Sub-acute exposure of male guppies (Poecilia reticulata) to environmentally relevant concentrations of PFOA and GenX induces significant changes in the testis transcriptome and reproductive traits.

Poly- and perfluoroalkyl substances (PFAS) are frequently detected in the environment and are linked to adverse reproductive health outcomes in humans. Although legacy PFAS have been phased out due to their toxicity, alternative PFAS are increasingly used despite the fact that information on their toxic effects on reproductive traits is particularly scarce. Here, we exposed male guppies (Poecilia reticulata) for a short period (21 days) to an environmentally realistic concentration (1 ppb) of PFOA, a legacy PFAS, and its replacement compound, GenX, to assess their impact on reproductive traits and gene expression. Exposure to PFAS did not impair survival but instead caused sublethal effects. Overall, PFAS exposure caused changes in male sexual behaviour and had detrimental effects on sperm motility. Sublethal variations were also seen at the transcriptional level, with the modulation of genes involved in immune regulation, spermatogenesis, and oxidative stress. We also observed bioaccumulation of PFAS, which was higher for PFOA than for GenX. Our results offer a comprehensive comparison of these two PFAS and shed light on the toxicity of a newly emerging alternative to legacy PFAS. It is therefore evident that even at low concentrations and with short exposure, PFAS can have subtle yet significant effects on behaviour, fertility, and immunity. These findings underscore the potential ramifications of pollution under natural conditions and their impact on fish populations.

Possible glimpses into early speciation: the effect of ovarian fluid on sperm velocity accords with post-copulatory isolation between two guppy populations.

Identifying mechanisms of reproductive isolation is key to understanding speciation. Among the putative mechanisms underlying reproductive isolation, sperm-female interactions (post-mating-prezygotic barriers) are arguably the hardest to identify, not least because these are likely to operate at the cellular or molecular level. Yet sperm-female interactions offer great potential to prevent the transfer of genetic information between different populations at the initial stages of speciation. Here, we provide a preliminary test for the presence of a putative post-mating-prezygotic barrier operating between three populations of Trinidadian guppies (Poecilia reticulata), an internally fertilizing fish that inhabits streams with different levels of connectivity across Trinidad. We experimentally evaluate the effect of female ovarian fluid on sperm velocity (a predictor of competitive fertilization success) according to whether males and females were from the same (native) or different (foreign) populations. Our results reveal the potential for ovarian fluid to act as a post-mating-prezygotic barrier between two populations from different drainages, but also that the strength of this barrier is different among populations. This result may explain the previous finding that, in some populations, sperm from native males have precedence over foreign sperm, which could eventually lead to reproductive isolation between these populations.

Effect of male age on sperm traits and sperm competition success in the guppy (Poecilia reticulata).

Deleterious mutations can accumulate in the germline with age, decreasing the genetic quality of sperm and imposing a cost on female fitness. If these mutations also affect sperm competition ability or sperm production, then females will benefit from polyandry as it incites sperm competition and, consequently, minimizes the mutational load in the offspring. We tested this hypothesis in the guppy (Poecilia reticulata), a species characterized by polyandry and intense sperm competition, by investigating whether age affects post-copulatory male traits and sperm competition success. Females did not discriminate between old and young males in a mate choice experiment. While old males produced longer and slower sperm with larger reserves of strippable sperm, compared to young males, artificial insemination did not reveal any effect of age on sperm competition success. Altogether, these results do not support the hypothesis that polyandry evolved in response to costs associated with mating with old males in the guppy.

Nestlings' carotenoid feather ornament affects parental allocation strategy and reduces maternal survival.

In some birds, feather ornaments are expressed in nestlings well before sexual maturation, possibly in response to parental favouritism towards high-quality offspring. In species with synchronous hatching, in which nestling ornaments may vary more among than within broods, parents may use this information to adjust their parental allocation to the current brood accordingly. We tested this hypothesis in the rock sparrow, in which a sexually selected yellow feather ornament is also expressed in nestlings. We experimentally enlarged nestlings' breast patch in a group of broods and sham-manipulated another group of control broods. Nestlings with enlarged ornament were fed more frequently and defended more actively from a dummy predator than their control counterparts. Mothers from the enlarged group were more likely to lay a second clutch and showed a reduced survival to the next breeding season. These results provide one of the first evidences of differential parental allocation among different broods based directly on nestlings' ornamentation, and the first, to our knowledge, to show a reduction in maternal survival.

Moult speed affects structural feather ornaments in the blue tit.

Growing evidence suggests that structural feather colours honestly reflect individual quality or body condition but, contrary to pigment-based colours, it is not clear what mechanism links condition to reflectance in structural feather colours. We experimentally accelerated the moult speed of a group of blue tits (Cyanistes caeruleus) by exposing them to a rapidly decreasing photoperiod and compared the spectral characteristics of their structural feather colours with those of control birds. Blue tits were sexually dimorphic on the UV/blue crown and on the white cheek feathers. Moult speed, however, dramatically reduced brightness and the saturation only on the UV/blue crown feathers, whereas structural white on the cheek feathers was basically unaffected by moult speed. Given that the time available for moulting is usually confined to the period between the end of the breeding season and migration or wintering, UV/blue colours, but not structural white, may convey long-term information about an individual's performance during the previous breeding season. The trade-off between fast moulting and structural colour expression may represent a previously unrecognized selective advantage for early-breeding birds.

Moult speed constrains the expression of a carotenoid-based sexual ornament.

We investigated the effect of moult speed on the expression of a sexually selected, carotenoid-based feather ornament in the rock sparrow (Petronia petronia). We experimentally accelerated the moult speed of a group of birds by exposing them to a rapidly decreasing photoperiod and compared the area and the spectral characteristics of their ornaments with those of control birds. Birds with accelerated moulting rate showed a smaller yellow patch with lower yellow reflectance compared to their slow-moulting counterparts. Considering that the time available for moulting is usually constrained between the end of the breeding season and migration or wintering, carotenoid feather ornaments, whose expression is mediated by moult speed, may convey long term information about an individual's condition, potentially encompassing the previous breeding season. Furthermore, the observed trade-off between moult speed and ornament expression may represent a previously unrecognized selective advantage for early breeding birds.

Colourful male guppies produce faster and more viable sperm.

In guppies (Poecilia reticulata) precopulatory sexual selection (via female choice) and post-copulatory selection (via sperm competition) both favour males with relatively high levels of carotenoid (orange) pigmentation, suggesting that colourful males produce more competitive ejaculates. Here we test whether there is a positive association between male orange pigmentation and sperm quality. Our analysis of sperm quality focused on sperm swimming speeds (using CASA: computer-assisted sperm analysis to estimate three parameters of sperm velocity in vitro), sperm viability (proportion of live sperm per stripped ejaculate) and sperm lengths. We found that males with relatively large areas of orange pigmentation had significantly faster and more viable sperm than their less ornamented counterparts, suggesting a possible link between dietary carotenoid intake and sperm quality. By contrast, we found no relationship between sperm length (head length and total sperm length) and male phenotype. These findings, in conjunction with previous work showing that highly ornamented male guppies sire higher quality offspring, suggest that female preference for colourful males and sperm competition work in concert to favour intrinsically higher quality males.

Offspring sexual dimorphism and sex-allocation in relation to parental age and paternal ornamentation in the barn swallow.

We analysed the morphology of nestling barn swallows (Hirundo rustica) in relation to their sex, and laying and hatching order. In addition, we studied sex-allocation in relation to parentage, parental age and expression of a secondary sexual character of fathers. Molecular sexing was conducted using the sex chromosome-linked avian CHD1 gene. Sex of the offspring was not associated with laying or hatching order. None of nine morphological, serological and immunological variables varied in relation to offspring sex. Sexual dimorphism did not vary in relation to parental age and expression of a paternal secondary sexual character. The proportion of sons declined with brood size. Individual males and females had a similar proportion of sons during consecutive breeding years. The proportion of sons of individual females declined with age, but increased with the expression of a secondary sexual character of their current mate. The generalized lack of variation in sexual dimorphism among nestlings may suggest that barn swallows do not differentially invest in sons vs. daughters. Alternatively, male offspring may require different parental effort compared to their female siblings in order to attain the same morphological state. The lack of variation in offspring sexual dimorphism with paternal ornamentation suggests no adjustment of overall parental effort in relation to reproductive value of the two sexes. However, male-biased sex ratio among offspring of highly ornamented males may represent an adaptive sex-allocation strategy because the expression of male ornaments is heritable and highly ornamented males are at a sexual selection advantage.

Variation of female preference for male coloration in the eastern mosquitofish Gambusia holbrooki.

The preference for melanistic males was studied in two populations of eastern mosquitofish (Gambusia holbrooki, Pisces: Poeciliidae), one from Florida and one from northern Italy. Melanism in the eastern mosquitofish is a Y--linked character, expressed in males only. Melanistic males have black spots varying in size and number. In the Florida population, melanistic males are common, whereas in the Italian population they have never been observed. Females were male-deprived for at least 2 months before being tested in a dichotomous choice chamber. Italian females showed a significant preference for unpigmented males from their own population, whereas Florida females preferred melanistic males. When given the choice between males with few (< 10% of the body surface) and males with many (> 50%) black spots, Italian females preferred males with few black spots and Florida females those with many black spots. The preference of the Italian females for unpigmented males was confirmed in females reared from birth to maturity in the presence of only melanistic males. The preference of Florida females for melanistic males was also confirmed in females reared from birth to maturity in the presence of only unpigmented males. Altogether, these results demonstrate that in the eastern mosquitofish there is polymorphism in female preference and that this preference does not have an environmental basis.

Effects of chronic dietary cadmium on hepatic glutathione levels and glutathione peroxidase activity in starlings (Sturnus vulgaris).

The effects of chronic exposure to dietary cadmium on the levels of hepatic glutathione (GSH) and on the activity of the glutathione peroxidase enzymes (GSH-Px) were studied for the first time in starlings (Sturnus vulgaris). Thirty-three individuals (17 females and 16 males) were divided into three groups: One represented the untreated control and two were respectively fed with diets containing 10 and 50 ppm cadmium chloride (CdCl(2)). The total duration of treatment was 22 weeks. The three groups respectively accumulated mean hepatic Cd residues of 2.29, 75.71, and 208.49 ppm. Hepatic GSH increased in the treated groups respectively 24% and 52% in comparison to controls. Total GSH-Px activity in the liver was inhibited in the group fed with 50 ppm, due to inhibition of the selenium-dependent fraction of the enzyme, while the selenium-independent fraction did not change significantly. During the treatment, after 14 weeks of exposure to cadmium, the 50 ppm-treated group showed a 47% decrease of the activity of the selenium-dependent GSH-Px and a 50% increase of the somatic liver index in comparison with controls.

DNA fingerprinting data and the analysis of population genetic structure by comparing band-sharing patterns.

Genetic isolation among populations can be effectively investigated by multilocus DNA fingerprinting. If populations have diverged, it is expected that the mean proportion of bands shared by individuals from the same population, Bw, exceeds the corresponding mean, Bb, calculated from pairs of individuals from distinct populations. A problem arises in deciding whether any difference between Bw and Bb is statistically significant. In fact, any two band-sharing data (bij), contributing to Bw or Bb, are not independent if they share a common individual (like bij and bjl). This prevents a correct application of parametric tests, such as the Student's t-test. Recently, a modification of this test has been proposed that should avoid the independence problem. Using a large number of samples of fingerprints, simulated from an appropriate 'genetic' model, under a wide range of conditions, we compared the performances of the Student's t-test, the modified t-test and five new permutation tests, where individuals, rather than bij values, are permuted. We found that: (i) the Student's t-test can be very permissive, rejecting too often the null hypothesis when true, but is correct or conservative in certain cases; (ii) the modified t-test is extremely conservative when the null hypothesis is true and very inefficient otherwise; (iii) all five permutation tests are strictly correct, provided that individuals are ordered randomly on gels; and (iv) in this case, the permutation tests are equally efficient, and are not inferior to the Student's t-test when the latter is approximately correct and provides a fair benchmark.

Genetic variation and bill size dimorphism in a passerine bird, the reed bunting Emberiza schoeniclus.

In passerine birds morphological differentiation in bill size within species is not commonly observed. Bill size is usually associated with a trophic niche, and strong differences in it may reflect the process of genetic differentiation and, possibly, speciation. We used both mitochondrial DNA (mtDNA) and nuclear microsatellites to study genetic variation between two subspecies of reed bunting, Emberiza schoeniclus schoeniclus and E.s. intermedia, along their distributional boundary in western Europe. These two subspecies are characterized by a high dimorphism in bill size and, although breeding populations of the two subspecies are found very close to each other in northern Italy, apparently no interbreeding occurs. The observed morphological pattern between the two subspecies may be maintained by geographically varying selective forces or, alternatively, may be the result of a long geographical separation followed by a secondary contact. MtDNA sequences of cytochrome b and ND5 (515 bp) showed little variation and did not discriminate between the two subspecies, indicating a divergence time of less than 500 000 years. The analysis of four microsatellite loci suggested a clear, although weak, degree of genetic differentiation in the large- and small-billed populations, as indicated by FST and RST values and genetic distances. The correlation between bill size and genetic distance between populations remained significant after accounting for the geographical distances between sampling localities. Altogether, these results indicate a very recent genetic differentiation between the two bill morphs and suggest that a strong selection for large bills in the southern part of the breeding range is probably involved in maintaining the geographical differentiation of this species.

Genetic variation in North Africa and Eurasia: neolithic demic diffusion vs. Paleolithic colonisation.

The hypothesis that both genetic and linguistic similarities among Eurasian and North African populations are due to demic diffusion of neolithic farmers is tested against a wide database of allele frequencies. Demic diffusion of farming and languages from the Near East should have determined clines in areas defined by linguistic criteria; the alternative hypothesis of cultural transmission does not predict clines. Spatial autocorrelation analysis shows significant gradients in three of the four linguistic families supposedly affected by neolithic demic diffusion; the Afroasiatic family is the exception. Many such gradients are not observed when populations are jointly analyzed, regardless of linguistic classification. This is incompatible with the hypothesis that major cultural transformations in Eurasia (diffusion of related languages and spread of agriculture) took place without major demographic changes. The model of demic diffusion seems therefore to provide a mechanism explaining coevolution of linguistic and biological traits in much of the Old World. Archaeological, linguistic, and genetic evidence agree in suggesting a multidirectional process of gene flow from the Near East in the neolithic. However, the possibility should be envisaged that some allele frequency patterns can predate the neolithic and depend on the initial spread of Homo sapiens sapiens from Africa into Eurasia.

Genetic evidence on origin and dispersal of human populations speaking languages of the Nostratic macrofamily.

Contemporary patterns of allele frequencies allow inferences on past evolutionary processes. L.L. CavalliSforza [(1988) Munibe 6, 129-137] and C. Renfrew [(1991) Cambridge Archaeol. J. 1, 3-23] proposed that neolithic farmers from the Near East propagated a group of related ancestral languages, from which three or four linguistic families developed. Here we show that genetic variation among Indo-European, Elamo-Dravidian, and Altaic speakers (grouped by some linguists in the Nostratic macrofamily) supports this hypothesis, whereas the evidence on Afro-Asiatic speakers is ambiguous. Gene-frequency clines within these linguistic families suggest that language diffusion was largely associated with population movements rather than with purely cultural transmission. Archeological, linguistic, and genetic evidence can be reconciled by envisaging a process of population growth and multidirectional dispersal from the Near East as the main factor shaping genetic and linguistic diversity in Eurasia and perhaps in North Africa.