlates of male mating success. We found that sperm number (i.e., cloacal protuberance volume), but not sperm morphology, was associated with reproductive success in male red-backed fairy-wrens. Most notably, males with large numbers of sperm available for copulation achieved greater within-pair paternity success. Our results suggest that males use large sperm numbers as a defensive strategy to guard within-pair paternity success in a system where there is a high risk of sperm competition and female control of copulation. Finally, our work highlights the importance of accounting for socio-ecological factors that may influence male mating opportunities when examining the role of sperm traits in determining male reproductive success.

On the Origin and Evolution of Sperm Cells.

Sperm cells have intrigued biologists since they were first observed nearly 350 years ago by Antonie van Leeuwenhoek and Johan Ham [...].

Variation in female reproductive tract morphology across the reproductive cycle in the zebra finch.

BACKGROUND: In seasonally breeding birds, the reproductive tract undergoes a dramatic circannual cycle of recrudescence and regression, with oviduct size increasing 5-220 fold from the non-breeding to the breeding state. Opportunistically breeding birds can produce multiple clutches sequentially across an extended period in response primarily to environmental rather than seasonal cues. In the zebra finch, it has been shown that there is a significant reduction in gonadal morphology in non-breeding females. However, the scale of recrudescence and regression of reproductive tissue within a single breeding cycle is unknown and yet important to understand the cost of breeding, and the physiological readiness to breed in such flexible breeders. METHODS: We examined the reproductive tissue of breeding female zebra finches at six stages in the nesting cycle from pre-breeding to fledging offspring. We quantified the wet mass of the oviduct, the volume of the largest pre-ovulatory follicle, and the total number of pre-ovulatory follicles present on the ovary. RESULTS: Measures of the female reproductive tract were highest during nesting and laying stages and declined significantly in the later stages of the breeding cycle. Importantly, we found that the mass of reproductive tissue changes as much across a single reproductive event as that previously characterized between birds categorized as breeding and non-breeding. However, the regression of the ovary is less dramatic than that seen in seasonal breeders. This could reflect low-level maintenance of reproductive tissues in opportunistic breeders, but needs to be confirmed in wild non-breeding birds.

Sperm length variation among Afrotropical songbirds reflects phylogeny rather than adaptations to the tropical environment.

Sperm cells vary tremendously in size and shape across the animal kingdom. In songbirds (Aves: Passeri), sperm have a characteristic helical form but vary considerably in size. Most of our knowledge about sperm morphology in this group stems from studies of species in the Northern temperate zone, while little is known about the numerous species in the tropics. Here we examined sperm size in 125 Afrotropical songbird species with emphasis on the length of the major structural components (head, midpiece, flagellum), and total sperm length measured using light microscopy. Mean total sperm length varied from 51 µm to 212 µm across species. Those belonging to the Corvoidea superfamily had relatively short sperm with a small midpiece, while those of the three major Passeridan superfamilies Passeroidea, Muscicapoidea and Sylvioidea showed large interspecific variation in total sperm length and associated variation in midpiece length. These patterns are consistent with previous findings for temperate species in the same major clades. A comparative analysis with songbird species from the Northern temperate zone (N = 139) showed large overlap in sperm length ranges although certain temperate families (e.g. Parulidae, Emberizidae) typically have long sperm and certain Afrotropical families (e.g. Cisticolidae, Estrildidae) have relatively short sperm. Afrotropical and temperate species belonging to the same families showed no consistent contrasts in sperm length. Sperm length variation among Afrotropical and Northern temperate songbirds exhibits a strong phylogenetic signal with little or no evidence for any directional latitudinal effect among closely related taxa.

The Reproductive Microbiome: An Emerging Driver of Sexual Selection, Sexual Conflict, Mating Systems, and Reproductive Isolation.

All multicellular organisms host microbial communities in and on their bodies, and these microbiomes can have major influences on host biology. Most research has focussed on the oral, skin, and gut microbiomes, whereas relatively little is known about the reproductive microbiome. Here, we review empirical evidence to show that reproductive microbiomes can have significant effects on the reproductive function and performance of males and females. We then discuss the likely repercussions of these effects for evolutionary processes related to sexual selection and sexual conflict, as well as mating systems and reproductive isolation. We argue that knowledge of the reproductive microbiome is fundamental to our understanding of the evolutionary ecology of reproductive strategies and sexual dynamics of host organisms.

Molecular Diversification of the Seminal Fluid Proteome in a Recently Diverged Passerine Species Pair.

Seminal fluid proteins (SFPs) mediate an array of postmating reproductive processes that influence fertilization and fertility. As such, it is widely held that SFPs may contribute to postmating, prezygotic reproductive barriers between closely related taxa. We investigated seminal fluid (SF) diversification in a recently diverged passerine species pair (Passer domesticus and Passer hispaniolensis) using a combination of proteomic and comparative evolutionary genomic approaches. First, we characterized and compared the SF proteome of the two species, revealing consistencies with known aspects of SFP biology and function in other taxa, including the presence and diversification of proteins involved in immunity and sperm maturation. Second, using whole-genome resequencing data, we assessed patterns of genomic differentiation between house and Spanish sparrows. These analyses detected divergent selection on immunity-related SF genes and positive selective sweeps in regions containing a number of SF genes that also exhibited protein abundance diversification between species. Finally, we analyzed the molecular evolution of SFPs across 11 passerine species and found a significantly higher rate of positive selection in SFPs compared with the rest of the genome, as well as significant enrichments for functional pathways related to immunity in the set of positively selected SF genes. Our results suggest that selection on immunity pathways is an important determinant of passerine SF composition and evolution. Assessing the role of immunity genes in speciation in other recently diverged taxa should be prioritized given the potential role for immunity-related proteins in reproductive incompatibilities in Passer sparrows.

Evolution of female promiscuity in Passerides songbirds.

BACKGROUND: Female promiscuity is highly variable among birds, and particularly among songbirds. Comparative work has identified several patterns of covariation with social, sexual, ecological and life history traits. However, it is unclear whether these patterns reflect causes or consequences of female promiscuity, or if they are byproducts of some unknown evolutionary drivers. Moreover, factors that explain promiscuity at the deep nodes in the phylogenetic tree may be different from those important at the tips, i.e. among closely related species. Here we examine the relationships between female promiscuity and a broad set of predictor variables in a comprehensive data set (N = 202 species) of Passerides songbirds, which is a highly diversified infraorder of the Passeriformes exhibiting significant variation in female promiscuity. RESULTS: Female promiscuity was highly variable in all major clades of the Passerides phylogeny and also among closely related species. We found several significant associations with female promiscuity, albeit with fairly small effect sizes (all R2 ≤ 0.08). More promiscuous species had: 1) less male parental care, particularly during the early stages of the nesting cycle (nest building and incubation), 2) more short-term pair bonds, 3) greater degree of sexual dichromatism, primarily because females were drabber, 4) more migratory behaviour, and 5) stronger pre-mating sexual selection. In a multivariate model, however, the effect of sexual selection disappeared, while the other four variables showed additive effects and together explained about 16% of the total variance in female promiscuity. Female promiscuity showed no relationship with body size, life history variation, latitude or cooperative breeding. CONCLUSIONS: We found that multiple traits were associated with female promiscuity, but these associations were generally weak. Some traits, such as reduced parental care in males and more cryptic plumage in females, might even be responses to, rather than causes of, variation in female promiscuity. Hence, the high variation in female promiscuity among Passerides species remains enigmatic. Female promiscuity seems to be a rapidly evolving trait that often diverges between species with similar ecologies and breeding systems. A future challenge is therefore to understand what drives within-lineage variation in female promiscuity over microevolutionary time scales.

Sperm head abnormalities are more frequent in songbirds with more helical sperm: A possible trade-off in sperm evolution.

Sperm morphology varies enormously across the animal kingdom. Whilst knowledge of the factors that drive the evolution of interspecific variation in sperm morphology is accumulating, we currently have little understanding of factors that may constrain evolutionary change in sperm traits. We investigated whether susceptibility to sperm abnormalities could represent such a constraint in songbirds, a group characterized by a distinctive helical sperm head shape. Specifically, using 36 songbird species and data from light and scanning electron microscopy, we examined among-species correlations between the occurrence of sperm head abnormalities and sperm morphology, as well as the correlation between sperm head abnormalities and two indicators of sperm competition. We found that species with more helically shaped sperm heads (i.e., a wider helical membrane and more pronounced cell waveform) had a higher percentage of abnormal sperm heads than species with less helical sperm (i.e., relatively straight sperm) and that sperm head traits were better predictors of head abnormalities than total sperm length. In contrast, there was no correlation between sperm abnormalities and the level of sperm competition. Given that songbird species with more pronounced helical sperm have higher average sperm swimming speed, our results suggest an evolutionary trade-off between sperm performance and the structural integrity of the sperm head. As such, susceptibility to morphological abnormalities may constrain the evolution of helical sperm morphology in songbirds.

Varied effects of dietary carotenoid supplementation on oxidative damage in tissues of two waterfowl species.

Carotenoids are regarded as a cornerstone of avian vitality and coloration. Currently, the antioxidant potential of dietary carotenoids is debated for birds. Although some studies support a protective role, others report either no effect or pro-oxidant effects. However, the majority of research on this topic has not analyzed the oxidative status of a series of tissues in animals nor considered a range of carotenoid dosages. We investigated the effects of three levels of carotenoid supplementation on plasma, liver, adipose, heart and breast muscle oxidative damage in two congeneric species of waterfowl that exhibit marked differences in carotenoid coloration. After a 6-week depletion period, captive adult northern pintail (Anas acuta) and mallard (A. platyrhynchos) ducks of both sexes were fed either a carotenoid-depleted diet (<3 µg/g xanthophylls, lutein and zeaxanthin), a carotenoid-supplemented diet (50 µg/g) within physiological range, or a carotenoid-rich diet (100 µg/g) within pharmacological range for 22 to 32 weeks. We hypothesized that these dosages of dietary carotenoids would differentially affect oxidative damage between species and sexes and among the tissues examined. We found that dietary xanthophyll supplementation had no significant effect on tissue pro-oxidation in males and females from both species. Moreover, sex or species differences in oxidative stress were only observed in two tissues (plasma and heart). Significant correlations in the levels of oxidative damage were not observed among the tissues examined. In conclusion, the current study does not support a consistent antioxidant role for dietary carotenoids in the tissues of these two waterfowl species. Instead, our results align with the notion that carotenoids play complex, tissue- and species-specific roles in oxidative status in birds.

Identification and characterization of the zebra finch (Taeniopygia guttata) sperm proteome.

Spermatozoa exhibit remarkable variability in size, shape, and performance. Our understanding of the molecular basis of this variation, however, is limited, especially in avian taxa. The zebra finch (Taeniopygia guttata) is a model organism in the study of avian sperm biology and sperm competition. Using LC-MS based proteomics, we identify and describe 494 proteins of the zebra finch sperm proteome (ZfSP). Gene ontology and associated bioinformatics analyses revealed a rich repertoire of proteins essential to sperm structure and function, including proteins linked to metabolism and energetics, as well as tubulin binding and microtubule related functions. The ZfSP also contained a number of immunity and defense proteins and proteins linked to sperm motility and sperm-egg interactions. Additionally, while most proteins in the ZfSP appear to be evolutionarily constrained, a small subset of proteins are evolving rapidly. Finally, in a comparison with the sperm proteome of the domestic chicken, we found an enrichment of proteins linked to catalytic activity and cytoskeleton related processes. As the first described passerine sperm proteome, and one of only two characterized avian sperm proteomes, the ZfSP provides a significant step towards a platform for studies of the molecular basis of sperm function and evolution in birds. SIGNIFICANCE: Using highly purified spermatozoa and LC-MS proteomics, we characterise the sperm proteome of the Zebra finch; the main model species for the avian order Passeriformes, the largest and most diverse of the avian clades. As the first described passerine sperm proteome, and one of only two reported avian sperm proteomes, these results will facilitate studies of sperm biology and mechanisms of fertilisation in passerines, as well as comparative studies of sperm evolution and reproduction across birds and other vertebrates.

Sperm head morphology is associated with sperm swimming speed: A comparative study of songbirds using electron microscopy.

Sperm exhibit extraordinary levels of morphological diversification across the animal kingdom. In songbirds, sperm have a helically shaped head incorporating a distinct acrosomal membrane or "helical keel," the form and extent of which varies across species. The functional significance of this helical shape, however, remains unknown. Using scanning electron microscopy, we quantified inter- and intraspecific variation in sperm head morphology across 36 songbird species (Passeriformes: Passerida). Using phylogenetic comparative methods, we investigated the relationship between sperm head morphology and both sperm swimming speed and the frequency of extra-pair young (EPY). We found that species whose sperm had a relatively more pronounced helical form (i.e., long acrosome, short nucleus, wide helical membrane, and a more pronounced waveform along the sperm head "core") had faster-swimming sperm. We found no evidence of a relationship between interspecific variation in sperm head morphology and EPY, although we did find that among- and within-male variation in sperm head traits were negatively correlated with EPY. Applying principles of fluid mechanics, we discuss how the helical form of the sperm head may influence swimming speed, and suggest that further studies considering aspects of sperm morphology beyond sperm length are needed to improve our understanding of sperm structure-function relationships.

Experimental heatwaves negatively impact sperm quality in the zebra finch.

For sexually reproducing species, functionally competent sperm are critical to reproduction. While high atmospheric temperatures are known to influence the timing of breeding, incubation and reproductive success in birds, the effect of temperature on sperm quality remains largely unexplored. Here, we experimentally investigated the impact of ecologically relevant extreme temperatures on cloacal temperature and sperm morphology and motility in zebra finches Taeniopygia guttata We periodically sampled males exposed to 30°C or 40°C temperatures daily for 14 consecutive days. Following a 12-day (23°C) recovery period, birds were again exposed to heat, but under the alternate treatment (e.g. birds initially exposed to 40°C were exposed to 30°C). Elevated temperatures led to an increase in cloacal temperature and a reduction in the proportion of sperm with normal morphology; these effects were most notable under 40°C conditions, and were influenced by the duration of heat exposure and prior exposure to high temperature. Our findings highlight the potential role of temperature in determining male fertility in birds, and perhaps also in constraining the timing of avian breeding. Given the increased frequency of heatwaves in a warming world, our results suggest the need for further work on climatic influences on sperm quality and male fertility.

Differential sperm-egg interactions in experimental pairings between two subspecies and their hybrids in a passerine bird.

Speciation research has largely overlooked reproductive barriers acting between copulation and the formation of the zygote (i.e., postmating, prezygotic [PMPZ] barriers), especially in internally fertilizing vertebrates. Nonetheless, it is becoming clear that PMPZ reproductive barriers can play a role in the formation and maintenance of species boundaries. We investigated sperm-egg interactions in the recently diverged subspecies pairs of the long-tailed finch, Poephila acuticauda acuticauda and P. a. hecki, to explore potential PMPZ barriers. Specifically, we compared the number of sperm reaching the perivitelline layer (PVL) of the ova, and hence the site of fertilization, in both intra- and inter-subspecies pairings and pairings of F1 hybrid adults with one parental subspecies. Although we found no difference in PVL sperm number among intra- and inter-subspecific pairs, a significantly lower number of sperm reached the site of fertilization in a backcross pairing. As low numbers of PVL sperm appear to be associated with low fertilization success in birds, our findings offer insight into the potential role of postcopulatory processes in limiting gene flow between the subspecies and may help explain the relatively narrow hybrid zone that exists in the wild in this species. Though further work is needed to gain a comprehensive understanding of the morphological, physiological, and molecular mechanisms underlying our results, our study supports the role of PMPZ reproductive barriers in avian speciation, even in recently diverged taxa, that may not yet be fully genetically incompatible.