Female Reproductive Fluid Increases the Opportunities for Postmating Sexual Selection by Prolonging the Egg Fertilization Window.

AbstractFemale reproductive fluid (the fluid that surrounds the eggs) has attracted increasing attention for its role in fertilization and postmating sexual selection through its effects on sperm traits. Surprisingly, however, only a few studies have investigated the effects of the female reproductive fluid on the eggs. Yet these effects might offer great potential to affect fertilization dynamics by, for example, increasing the opportunities for postmating sexual selection. Here, we determined whether the female reproductive fluid, by extending the egg fertilization window (the time available for egg fertilization), could also increase the opportunities for multiple paternity. Using the zebrafish (Danio rerio), we first tested the prediction that female reproductive fluid increases the egg fertilization window; then, using a split-brood design with the sperm of two males added at different time points after egg activation, we tested whether the degree of multiple paternity varies in the presence or absence of female reproductive fluid. Our results reveal the potential of female reproductive fluid to increase multiple paternity through its effects on the egg fertilization window, thus broadening our knowledge of how female mechanisms affect postmating sexual selection in externally fertilizing species.

Sperm production is negatively associated with muscle and sperm telomere length in a species subjected to strong sperm competition.

Life-history theory suggests that ageing is one of the costs of reproduction. Accordingly, a higher reproductive allocation is expected to increase the deterioration of both the somatic and the germinal lines through enhanced telomere attrition. In most species, males' reproductive allocation mainly regards traits that increase mating and fertilization success, that is sexually selected traits. In this study, we tested the hypothesis that a higher investment in sexually selected traits is associated with a reduced relative telomere length (RTL) in the guppy (Poecilia reticulata), an ectotherm species characterized by strong pre- and postcopulatory sexual selection. We first measured telomere length in both the soma and the sperm over guppies' lifespan to see whether there was any variation in telomere length associated with age. Second, we investigated whether a greater investment in pre- and postcopulatory sexually selected traits is linked to shorter telomere length in both the somatic and the sperm germinal lines, and in young and old males. We found that telomeres lengthened with age in the somatic tissue, but there was no age-dependent variation in telomere length in the sperm cells. Telomere length in guppies was significantly and negatively correlated with sperm production in both tissues and life stages considered in this study. Our findings indicate that telomere length in male guppies is strongly associated with their reproductive investment (sperm production), suggesting that a trade-off between reproduction and maintenance is occurring at each stage of males' life in this species.

Experimental nest cooling reveals dramatic effects of heatwaves on reproduction in a Mediterranean bird of prey.

Future climatic scenarios forecast increases in average temperatures as well as in the frequency, duration, and intensity of extreme events, such as heatwaves. Whereas behavioral adjustments can buffer direct physiological and fitness costs of exposure to excessive temperature in wild animals, these may prove more difficult during specific life stages when vagility is reduced (e.g., early developmental stages). By means of a nest cooling experiment, we tested the effects of extreme temperatures on different stages of reproduction in a cavity-nesting Mediterranean bird of prey, the lesser kestrel (Falco naumanni), facing a recent increase in the frequency of heatwaves during its breeding season. Nest temperature in a group of nest boxes placed on roof terraces was reduced by shading them from direct sunlight in 2 consecutive years (2021 and 2022). We then compared hatching failure, mortality, and nestling morphology between shaded and non-shaded (control) nest boxes. Nest temperature in control nest boxes was on average 3.9°C higher than in shaded ones during heatwaves, that is, spells of extreme air temperature (>37°C for ≥2 consecutive days) which hit the study area during the nestling-rearing phase in both years. Hatching failure markedly increased with increasing nest temperature, rising above 50% when maximum nest temperatures exceeded 44°C. Nestlings from control nest boxes showed higher mortality during heatwaves (55% vs. 10% in shaded nest boxes) and those that survived further showed impaired morphological growth (body mass and skeletal size). Hence, heatwaves occurring during the breeding period can have both strong lethal and sublethal impacts on different components of avian reproduction, from egg hatching to nestling growth. More broadly, these findings suggest that the projected future increases of summer temperatures and heatwave frequency in the Mediterranean basin and elsewhere in temperate areas may threaten the local persistence of even relatively warm-adapted species.

Fighting Pseudomonas aeruginosa Infections: Antibacterial and Antibiofilm Activity of D-Q53 CecB, a Synthetic Analog of a Silkworm Natural Cecropin B Variant.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium responsible for severe nosocomial infections and is considered a critical pulmonary pathogen for both immunocompromised and cystic fibrosis patients. Planktonic cells of P. aeruginosa possess intrinsic and acquired resistances, inactivating several classes of conventional antibiotics. Additionally, this bacterium can grow, forming biofilms, and complex structures, further hampering the action of multiple antibiotics. Here, we report the biological properties of D-Q53 CecB, an all-D enantiomer of the silkworm natural peptide Q53 CecB. Compared to the L-variant, D-Q53 CecB was resistant to in vitro degradation by humans and P. aeruginosa elastases and showed an enhanced bactericidal activity against P. aeruginosa planktonic bacteria. D-Q53 CecB was thermostable and maintained its antimicrobial activity at high salt concentrations and in the presence of divalent cations or fetal-bovine serum, although at reduced levels. Against different types of human cells, D-Q53 CecB showed cytotoxic phenomena at concentrations several folds higher compared to those active against P. aeruginosa. When L- and D-Q53 CecB were compared for their antibiofilm properties, both peptides were active in inhibiting biofilm formation. However, the D-enantiomer was extremely effective in inducing biofilm degradation, suggesting this peptide as a favorable candidate in an anti-Pseudomonas therapy.

Differential gene regulation in selected lines for high and low sperm production in male guppies.

In species where females mate with more than one male during the same reproductive event, males typically increase the number of sperm produced to boost their fertilization share. Sperm is not limitless, however, and theory predicts that their production will come at the cost of other fitness-related traits, such as body growth or immunocompetence, although these evolutionary trade-offs are notoriously difficult to highlight. To this end, we combined artificial selection for sperm production with a transcriptome analysis using Poecilia reticulata, a fish characterized by intense sperm competition in which the number of sperm transferred during mating is the most important predictor of fertilization success, yet sperm production is highly variable among males. We compared the brain and testes transcriptome in male guppies of lines artificially selected for high and low sperm production by identifying pivotal differentially expressed gene sets that may regulate spermatogenesis and immune function in this species. Despite the small differences in single genes' expression, gene set enrichment analysis showed coordinated gene expression differences associated with several pathways differentially regulated in the two selection lines. High sperm production males showed an upregulation of pathways related to immunosuppression and development of spermatozoa indicating a possible immunological cost of sperm production.

Insect Cecropins, Antimicrobial Peptides with Potential Therapeutic Applications.

The alarming escalation of infectious diseases resistant to conventional antibiotics requires urgent global actions, including the development of new therapeutics. Antimicrobial peptides (AMPs) represent potential alternatives in the treatment of multi-drug resistant (MDR) infections. Here, we focus on Cecropins (Cecs), a group of naturally occurring AMPs in insects, and on synthetic Cec-analogs. We describe their action mechanisms and antimicrobial activity against MDR bacteria and other pathogens. We report several data suggesting that Cec and Cec-analog peptides are promising antibacterial therapeutic candidates, including their low toxicity against mammalian cells, and anti-inflammatory activity. We highlight limitations linked to the use of peptides as therapeutics and discuss methods overcoming these constraints, particularly regarding the introduction of nanotechnologies. New formulations based on natural Cecs would allow the development of drugs active against Gram-negative bacteria, and those based on Cec-analogs would give rise to therapeutics effective against both Gram-positive and Gram-negative pathogens. Cecs and Cec-analogs might be also employed to coat biomaterials for medical devices as an approach to prevent biomaterial-associated infections. The cost of large-scale production is discussed in comparison with the economic and social burden resulting from the progressive diffusion of MDR infectious diseases.

Differential gene expression in the evolution of sex pheromone communication in New Zealand's endemic leafroller moths of the genera Ctenopseustis and Planotortrix.

BACKGROUND: Sex pheromone communication in moths has attracted the attention of evolutionary biologists due to the vast array of pheromone compounds used, addressing questions of how this diversity arose and how male reception has evolved in step with the female signal. Here we examine the role of changing gene expression in the evolution of mate recognition systems in leafroller moths, particularly focusing on genes involved in the biosynthetic pathways of sex pheromones in female pheromone glands and the peripheral reception repertoire in the antennae of males. From tissue-specific transcriptomes we mined and compared a database of genes expressed in the pheromone glands and antennae of males and females of four closely related species of leafroller moths endemic to New Zealand, Ctenopseutis herana and C. obliquana, and Planotortrix excessana and P. octo. The peculiarity of this group, compared to other Lepidoptera, is the use of (Z)-5-tetradecenyl acetate, (Z)-7-tetradecenyl acetate, and (Z)-8-tetradecenyl acetate as sex pheromone components. RESULTS: We identify orthologues of candidate genes from the pheromone biosynthesis pathway, degradation and transport, as well as genes of the periphery olfactory repertoire, including large families of binding proteins, receptors and odorant degrading enzymes. The production of distinct pheromone blends in the sibling species is associated with the differential expression of two desaturase genes, deast5 and desat7, in the pheromone glands. In male antennae, three odorant receptors, OR74, OR76a and OR30 are over-expressed, but their expression could not be clearly associated with the detection of species-specific pheromones components. In addition these species contain duplications of all three pheromone binding proteins (PBPs) that are also differentially expressed among species. CONCLUSIONS: While in females differences in the expression of desaturases may be sufficient to explain pheromone blend differences among these New Zealand leafroller species, in males differential expression of several genes, including pheromone binding proteins, may underpin differences in the response by males to changing pheromone components among the species.

Pre-invasion history and demography shape the genetic variation in the insecticide resistance-related acetylcholinesterase 2 gene in the invasive Colorado potato beetle.

BACKGROUND: Invasive pest species offers a unique opportunity to study the effects of genetic architecture, demography and selection on patterns of genetic variability. Invasive Colorado potato beetle (Leptinotarsa decemlineata) populations have experienced a rapid range expansion and intense selection by insecticides. By comparing native and invasive beetle populations, we studied the origins of organophosphate (OP) resistance-associated mutations in the acetylcholinesterase 2 (AChE2) gene, and the role of selection and demography on its genetic variability. RESULTS: Analysis of three Mexican, two US and five European populations yielded a total of 49 haplotypes. Contrary to the expectations all genetic variability was associated with a point mutation linked to insecticide resistance (S291G), this mutation was found in 100% of Mexican, 95% of US and 71% of European beetle sequences analysed. Only two susceptible haplotypes, genetically very differentiated, were found, one in US and one in Europe. The genetic variability at the AChE2 gene was compared with two other genes not directly affected by insecticide selection, diapause protein 1 and juvenile hormone esterase. All three genes showed reduction in genetic variability indicative of a population bottleneck associated with the invasion. CONCLUSIONS: Stochastic effects during invasion explain most of the observed patterns of genetic variability at the three genes investigated. The high frequency of the S291G mutation in the AChE2 gene among native populations suggests this mutation is the ancestral state and thus, either a pre-adaptation of the beetle for OP resistance or the AChE2 is not the major gene conferring OP resistance. The long historical association with host plant alkaloids together with recombination may have contributed to the high genetic variation at this locus. The genetic diversity in the AChE2 locus of the European beetles, in turn, strongly reflects founder effects followed by rapid invasion. Our results suggest that despite the long history of insecticide use in this species, demographic events together with pre-invasion history have been strongly influential in shaping the genetic diversity of the AChE2 gene in the invasive beetle populations.

Transcriptome sequencing and de novo annotation of the critically endangered Adriatic sturgeon.

BACKGROUND: Sturgeons are a group of Condrostean fish with very high evolutionary, economical and conservation interest. The eggs of these living fossils represent one of the most high prized foods of animal origin. The intense fishing pressure on wild stocks to harvest caviar has caused in the last decades a dramatic decline of their distribution and abundance leading the International Union for Conservation of Nature to list them as the more endangered group of species. As a direct consequence, world-wide efforts have been made to develop sturgeon aquaculture programmes for caviar production. In this context, the characterization of the genes involved in sex determination could provide relevant information for the selective farming of the more profitable females. RESULTS: The 454 sequencing of two cDNA libraries from the gonads and brain of one male and one female full-sib A. naccarii, yielded 182,066 and 167,776 reads respectively, which, after strict quality control, were iterative assembled into more than 55,000 high quality ESTs. The average per-base coverage reached by assembling the two libraries was 4X. The multi-step annotation process resulted in 16% successfully annotated sequences with GO terms. We screened the transcriptome for 32 sex-related genes and highlighted 7 genes that are potentially specifically expressed, 5 in male and 2 in females, at the first life stage at which sex is histologically identifiable. In addition we identified 21,791 putative EST-linked SNPs and 5,295 SSRs. CONCLUSIONS: This study represents the first large massive release of sturgeon transcriptome information that we organized into the public database AnaccariiBase, which is freely available at http://compgen.bio.unipd.it/anaccariibase/. This transcriptomic data represents an important source of information for further studies on sturgeon species. The hundreds of putative EST-linked molecular makers discovered in this study will be invaluable for sturgeon reintroduction and breeding programs.

The Genomics and Population Genomics of the Light Brown Apple Moth, Epiphyas postvittana, an Invasive Tortricid Pest of Horticulture.

The light brown apple moth, Epiphyas postvittana is an invasive, polyphagous pest of horticultural systems around the world. With origins in Australia, the pest has subsequently spread to New Zealand, Hawaii, California and Europe, where it has been found on over 500 plants, including many horticultural crops. We have produced a genomic resource, to understand the biological basis of the polyphagous and invasive nature of this and other lepidopteran pests. The assembled genome sequence encompassed 598 Mb and has an N50 of 301.17 kb, with a BUSCO completion rate of 97.9%. Epiphyas postvittana has 34% of its assembled genome represented as repetitive sequences, with the majority of the known elements made up of longer DNA transposable elements (14.07 Mb) and retrotransposons (LINE 17.83 Mb). Of the 31,389 predicted genes, 28,714 (91.5%) were assigned to 11,438 orthogroups across the Lepidoptera, of which 945 were specific to E. postvittana. Twenty gene families showed significant expansions in E. postvittana, including some likely to have a role in its pest status, such as cytochrome p450s, glutathione-S-transferases and UDP-glucuronosyltransferases. Finally, using a RAD-tag approach, we investigated the population genomics of this pest, looking at its likely patterns of invasion.

Sequence variation and regulatory variation in acetylcholinesterase genes contribute to insecticide resistance in different populations of Leptinotarsa decemlineata.

Although insect herbivores are known to evolve resistance to insecticides through multiple genetic mechanisms, resistance in individual species has been assumed to follow the same mechanism. While both mutations in the target site insensitivity and increased amplification are known to contribute to insecticide resistance, little is known about the degree to which geographic populations of the same species differ at the target site in a response to insecticides. We tested structural (e.g., mutation profiles) and regulatory (e.g., the gene expression of Ldace1 and Ldace2, AChE activity) differences between two populations (Vermont, USA and Belchow, Poland) of the Colorado potato beetle, Leptinotarsa decemlineata in their resistance to two commonly used groups of insecticides, organophosphates, and carbamates. We established that Vermont beetles were more resistant to azinphos-methyl and carbaryl insecticides than Belchow beetles, despite a similar frequency of resistance-associated alleles (i.e., S291G) in the Ldace2 gene. However, the Vermont population had two additional amino acid replacements (G192S and F402Y) in the Ldace1 gene, which were absent in the Belchow population. Moreover, the Vermont population showed higher expression of Ldace1 and was less sensitive to AChE inhibition by azinphos-methyl oxon than the Belchow population. Therefore, the two populations have evolved different genetic mechanisms to adapt to organophosphate and carbamate insecticides.

Imminent risk of predation reduces the relative strength of postcopulatory sexual selection in the guppy.

Fifty years of research on sperm competition has led to a very good understanding of the interspecific variation in sperm production traits. The reasons why this variation is often very large within populations have been less investigated. We suggest that the interaction between fluctuating environmental conditions and polyandry is a key phenomenon explaining such variation. We focus here on imminent predation risk (IPR). IPR impacts significantly several aspects of prey behaviour and reproduction, and it is expected to influence the operation of sexual selection before and after mating. We estimated the effect of IPR on the male opportunity for pre- and postcopulatory sexual selection in guppies (Poecilia reticulata), a livebearing fish where females prefer colourful males and mate multiply. We used a repeated-measures design, in which males were allowed to mate with different females either under IPR or in a predator-free condition. We found that IPR increased the total opportunity for sexual selection and reduced the relative contribution of postcopulatory sexual selection to male reproductive success. IPR is inherently variable and our results suggest that interspecific reproductive interference by predators may contribute towards maintaining the variation in sperm production within populations. This article is part of the theme issue 'Fifty years of sperm competition'.

Maternal predator-exposure affects offspring size at birth but not telomere length in a live-bearing fish.

The perception of predation risk could affect prey phenotype both within and between generations (via parental effects). The response to predation risk could involve modifications in physiology, morphology, and behavior and can ultimately affect long-term fitness. Among the possible modifications mediated by the exposure to predation risk, telomere length could be a proxy for investigating the response to predation risk both within and between generations, as telomeres can be significantly affected by environmental stress. Maternal exposure to the perception of predation risk can affect a variety of offspring traits but the effect on offspring telomere length has never been experimentally tested. Using a live-bearing fish, the guppy (Poecilia reticulata), we tested if the perceived risk of predation could affect the telomere length of adult females directly and that of their offspring with a balanced experimental setup that allowed us to control for both maternal and paternal contribution. We exposed female guppies to the perception of predation risk during gestation using a combination of both visual and chemical cues and we then measured female telomere length after the exposure period. Maternal effects mediated by the exposure to predation risk were measured on offspring telomere length and body size at birth. Contrary to our predictions, we did not find a significant effect of predation-exposure neither on female nor on offspring telomere length, but females exposed to predation risk produced smaller offspring at birth. We discuss the possible explanations for our findings and advocate for further research on telomere dynamics in ectotherms.

Molecular and biochemical responses of vitellogenin in the mussel Mytilus galloprovincialis exposed to the glyphosate-based herbicide Roundup® Power 2.0.

Glyphosate-based herbicides (GBHs) occur in aquatic ecosystems at concentrations of hundreds of micrograms per liter. As formulation adjuvants are suspected to be endocrine-disrupting chemicals, we assessed the effects of the recent GBH formulation Roundup® Power 2.0 on vitellogenin (VTG) in Mytilus galloprovincialis. Mussels were exposed for 7, 14, and 21 days to two concentrations of the commercial formulation, corresponding to 100 and 1000 µg/L of glyphosate. The expression of the vtg gene in gonads of females and males, as well as the levels of alkali labile phosphates (ALP) in gonads and non-gonadal tissues from the two sexes were measured. No significant alterations were observed in vtg expression values during the exposure. Conversely, a significant reduction in gonadal ALP levels was observed in females exposed for 21 days and in males exposed for 7 days. In addition, ALP levels increased significantly in gonads from males exposed for 21 days to the two concentrations of Roundup®. As for non-gonadal tissues, ALP levels did not change significantly in females, whereas ALP levels decreased significantly in non-gonadal tissues from males exposed for 21 days to the lowest concentration tested. An overall statistically significant difference in ALP levels was found between females and males. Although preliminary, our study suggests that GBH can affect reproduction-related parameters in mussels.

Testosterone-mediated effects on fitness-related phenotypic traits and fitness.

The physiological and behavioral mechanisms underlying life-history trade-offs are a continued source of debate. Testosterone (T) is one physiological factor proposed to mediate the trade-off between reproduction and survival. We use phenotypic engineering and multiple laboratory and field fitness-related phenotypic traits to test the effects of elevated T between two bank vole Myodes glareolus groups: dominant and subordinate males. Males with naturally high T levels showed higher social status (laboratory dominance) and mobility (distance between capture sites) than low-T males, and the effect of T on immune response was also T group specific, suggesting that behavioral strategies may exist in male bank voles due to the correlated responses of T. Exogenous T enhanced social status, mate searching (polygon of capture sites), mobility, and reproductive success (relative measure of pups sired). However, exogenous T also resulted in the reduction of immune function, but only in males from the high-T group. This result may be explained either by the immunosuppression costs of T or by differential sensitivity of different behavioral strategies to steroids. Circulating T levels were found to be heritable; therefore, female bank voles would derive indirect genetic benefits via good genes from mating with males signaling dominance.

Island selection on mammalian life-histories: genetic differentiation in offspring size.

BACKGROUND: Since Darwin's pioneering work, evolutionary changes in isolated island populations of vertebrates have continued to provide the strongest evidence for the theory of natural selection. Besides macro-evolutionary changes, micro-evolutionary changes and the relative importance of natural selection vs. genetic drift are under intense investigation. Our study focuses on the genetic differentiation in morphological and life-history traits in insular populations of a small mammal the bank vole Myodes glareolus. RESULTS: Our results do not support the earlier findings for larger adult size or lower reproductive effort in insular populations of small mammals. However, the individuals living on islands produced larger offspring than individuals living on the mainland. Genetic differentiation in offspring size was further confirmed by the analyses of quantitative genetics in lab. In insular populations, genetic differentiation in offspring size simultaneously decreases the additive genetic variation (VA) for that trait. Furthermore, our analyses of differentiation in neutral marker loci (Fst) indicate that VA is less than expected on the basis of genetic drift alone, and thus, a lower VA in insular populations could be caused by natural selection. CONCLUSION: We believe that different selection pressures (e.g. higher intraspecific competition) in an insular environment might favour larger offspring size in small mammals. Island selection for larger offspring could be the preliminary mechanism in a process which could eventually lead to a smaller litter size and lower reproductive effort frequently found in insular vertebrates.

A model species for agricultural pest genomics: the genome of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The Colorado potato beetle is one of the most challenging agricultural pests to manage. It has shown a spectacular ability to adapt to a variety of solanaceaeous plants and variable climates during its global invasion, and, notably, to rapidly evolve insecticide resistance. To examine evidence of rapid evolutionary change, and to understand the genetic basis of herbivory and insecticide resistance, we tested for structural and functional genomic changes relative to other arthropod species using genome sequencing, transcriptomics, and community annotation. Two factors that might facilitate rapid evolutionary change include transposable elements, which comprise at least 17% of the genome and are rapidly evolving compared to other Coleoptera, and high levels of nucleotide diversity in rapidly growing pest populations. Adaptations to plant feeding are evident in gene expansions and differential expression of digestive enzymes in gut tissues, as well as expansions of gustatory receptors for bitter tasting. Surprisingly, the suite of genes involved in insecticide resistance is similar to other beetles. Finally, duplications in the RNAi pathway might explain why Leptinotarsa decemlineata has high sensitivity to dsRNA. The L. decemlineata genome provides opportunities to investigate a broad range of phenotypes and to develop sustainable methods to control this widely successful pest.

Quantitative measure of sexual selection with respect to the operational sex ratio: a comparison of selection indices.

Despite numerous indices proposed to predict the evolution of mating systems, a unified measure of sexual selection has remained elusive. Three previous studies have compared indices of sexual selection under laboratory conditions. Here, we use a genetic study to compare the most widely used measures of sexual selection in natural populations. We explored the mating and reproductive successes of male and female bank voles, Clethrionomys glareolus, across manipulated operational sex ratios (OSRs) by genotyping all adult and pup bank voles on 13 islands using six microsatellite loci. We used Bateman's principles (Is and I and Bateman gradients) and selection coefficients (s' and beta') to evaluate, for the first time, the genetic mating system of bank voles and compared these measures with alternative indices of sexual selection (index of monopolization and Morisita's index) across the OSRs. We found that all the sexual selection indices show significant positive intercorrelations for both males and females, suggesting that Bateman's principles are an accurate and a valid measure of the mating system. The Bateman gradient, in particular, provides information over and above that of other sexual selection indices. Male bank voles show a greater potential for sexual selection than females, and Bateman gradients indicate a polygynandrous mating system. Selection coefficients reveal strong selection gradients on male bank vole plasma testosterone level rather than body size.

Negative density-distribution relationship in butterflies.

BACKGROUND: Because "laws of nature" do not exist in ecology, much of the foundations of community ecology rely on broad statistical generalisations. One of the strongest generalisations is the positive relationship between density and distribution within a given taxonomic assemblage; that is, locally abundant species are more widespread than locally sparse species. Several mechanisms have been proposed to create this positive relationship, and the testing of these mechanisms is attracting increasing attention. RESULTS: We report a strong, but counterintuitive, negative relationship between density and distribution in the butterfly fauna of Finland. With an exceptionally comprehensive data set (data includes all 95 resident species in Finland and over 1.5 million individuals), we have been able to submit several of the mechanisms to powerful direct empirical testing. Without exception, we failed to find evidence for the proposed mechanisms creating a positive density-distribution relationship. On the contrary, we found that many of the mechanisms are equally able to generate a negative relationship. CONCLUSION: We suggest that one important determinant of density-distribution relationships is the geographical location of the study: on the edge of a distribution range, suitable habitat patches are likely to be more isolated than in the core of the range. In such a situation, only the largest and best quality patches are likely to be occupied, and these by definition can support a relatively dense population leading to a negative density-distribution relationship. Finally, we conclude that generalizations about the positive density-distribution relationship should be made more cautiously.

Directional postcopulatory sexual selection is associated with female sperm storage in Trinidadian guppies.

Female sperm storage (FSS) is taxonomically widespread and often associated with intense sperm competition, yet its consequences on postcopulatory sexual selection (PCSS) are poorly known. Theory predicts that FSS will reduce the strength of PCSS, because sperm characteristics favored before and after FSS may be traded-off, and opportunities for nondirectional PCSS should increase. We explored these questions in the guppy (Poecilia reticulata), by allowing females to mate multiply and by comparing the paternity pattern in two successive broods. Contrary to predictions, the variance in male fertilization success increased after FSS, driven by a change in male paternity share across broods. This change was positively associated with sperm velocity (measured before FSS) but not with the duration of FSS, indirectly suggesting that faster sperm were better in entering female storage organs, rather than in persisting within them. Other male traits, such as male size and orange color, heterozygosity, and relatedness to the female, did not influence paternity after FSS. These results indicate that processes associated with FSS tend to reinforce the strength of PCSS in guppies, rather than weaken it. Further work is necessary to test whether this pattern changes in case of more prolonged FSS.