Divergence in the internal genital morphology of females and correlated divergence in male intromittent structures among populations of a millipede.

Our understanding of genital evolution comes largely from studies of male genitalia. Females have received far less attention because of the difficulties inherent in quantifying the shapes of their internal genital structures. Here we combine advances in micro-computed tomography with a new landmark free method of quantifying three-dimensional trait shape, to document patterns of divergence in female genital shape, and the correlated divergence of male genitalia among populations of the millipede Antichiropus variabilis. We used single-nucleotide polymorphisms to estimate levels of neutral genetic divergence among seven populations of millipede. Genetic divergence was high and correlated with geographic distance. Comparing phenotypic divergence in genital shape to neutral genetic divergence, we found that genital shape for both females and males has diverged more than would be expected from random drift, consistent with a pattern of directional selection. While there was significant covariation between female and male genital shape across populations, the magnitude of divergence in genital shape between the sexes was not correlated. Our results demonstrate the utility of using three-dimensional scanning technologies to examine female genital traits and add to a small but growing number of studies showing that like male genitalia, female genitalia can be under strong directional selection.

Daily temperature cycles prolong lifespan and have sex-specific effects on peripheral clock gene expression in Drosophila melanogaster.

Circadian rhythms optimize health by coordinating the timing of physiological processes to match predictable daily environmental challenges. The circadian rhythm of body temperature is thought to be an important modulator of molecular clocks in peripheral tissues, but how daily temperature cycles affect physiological function is unclear. Here, we examined the effect of constant temperature (Tcon, 25°C) and cycling temperature (Tcyc, 28°C:22°C during light:dark) paradigms on lifespan of Drosophila melanogaster, and the expression of clock genes, heat shock protein 83 (Hsp83), Frost (Fst) and senescence marker protein-30 (smp-30). Male and female D. melanogaster housed at Tcyc had longer median lifespans than those housed at Tcon. Tcyc induced robust Hsp83 rhythms and rescued the age-related decrease in smp-30 expression that was observed in flies at Tcon, potentially indicating an increased capacity to cope with age-related cellular stress. Ageing under Tcon led to a decrease in the amplitude of expression of all clock genes in the bodies of male flies, except for cyc, which was non-rhythmic, and for per and cry in female flies. Strikingly, housing under Tcyc conditions rescued the age-related decrease in amplitude of all clock genes, and generated rhythmicity in cyc expression, in the male flies, but not the female flies. The results suggest that ambient temperature rhythms modulate D. melanogaster lifespan, and that the amplitude of clock gene expression in peripheral body clocks may be a potential link between temperature rhythms and longevity in male D. melanogaster. Longevity due to Tcyc appeared predominantly independent of clock gene amplitude in female D. melanogaster.

Genome-wide SNPs detect no evidence of genetic population structure for reef manta rays (Mobula alfredi) in southern Mozambique.

Little is known about the extent of genetic connectivity along continuous coastlines in manta rays, or whether site visitation is influenced by relatedness. Such information is pertinent to defining population boundaries and understanding localized dispersal patterns and behaviour. Here, we use 3057 genome-wide single-nucleotide polymorphisms (SNPs) to evaluate population genetic structure and assess the levels of relatedness at aggregation sites of reef manta rays (Mobula alfredi) in southern Mozambique (n = 114). Contrary to indications of limited dispersal along the southern Mozambican coastline inferred from photo-identification and telemetry studies, our results show no evidence of population structure (non-significant FST < 0.001) for M. alfredi along this coast. We also found no evidence that individuals sampled at the same site were more related than expected by chance for males, females or across both sexes, suggesting that kinship may not influence visitation patterns at these sites. We estimated the effective population size (Ne) of this population to be 375 (95% CI = 369-380). Comparison to a distant eastern Indian Ocean site (Western Australia, n = 15) revealed strong genetic differentiation between Mozambique and Western Australia (FST = 0.377), identifying the Indian Ocean basin as a barrier to dispersal. Our findings show that genetic connectivity in M. alfredi extends for several hundred kilometres along continuous coastlines. We therefore recommend that the population in Mozambique be considered a discrete management unit, and future conservation plans should prioritize integrated strategies along the entire southern coastline.

Post-ejaculation thermal stress causes changes to the RNA profile of sperm in an external fertilizer.

Sperm cells experience considerable post-ejaculation environmental variation. However, little is known about whether this affects their molecular composition, probably owing to the assumption that sperm are transcriptionally quiescent. Nevertheless, recent evidence shows sperm have distinct RNA profiles that affect fertilization and embryo viability. Moreover, RNAs are expected to be highly sensitive to extracellular changes. One such group of RNAs are heat shock protein (hsp) transcripts, which function in stress responses and are enriched in sperm. Here, we exploit the experimental tractability of the mussel Mytilus galloprovincialis by exposing paired samples of ejaculated sperm to ambient (19°C) and increased (25°C) temperatures, then measure (i) sperm motility phenotypes, and (ii) messenger RNA (mRNA) levels of two target genes (hsp70 and hsp90) and several putative reference genes. We find no phenotypic changes in motility, but reduced mRNA levels for hsp90 and the putative reference gene gapdh at 25°C. This could reflect either decay of specific RNAs, or changes in translation and degradation rates of transcripts to maintain sperm function under stress. These findings represent, to our knowledge, the first evidence for changes in sperm RNA profiles owing to post-ejaculation environments, and suggest that sperm may be more vulnerable to stress from rising temperatures than currently thought.

Experimental evidence for accelerated adaptation to desiccation through sexual selection on males.

The impact of sexual selection on the adaptive process remains unclear. On the one hand, sexual selection might hinder adaptation by favouring costly traits and preferences that reduce nonsexual fitness. On the other hand, condition dependence of success in sexual selection may accelerate adaptation. Here, we used replicate populations of Drosophila melanogaster to artificially select on male desiccation resistance while manipulating the opportunity for precopulatory sexual selection in a factorial design. Following five generations of artificial selection, we measured the desiccation resistance of males and females to test whether the addition of sexual selection accelerated adaptation. We found a significant interaction between the effects of natural selection and sexual selection: desiccation resistance was highest in populations where sexual selection was allowed to operate. Despite only selecting on males, we also found a correlated response in females. These results provide empirical support for the idea that sexual selection can accelerate the rate of adaptation.

Ocean acidification during prefertilization chemical communication affects sperm success.

Ocean acidification (OA) poses a major threat to marine organisms, particularly during reproduction when externally shed gametes are vulnerable to changes in seawater pH. Accordingly, several studies on OA have focused on how changes in seawater pH influence sperm behavior and/or rates of in vitro fertilization. By contrast, few studies have examined how pH influences prefertilization gamete interactions, which are crucial during natural spawning events in most externally fertilizing taxa. One mechanism of gamete interaction that forms an important component of fertilization in most taxa is communication between sperm and egg-derived chemicals. These chemical signals, along with the physiological responses in sperm they elicit, are likely to be highly sensitive to changes in seawater chemistry. In this study, we experimentally tested this possibility using the blue mussel, Mytilus galloprovincialis, a species in which females have been shown to use egg-derived chemicals to promote the success of sperm from genetically compatible males. We conducted trials in which sperm were allowed to swim in gradients of egg-derived chemicals under different seawater CO2 (and therefore pH) treatments. We found that sperm had elevated fertilization rates after swimming in the presence of egg-derived chemicals in low pH (pH 7.6) compared with ambient (pH 8.0) seawater. This observed effect could have important implications for the reproductive fitness of external fertilizers, where gamete compatibility plays a critical role in modulating reproduction in many species. For example, elevated sperm fertilization rates might disrupt the eggs' capacity to avoid fertilizations by genetically incompatible sperm. Our findings highlight the need to understand how OA affects the multiple stages of sperm-egg interactions and to develop approaches that disentangle the implications of OA for female, male, and population fitness.

Population Genomics of Bettongia lesueur: Admixing Increases Genetic Diversity with no Evidence of Outbreeding Depression.

Small and isolated populations are subject to the loss of genetic variation as a consequence of inbreeding and genetic drift, which in turn, can affect the fitness and long-term viability of populations. Translocations can be used as an effective conservation tool to combat this loss of genetic diversity through establishing new populations of threatened species, and to increase total population size. Releasing animals from multiple genetically diverged sources is one method to optimize genetic diversity in translocated populations. However, admixture as a conservation tool is rarely utilized due to the risks of outbreeding depression. Using high-resolution genomic markers through double-digest restriction site-associated sequencing (ddRAD-seq) and life history data collected over nine years of monitoring, this study investigates the genetic and fitness consequences of admixing two genetically-distinct subspecies of Bettongia lesueur in a conservation translocation. Using single nucleotide polymorphisms (SNPs) identified from 215 individuals from multiple generations, we found an almost 2-fold increase in genetic diversity in the admixed translocation population compared to the founder populations, and this was maintained over time. Furthermore, hybrid class did not significantly impact on survivorship or the recruitment rate and therefore we found no indication of outbreeding depression. This study demonstrates the beneficial application of mixing multiple source populations in the conservation of threatened species for minimizing inbreeding and enhancing adaptive potential and overall fitness.

It's not all black and white: investigating colour polymorphism in manta rays across Indo-Pacific populations.

Intraspecific colour polymorphisms have been the focus of numerous studies, yet processes affecting melanism in the marine environment remain poorly understood. Arguably, the most prominent example of melanism in marine species occurs in manta rays (Mobula birostris and Mobula alfredi). Here, we use long-term photo identification catalogues to document the frequency variation of melanism across Indo-Pacific manta ray populations and test for evidence of selection by predation acting on colour morph variants. We use mark-recapture modelling to compare survivorship of typical and melanistic colour morphs in three M. alfredi populations and assess the relationship between frequency variation and geographical distance. While there were large differences in melanism frequencies among populations of both species (0-40.70%), apparent survival estimates revealed no difference in survivorship between colour morphs. We found a significant association between phenotypic and geographical distance in M. birostris, but not in M. alfredi. Our results suggest that melanism is not under selection by predation in the tested M. alfredi populations, and that frequency differences across populations of both species are a consequence of neutral genetic processes. As genetic colour polymorphisms are often subjected to complex selection mechanisms, our findings only begin to elucidate the underlying evolutionary processes responsible for the maintenance and frequency variation of melanism in manta ray populations.

Mixing Genetically and Morphologically Distinct Populations in Translocations: Asymmetrical Introgression in A Newly Established Population of the Boodie (Bettongia lesueur).

The use of multiple source populations provides a way to maximise genetic variation and reduce the impacts of inbreeding depression in newly established translocated populations. However, there is a risk that individuals from different source populations will not interbreed, leading to population structure and smaller effective population sizes than expected. Here, we investigate the genetic consequences of mixing two isolated, morphologically distinct island populations of boodies (Bettongia lesueur) in a translocation to mainland Australia over three generations. Using 18 microsatellite loci and the mitochondrial D-loop region, we monitored the released animals and their offspring between 2010 and 2013. Despite high levels of divergence between the two source populations (FST = 0.42 and ϕST = 0.72), there was clear evidence of interbreeding between animals from different populations. However, interbreeding was non-random, with a significant bias towards crosses between the genetically smaller-sized Barrow Island males and the larger-sized Dorre Island females. This pattern of introgression was opposite to the expectation that male-male competition or female mate choice would favour larger males. This study shows how mixing diverged populations can bolster genetic variation in newly established mammal populations, but the ultimate outcome can be difficult to predict, highlighting the need for continued genetic monitoring to assess the long-term impacts of admixture.

A genome-wide search for local adaptation in a terrestrial-breeding frog reveals vulnerability to climate change.

Terrestrial-breeding amphibians are likely to be vulnerable to warming and drying climates, as their embryos require consistent moisture for successful development. Adaptation to environmental change will depend on sufficient genetic variation existing within or between connected populations. Here, we use Single Nucleotide Polymorphism (SNP) data to investigate genome-wide patterns in genetic diversity, gene flow and local adaptation in a terrestrial-breeding frog (Pseudophryne guentheri) subject to a rapidly drying climate and recent habitat fragmentation. The species was sampled across 12 central and range-edge populations (192 samples), and strong genetic structure was apparent, as were high inbreeding coefficients. Populations showed differences in genetic diversity, and one population lost significant genetic diversity in a decade. More than 500 SNP loci were putatively under directional selection, and 413 of these loci were correlated with environmental variables such as temperature, rainfall, evaporation and soil moisture. One locus showed homology to a gene involved in the activation of maturation in Xenopus oocytes, which may facilitate rapid development of embryos in drier climates. The low genetic diversity, strong population structuring and presence of local adaptation revealed in this study shows why management strategies such as targeted gene flow may be necessary to assist isolated populations to adapt to future climates.

Evaluating the genetic architecture of quantitative traits via selection followed by inbreeding.

The deleterious mutation model proposes that quantitative trait variation should be dominated by rare, partially recessive, deleterious mutations. Following artificial selection on a focal trait, the ratio of the difference in inbreeding effects between control and selected populations (ΔB), to the difference in trait means caused by directional selection (ΔM), can inform the extent to which deleterious mutations cause quantitative trait variation. Here, we apply the ΔB/ΔM ratio test to two quantitative traits (male mating success and body size) in Drosophila melanogaster. For both traits, ΔB/ΔM ratios suggested that intermediate-frequency alleles, rather than rare, partially recessive alleles (i.e. deleterious mutations), caused quantitative trait variation. We discuss these results in relation to viability data, exploring how differences between regimens in segregating (measured through inbreeding) and fixed (measured through population crosses) mutational load could affect the ratio test. Finally, we present simulations that test the statistical power of the ratio test, providing guidelines for future research.

Molecular evidence supports a genic capture resolution of the lek paradox.

The genic capture hypothesis, where sexually selected traits capture genetic variation in condition and the condition reflects genome-wide mutation load, stands to explain the presence of abundant genetic variation underlying sexually selected traits. Here we test this hypothesis by applying bidirectional selection to male mating success for 14 generations in replicate populations of Drosophila melanogaster. We then resequenced the genomes of flies from each population. Consistent with the central predictions of the genic capture hypothesis, we show that genetic variance decreased with success selection and increased with failure selection, providing evidence for purifying sexual selection. This pattern was distributed across the genome and no consistent molecular pathways were associated with divergence, consistent with condition being the target of selection. Together, our results provide molecular evidence suggesting that strong sexual selection erodes genetic variation, and that genome-wide mutation-selection balance contributes to its maintenance.

Multivariate Sexual Selection on Ejaculate Traits under Sperm Competition.

The widespread prevalence of sperm competition means that ejaculates face intense sexual selection. However, prior investigations of sexual selection on gametes have been hampered by two difficulties: (1) deriving estimates of relative fitness from sperm competition trials that are comparable across rival male and female genotypes and (2) obtaining measures of competitive fertilization success that are not confounded by postzygotic effects. Here, we exploit the experimental tractability of a broadcast spawning marine invertebrate to overcome these challenges and characterize multivariate sexual selection on sperm traits when multiple ejaculates compete. In multimale spawning events, we tracked real-time success of sperm using fluorescent tags that are visible inside fertilized eggs. We then used multivariate selection analyses to identify patterns of linear and nonlinear sexual selection on multiple sperm morphology and motility traits. Specifically, we found nonlinear selection against divergent combinations of sperm length, velocity, and swimming path linearity. These patterns likely reflect the way different swimming strategies allow sperm to locate and track eggs. Our results demonstrate that there are overall patterns of selection on ejaculates across a biologically realistic range of ejaculate-ejaculate and ejaculate-female interactions; therefore, there is the potential for adaptive evolution of ejaculate traits under sperm competition.

Evolutionary divergence in competitive mating success through female mating bias for good genes.

Despite heritable variation for univariate sexually selected traits, recent analyses exploring multivariate traits find evidence consistent with the lek paradox in showing no genetic variation available to choosy females, and therefore no genetic benefits of choice. We used the preferences of Drosophila melanogaster females to exert bidirectional selection on competitive male mating success to test for the presence and nature of genetic variation underlying this multivariate trait. Male mating success diverged between selection regimens, and flies from success-selected lines had a smaller burden of deleterious, recessive mutations that affect egg-to-adult viability, were better sperm competitors (sperm offence), and did not demonstrate reduced desiccation resistance or components of female fitness (traits thought to trade off with attractiveness) relative to flies from failure-selected populations. Mating success remained subject to inbreeding depression in success-selected lines, suggesting that variation in mating success remains, thanks to numerous genes of small effect. Together, our results provide unique evidence for the evolutionary divergence in male mating success, demonstrating that genetic variation is not exhausted along the axis of precopulatory sexual selection and that female mating biases align with the avoidance of bad genes.

Genomic signatures of local adaptation reveal source-sink dynamics in a high gene flow fish species.

Understanding source-sink dynamics is important for conservation management, particularly when climatic events alter species' distributions. Following a 2011 'marine heatwave' in Western Australia, we observed high recruitment of the endemic fisheries target species Choerodon rubescens, towards the cooler (southern) end of its distribution. Here, we use a genome wide set of 14 559 single-nucleotide polymorphisms (SNPs) to identify the likely source population for this recruitment event. Most loci (76%) showed low genetic divergence across the species' range, indicating high levels of gene flow and confirming previous findings using neutral microsatellite markers. However, a small proportion of loci showed strong patterns of differentiation and exhibited patterns of population structure consistent with local adaptation. Clustering analyses based on these outlier loci indicated that recruits at the southern end of C. rubescens' range originated 400 km to the north, at the centre of the species' range, where average temperatures are up to 3 °C warmer. Survival of these recruits may be low because they carry alleles adapted to an environment different to the one they now reside in, but their survival is key to establishing locally adapted populations at and beyond the range edge as water temperatures increase with climate change.

Restricted gene flow and local adaptation highlight the vulnerability of high-latitude reefs to rapid environmental change.

Global climate change poses a serious threat to the future health of coral reef ecosystems. This calls for management strategies that are focused on maximizing the evolutionary potential of coral reefs. Fundamental to this is an accurate understanding of the spatial genetic structure in dominant reef-building coral species. In this study, we apply a genotyping-by-sequencing approach to investigate genome-wide patterns of genetic diversity, gene flow, and local adaptation in a reef-building coral, Pocillopora damicornis, across 10 degrees of latitude and a transition from temperate to tropical waters. We identified strong patterns of differentiation and reduced genetic diversity in high-latitude populations. In addition, genome-wide scans for selection identified a number of outlier loci putatively under directional selection with homology to proteins previously known to be involved in heat tolerance in corals and associated with processes such as photoprotection, protein degradation, and immunity. This study provides genomic evidence for both restricted gene flow and local adaptation in a widely distributed coral species, and highlights the potential vulnerability of leading-edge populations to rapid environmental change as they are locally adapted, reproductively isolated, and have reduced levels of genetic diversity.

Phylogenomics provides new insight into evolutionary relationships and genealogical discordance in the reef-building coral genus Acropora.

Understanding the genetic basis of reproductive isolation is a long-standing goal of speciation research. In recently diverged populations, genealogical discordance may reveal genes and genomic regions that contribute to the speciation process. Previous work has shown that conspecific colonies of Acropora that spawn in different seasons (spring and autumn) are associated with highly diverged lineages of the phylogenetic marker PaxC Here, we used 10 034 single-nucleotide polymorphisms to generate a genome-wide phylogeny and compared it with gene genealogies from the PaxC intron and the mtDNA Control Region in 20 species of Acropora, including three species with spring- and autumn-spawning cohorts. The PaxC phylogeny separated conspecific autumn and spring spawners into different genetic clusters in all three species; however, this pattern was not supported in two of the three species at the genome level, suggesting a selective connection between PaxC and reproductive timing in Acropora corals. This genome-wide phylogeny provides an improved foundation for resolving phylogenetic relationships in Acropora and, combined with PaxC, provides a fascinating platform for future research into regions of the genome that influence reproductive isolation and speciation in corals.

Egg chemoattractants moderate intraspecific sperm competition.

Interactions among eggs and sperm are often assumed to generate intraspecific variation in reproductive fitness, but the specific gamete-level mechanisms underlying competitive fertilization success remain elusive in most species. Sperm chemotaxis-the attraction of sperm by egg-derived chemicals-is a ubiquitous form of gamete signaling, occurring throughout the animal and plant kingdoms. The chemical cues released by eggs are known to act at the interspecific level (e.g., facilitating species recognition), but recent studies have suggested that they could have roles at the intraspecific level by moderating sperm competition. Here, we exploit the experimental tractability of a broadcast spawning marine invertebrate to test this putative mechanism of gamete-level sexual selection. We use a fluorescently labeled mitochondrial dye in mussels to track the real-time success of sperm as they compete to fertilize eggs, and provide the first direct evidence in any species that competitive fertilization success is moderated by differential sperm chemotaxis. Furthermore, our data are consistent with the idea that egg chemoattractants selectively attract ejaculates from genetically compatible males, based on relationships inferred from both nuclear and mitochondrial genetic markers. These findings for a species that exhibits the ancestral reproductive strategy of broadcast spawning have important implications for the numerous species that also rely on egg chemoattractants to attract sperm, including humans, and have potentially important implications for our understanding of the evolutionary cascade of sexual selection.

Fluorescent sperm offer a method for tracking the real-time success of ejaculates when they compete to fertilise eggs.

Despite intensive research effort, many uncertainties remain in the field of gamete-level sexual selection, particularly in understanding how sperm from different males interact when competing for fertilisations. Here, we demonstrate the utility of broadcast spawning marine invertebrates for unravelling these mysteries, highlighting their mode of reproduction and, in some species, unusual patterns of mitochondrial inheritance. We present a method utilising both properties in the blue mussel, Mytilus galloprovincialis. In mytilids and many other bivalves, both sperm and egg mitochondria are inherited. We exploit this, using the vital mitochondrial dye MitoTracker, to track the success of sperm from individual males when they compete with those from rivals to fertilise eggs. We confirm that dying mitochondria has no adverse effects on in vitro measures of sperm motility (reflecting mitochondrial energetics) or sperm competitive fertilisation success. Therefore, we propose the technique as a powerful and logistically tractable tool for sperm competition studies. Importantly, our method allows the competitive fertilisation success of sperm from any male to be measured directly and disentangled from confounding effects of post-fertilisation embryo survival. Moreover, the mitochondrial dye has broader applications in taxa without paternal mitochondrial inheritance, for example by tracking the dynamics of competing ejaculates prior to fertilisation.

Isolation by resistance across a complex coral reef seascape.

A detailed understanding of the genetic structure of populations and an accurate interpretation of processes driving contemporary patterns of gene flow are fundamental to successful spatial conservation management. The field of seascape genetics seeks to incorporate environmental variables and processes into analyses of population genetic data to improve our understanding of forces driving genetic divergence in the marine environment. Information about barriers to gene flow (such as ocean currents) is used to define a resistance surface to predict the spatial genetic structure of populations and explain deviations from the widely applied isolation-by-distance model. The majority of seascape approaches to date have been applied to linear coastal systems or at large spatial scales (more than 250 km), with very few applied to complex systems at regional spatial scales (less than 100 km). Here, we apply a seascape genetics approach to a peripheral population of the broadcast-spawning coral Acropora spicifera across the Houtman Abrolhos Islands, a high-latitude complex coral reef system off the central coast of Western Australia. We coupled population genetic data from a panel of microsatellite DNA markers with a biophysical dispersal model to test whether oceanographic processes could explain patterns of genetic divergence. We identified significant variation in allele frequencies over distances of less than 10 km, with significant differentiation occurring between adjacent sites but not between the most geographically distant ones. Recruitment probabilities between sites based on simulated larval dispersal were projected into a measure of resistance to connectivity that was significantly correlated with patterns of genetic divergence, demonstrating that patterns of spatial genetic structure are a function of restrictions to gene flow imposed by oceanographic currents. This study advances our understanding of the role of larval dispersal on the fine-scale genetic structure of coral populations across a complex island system and applies a methodological framework that can be tailored to suit a variety of marine organisms with a range of life-history characteristics.