Extra-pair paternity is not repeatable between years in Pied Flycatchers (Ficedula hypoleuca): a role for social context

Extra-pair paternity (EPP) is the consequence of the interactions between a social pair and extra-pair males during the female's fertile phase in a specific social context and ecological environment. Although EPP occurs in many avian species, there is a lack of understanding of how environmental factors may impact its frequency. Accordingly, the relative importance of individual characteristics on the one hand, and of their immediate social environment on the other, may affect EPP repeatability and thereby the capacity of this trait to respond to selection. If EPP is an individual genetically based trait that may respond to selection, we expect it to be repeatable across breeding seasons. To check this possibility, we analysed the within-individual repeatability in EPP of breeding males and females in two natural populations of Pied Flycatchers Ficedula hypoleuca in central Spain during three field seasons. We then studied the relationship between EPP and individual male and female traits (only some of which were themselves repeatable), as well as key context variables such as breeding synchrony and population density. Our results showed no repeatability for EPP in either sex. We found a positive association with laying date and stronger associations of EPP with male plumage and morphological traits than with female characteristics. We suggest that the variable social environment is an important modulator of EPP patterns, and that differences between field EPP studies in this respect may explain contradictory results in the same species, even in the same populations in different years.     

Screening for ferrochelatase mutations: molecular heterogeneity of erythropoietic protoporphyria

The DNA of 21 patients from 19 unrelated families with erythropoietic protoporphyria (EPP) were screened for the 6 ferrochelatase point mutations so far described. The mutation previously described by us (A ? t transversion at position ?3 of the donor site of intron 10, causing exon 10 skipping) was detected in two additional unrelated EPP patients: in these patients, cDNA lacking exon 10 was also detected. The mutation described by Nakahashi et al. as responsible for exon 2 skipping (C ? T transition at position ?23 of the acceptor site of intron 1), although also observed in some normal individuals, was invariably observed in all EPP patients tested and may thus play some role in the pathognesis of EPP. Thus, it does not appear that this mutation is the primary mechanism underlying exon 2 skipping. None of the other four previously described mutations were detected. These data demonstrate the heterogeneity of the ferrochelatase locus and of the genetic defect in EPP.     

Systematic analysis of molecular defects in the ferrochelatase gene from patients with erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP; MIM 177000) is an inherited disorder caused by partial deficiency of ferrochelatase (FECH), the last enzyme in the heme biosynthetic pathway. In EPP patients, the FECH deficiency causes accumulation of free protoporphyrin in the erythron, associated with a painful skin photosensitivity. In rare cases, the massive accumulation of protoporphyrin in hepatocytes may lead to a rapidly progressive liver failure. The mode of inheritance in EPP is complex and can be either autosomal dominant with low clinical penetrance, as it is in most cases, or autosomal recessive. To acquire an in-depth knowledge of the genetic basis of EPP, we conducted a systematic mutation analysis of the FECH gene, following a procedure that combines the exon-by-exon denaturing-gradient-gel-electrophoresis screening of the FECH genomic DNA and direct sequencing. Twenty different mutations, 15 of which are newly described here, have been characterized in 26 of 29 EPP patients of Swiss and French origin. All the EPP patients, including those with liver complications, were heterozygous for the mutations identified in the FECH gene. The deleterious effect of all missense mutations has been assessed by bacterial expression of the respective FECH cDNAs generated by site-directed mutagenesis. Mutations leading to a null allele were a common feature among three EPP pedigrees with liver complications. Our systematic molecular study has resulted in a significant enlargement of the mutation repertoire in the FECH gene and has shed new light on the hereditary behavior of EPP.     

Loss-of-Function Ferrochelatase and Gain-of-Function Erythroid-Specific 5-Aminolevulinate Synthase Mutations Causing Erythropoietic Protoporphyria and X-Linked Protoporphyria in North American Patients Reveal Novel Mutations and a High Prevalence of X-Linked Protoporphyria

Erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP) are inborn errors of heme biosynthesis with the same phenotype but resulting from autosomal recessive loss-of-function mutations in the ferrochelatase (FECH) gene and gain-of-function mutations in the X-linked erythroid-specific 5-aminolevulinate synthase (ALAS2) gene, respectively. The EPP phenotype is characterized by acute, painful, cutaneous photosensitivity and elevated erythrocyte protoporphyrin levels. We report the FECH and ALAS2 mutations in 155 unrelated North American patients with the EPP phenotype. FECH sequencing and dosage analyses identified 140 patients with EPP: 134 with one loss-of-function allele and the common IVS3-48T>C low expression allele, three with two loss-of-function mutations and three with one loss-of-function mutation and two low expression alleles. There were 48 previously reported and 23 novel FECH mutations. The remaining 15 probands had ALAS2 gain-of-function mutations causing XLP: 13 with the previously reported deletion, c.1706_1709delAGTG, and two with novel mutations, c.1734delG and c.1642C>T(p.Q548X). Notably, XLP represented ~10% of EPP phenotype patients in North America, two to five times more than in Western Europe. XLP males had twofold higher erythrocyte protoporphyrin levels than EPP patients, predisposing to more severe photosensitivity and liver disease. Identification of XLP patients permits accurate diagnosis and counseling of at-risk relatives and asymptomatic heterozygotes.     

Genetic diversity does not explain variation in extra‐pair paternity in multiple populations of a songbird

Many songbirds are socially monogamous but genetically polyandrous, mating with individuals outside their pair bonds. Extra‐pair paternity (EPP) varies within and across species, but reasons for this variation remain unclear. One possible source of variation is population genetic diversity, which has been shown in interspecific meta‐analyses to correlate with EPP but which has limited support from intraspecific tests. Using eight populations of the genetically polyandrous red‐winged blackbird (Agelaius phoeniceus), including an island population, we investigated whether population‐level differences in genetic diversity led to differences in EPP. We first measured genetic diversity over 10 microsatellite loci and found, as predicted, low genetic diversity in the island population. Additional structure analyses with multilocus genotypes and mtDNA showed the island population to be distinct from the continental populations. However, the island population's EPP rate fell in the middle of the continental populations' distribution, whereas the continental populations themselves showed significant variation in EPP. This result suggests that genetic diversity by itself is not a predictor of EPP rate. We discuss reasons for the departure from previous results, including hypotheses for EPP that do not solely implicate female‐driven behaviour.     

Molecular defects in erythropoietic protoporphyria with terminal liver failure

We identified two additional mutations in the ferrochelatase gene in two Swiss patients with erythropoietic protoporphyria (EPP). Ferrochelatase cDNA from patients was amplified by the polymerase chain reaction (PCR) and subjected to mutation analysis by sequencing PCR products either directly or after subcloning. The first patient, who underwent liver transplantation because of terminal liver failure, was identified as having a single point mutation (C to T) at nucleotide 175 that resulted in a Gln to stop codon conversion in one allele of the gene. In the second case, in which the patient has so far no liver involvement, a two-base deletion (T899G900) was found in one allele. Frameshift as a result of the deletion creates a stop codon. This study presents two new genotypes of EPP, including one with liver failure, a rare and fatal form of EPP.     

Large brain size is associated with low extra‐pair paternity across bird species

BackgroundGaining extrapair copulations (EPCs) is a complicated behavior process. The interaction between males and females to procure EPCs may be involved in brain function evolution and lead to a larger brain. Thus, we hypothesized that extrapair paternity (EPP) rate can be predicted by relative brain size in birds. Past work has implied that the EPP rate is associated with brain size, but empirical evidence is rare.MethodsWe collated data from published references on EPP levels and brain size of 215 bird species to examine whether the evolution of EPP rate can be predicted by brain size using phylogenetically generalized least square (PGLS) models and phylogenetic path analyses.ResultsWe found that EPP rates (both the percentage EP offspring and percentage of broods with EP offspring) are negatively associated with relative brain size. We applied phylogenetic path analysis to test the causal relationship between relative brain size and EPP rate. Best‐supported models (ΔCICc < 2) suggested that large brain lead to reduced EPP rate, which failed to support the hypothesis that high rates of EPP cause the evolution of larger brains.ConclusionThis study indicates that pursuing EPCs may be a natural instinct in birds and the interaction between males and females for EPCs may lead to large brains, which in turn may restrict their EPC level for both sexes across bird species.     

Tests of spatial and temporal factors influencing extra-pair paternity in red-winged blackbirds

Extra-pair paternity (EPP) is a widespread and highly variable reproductive phenomenon in birds. We tested the effects of habitat, spatial factors, and timing of breeding on the occurrence of EPP in red-winged blackbirds (Agelaius phoeniceus). We used PCR-amplified microsatellites to assess the paternity of 1479 nestlings from 537 broods on 235 territories over four breeding seasons. Over 4 years, 40% of nestlings were extra-pair. At least 27% of actual sires were non-neighbours, suggesting that males or females interacted over longer distances than in other populations of red-winged blackbirds. The level of EPP was significantly clumped within broods and males but not within females across broods. EPP was negatively related to the area of a male's territory. The spatial proximity of a female's nest to the territory boundary had no effect on total EPP, but tended to increase the probability of an EPP by a nearby male. We found no influence on EPP of the type of habitat on the territory or the level of nesting activity nearby. The time in the season when a nest was started and the synchrony of breeding also had no significant effect on the level of EPP. The age of the male, the age of his neighbours, and the interaction between the two had no effect on total EPP. However, older males were less likely to have an offspring sired by a neighbour on their territory. Males with older neighbours were also less likely to have offspring sired by a neighbour, particularly if they were new territory owners. The high variability in who gained and lost paternity, and the limited impact of spatial and temporal factors influencing it, have some interesting implications for theories seeking to explain mating patterns.     

Multiple hypotheses explain variation in extra‐pair paternity at different levels in a single bird family

Extra‐pair paternity (EPP), where offspring are sired by a male other than the social male, varies enormously both within and among species. Trying to explain this variation has proved difficult because the majority of the interspecific variation is phylogenetically based. Ideally, variation in EPP should be investigated in closely related species, but clades with sufficient variation are rare. We present a comprehensive multifactorial test to explain variation in EPP among individuals in 20 populations of nine species over 89 years from a single bird family (Maluridae). Females had higher EPP in the presence of more helpers, more neighbours or if paired incestuously. Furthermore, higher EPP occurred in years with many incestuous pairs, populations with many helpers and species with high male density or in which males provide less care. Altogether, these variables accounted for 48% of the total and 89% of the interspecific and interpopulation variation in EPP. These findings indicate why consistent patterns in EPP have been so challenging to detect and suggest that a single predictor is unlikely to account for the enormous variation in EPP across levels of analysis. Nevertheless, it also shows that existing hypotheses can explain the variation in EPP well and that the density of males in particular is a good predictor to explain variation in EPP among species when a large part of the confounding effect of phylogeny is excluded.     

Weather conditions affect levels of extra-pair paternity in the reed bunting Emberiza schoeniclus

Extra-pair paternity (EPP) is common in many socially monogamous birds, but large variations in frequency of EPP are found both between and within species. Local ecological factors can affect the costs and benefits of extra-pair mating behaviour, and may therefore influence the chance that individuals engage in extra-pair copulations (EPCs). We investigated the effect of weather conditions during the peak fertile period of the female on the levels of EPP in reed buntings Emberiza schoeniclus . The reed bunting is a socially monogamous passerine, with extremely high levels of EPP (50% of offspring in 80% of broods). We found that higher daily minimum temperatures and more rainfall during the peak fertile period were associated with lower proportions of EPP. As during adverse weather conditions individuals have to invest more in self maintenance, we suggest that during long periods of rain the extra-pair mating behaviour of all individuals will be restricted, leading to lower proportions of EPP. During cold mornings, time-consuming activities such as mate guarding are likely to be more strongly affected than less time-consuming activities such as EPCs, leading to higher proportions of EPP.     

Extra-pair paternity and sexual dimorphism in birds

Differences in the strength of sexual selection between males and females can lead to sexual dimorphism. Extra-pair paternity (EPP) can increase the variance in male reproductive success and hence the opportunity for sexual selection. Previous research on birds suggests that EPP drives the evolution of dimorphism in plumage colour and in body size. Because EPP increases the intensity of sexual selection in males, it should lead to increased dimorphism in species with larger or more colourful males, but decreased dimorphism in species with larger or more colourful females. We explored the covariation between EPP and sexual dimorphism in wing length and plumage colouration in 401 bird species, while controlling for other, potentially confounding variables. Wing length dimorphism was associated positively with the frequency of EPP, but also with social polygamy, sex bias in parental behaviour and body size and negatively with migration distance. The frequency of EPP was the only predictor of plumage colour dimorphism. In support of our prediction, high EPP levels were associated with sexual dichromatism, positively in species in which males are more colourful and negatively in those in which females are more colourful. Contrary to our prediction, high EPP rates were associated with increased wing length dimorphism in species with both male- and female-biased dimorphism. The results support a role for EPP in the evolution of both size and plumage colour dimorphism. The two forms of dimorphism were weakly correlated and predicted by different reproductive, social and life-history traits, suggesting an independent evolution.     

A genotype-phenotype correlation between null-allele mutations in the ferrochelatase gene and liver complication in patients with erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP), an inborn error of heme metabolism, causes in the majority of the patients only a symptom of photosensitivity. However, around 2% of the EPP sufferers develop liver complication in the form of liver cirrhosis and progressive liver failure. Mutations in the human ferrochelatase (FECH) gene causing EPP are highly heterogeneous and mostly family-specific. Actually, 62 FECH mutations have been published, 48 of them are "null allele" mutations inducing the formation of a truncated protein. The remaining 14 are missense mutations. In contrast to the null allele mutations, the latter lead to substitution of a single amino acid residue in the protein molecule and generate an enzyme that, although functionally impaired, is in its full length. In order to study the association between "null allele" mutation and liver complication, we combined our data with those in the literature. A total of 112 EPP patients were counted among 93 EPP families with a known FECH mutation. All 18 EPP patients who had severe liver complication carried a "null allele" mutation. In contrast, none of the 20 patients who carried a missense mutation had developed liver complication till the time of study (Fisher's exact test, p<0.05). High protoporphyrin blood concentration are considered to be a sign of an increased risk of liver disease. No correlation of protoporphyrin blood level with the type of mutation, was found, if patients with overt liver disease were excluded from the sample. Furthermore, no significant association of the liver complication with the location of the mutation within the FECH gene was found (Fisher exact test p = 0.46). These available data indicate a significant genotype-phenotype correlation between "null allele" mutation and protoporphyrin related liver disease in EPP. Although the risk for a EPP patient with a missense mutation to develop liver disease cannot be totally eliminated based on these data, it is comparably low.     

High rates of extra‐pair paternity in two equatorial populations of rufous‐collared sparrow,Zonotrichia capensis

The latitudinal increase in extra‐pair paternity (EPP) rates in birds suggests broad selective benefits to low EPP rates in the tropics. However, we have few EPP data from tropical birds, particularly from species with close relatives at high latitudes. Here, we report EPP rates in two resident equatorial populations of rufous‐collared sparrow Zonotrichia capensis, a genus well‐represented at high latitudes. We found 64% and 60% of broods contained extra‐pair offspring, and 42% and 52% of all young were extra‐pair. EPP rates were similar in these populations, despite clear differences in elevation, temperature, rainfall, and breeding season length. These findings provide evidence that EPP rates in tropical birds can be as high as those observed in temperate birds, and suggest that the selective pressures acting on EPP rates vary markedly across tropical birds.     

Extra-pair paternity in the strongly monogamous Wandering Albatross Diomedea exulans has no apparent benefits for females

Although 92% of avian species are socially monogamous, extra-pair copulation (EPC), resulting in extra-pair paternity (EPP), is a common reproductive strategy in birds. Among seabirds, in which the rate of social monogamy reaches 100%, Procellariiformes (albatrosses and petrels) show low EPP rates, with the noticeable exception of the only albatross investigated in this regard, the Waved Albatross Phoebastria irrorata . This species, in which forced copulations are known to occur, showed a surprisingly high rate of EPP (25% of chicks). We investigate here EPP rates in another albatross species, the Wandering Albatross Diomedea exulans , subject to a demographic survey conducted for 38 years. We combined data on pair bonds with analysis of ten microsatellite loci and found that 10.7% of 75 chicks had an extra-pair sire. Although there was some evidence for inbreeding avoidance, within-pair and extra-pair chicks showed similar levels of heterozygosity, and the incidence of EPP was independent of age, experience or past reproductive success. Hence, we found no evidence that females benefit from EPCs. Owing to the male-biased sex ratio in adults, widowed and divorced males required more time to find a new mate (+28 and +72%, respectively) than did females. Combined with high sexual size dimorphism, this phenomenon might promote the forced copulations observed in this species. Our data therefore suggest that EPC is beneficial to unpaired males but occurs at random in females, consistent with the hypothesis that EPP results solely from forced EPCs. However, the importance of the latter for EPP and the part played by solitary males require further investigation.     

Extra-pair paternity does not result in differential sexual selection in the mutually ornamented black swan (Cygnus atratus)

We studied patterns of parentage in 85 broods (332 cygnets) of black swans during three breeding seasons, using a set of eight polymorphic microsatellite markers. We detected both intraspecific brood parasitism (IBP; < 5% of cygnets per year) and extra-pair paternity (EPP). In these years, 10-17% (mean = 15.1%) of cygnets resulted from EPP, and 27-40% (mean 37.6%) of broods contained at least one extra-pair cygnet. Compared with levels of EPP in closely related species with similar life histories, these values are unexpectedly high. EPP in black swans appears unrelated to ecological factors (breeding density and synchrony) or genetic factors (genetic similarity between pair members or genetic quality of the offspring). We found no evidence that a mutual sexual feather ornament known to play a role in social mate choice in black swans (curled wing feathers) is involved in extra-pair mate choice. EPP does not lead to greater variance in reproductive success in males, relative to females in this species. We therefore suggest that EPP does not result in differential sexual selection on males and females, explaining why they are ornamented to the same degree.     

Extra‐pair paternity in birds

Since the first molecular study providing evidence for mating outside the pair bond in birds over 30 years ago, >500 studies have reported rates of extra‐pair paternity (EPP) in >300 bird species. Here, we give a detailed overview of the current literature reporting EPP in birds and highlight the sampling biases and patterns in the data set with respect to taxonomy, avian phylogeny and global regions, knowledge of which will be crucial for correct interpretation of results in future comparative studies. Subsequently, we use this comprehensive dataset to simultaneously test the role of several ecological and life history variables. We do not find clear evidence that variation in EPP across socially monogamous species can be explained by latitude, density (coloniality), migration, generation length, genetic structuring (dispersal distance), or climatic variability, after accounting for phylogeny. These results contrast previous studies, most likely due to the large heterogeneity within species in both EPP and the predictor of interest, indicating that using species averages might be unreliable. Despite the absence of broadscale ecological drivers in explaining interspecific variation in EPP, we suggest that certain behaviours and ecological variables might facilitate or constrain EPP, as indicated by our finding that EPP was negatively associated with latitude within noncolonial species, suggesting a role of breeding synchrony. Thus, rather than focussing on general explanations for variation in EPP across all species, a future focus should be on how various aspects of ecology or life history might have driven variation in EPP among groups of species or populations of the same species. Hence, we argue that variation in EPP can be partly explained when taking the right perspective. This comprehensive overview, and particularly the dataset provided herein will create a foundation for further studies.     

Extrapair paternity, migration, and breeding synchrony in birds

To understand interspecific patterns in the strength of sexualselection, variation in the costs and benefits of exercisingmate choice needs to be evaluated. One manifestation of sexualselection in birds is the occurrence of greatly variable levelsof extrapair paternity (EPP). A proposed general explanationfor this variation is that the benefits to females in seekingextrapair copulations vary in a predictable manner accordingto the degree of breeding synchrony because females are betterable to assess potential extrapair partners when males are simultaneouslyin breeding condition. This hypothesis predicts a latitudinaltrend in EPP because birds tend to breed more synchronouslyaway from the equator. Expanding on previous geographicallyand taxonomically restricted tropical/temperate comparisons,we used phylogenetically independent standardized linear contraststo show that this positive relationship persists when all birdspecies for which EPP estimates currently exist are considered.However, if a third factor covaries with latitude in the sameway as breeding synchrony and EPP, this relationship need notbe causal. Migration could also account for latitudinal variationin the benefits to females of pursuing EPP, if migration isassociated with (1) hasty or (2) inaccurate mate choice, (3)facilitated assessment of male quality through the condition-dependenceof arrival time, or (4) increased genetic variance in male quality.We show that migration distance is positively related to theproportion of EPP and that migration can statistically explainthe latitudinal trend in EPP, even when confounding factorsare simultaneously controlled. Hence, alternative explanationsfor latitudinal variation in EPP may be feasible, and carefulintraspecific tests are needed to assess their relative importanceand their implications for geographical variation in life-historyevolution.     

Jacobsen syndrome

Erythropoietic protoporphyria (EPP) is an inherited disorder of the haem metabolic pathway characterised by accumulation of protoporphyrin in blood, erythrocytes and tissues, and cutaneous manifestations of photosensitivity. EPP has been reported worldwide, with prevalence between 1:75,000 and 1:200,000. It usually manifests in early infancy upon the first sun exposures. EPP is characterised by cutaneous manifestations of acute painful photosensitivity with erythema and oedema, sometimes with petechiae, together with stinging and burning sensations upon exposure to sunlight, without blisters. These episodes have a variable severity depending on the exposure duration and may result in chronic permanent lesions on exposed skin. As protoporphyrin is a lipophilic molecule that is excreted by the liver, EPP patients are at risk of cholelithiasis with obstructive episodes, and chronic liver disease that might evolve to rapid acute liver failure. In most patients, EPP results from a partial deficiency of the last enzyme of the haem biosynthetic pathway, ferrochelatase, EC 4.99.1.1/FECH (encoded by the FECH gene). EPP appears to be inherited as an autosomal dominant disease, the clinical expression of which is modulated by the presence of the hypomorphic FECH IVS3-48C allele trans, but recessive inheritance with two mutated FECH alleles has also been described. In about 2% of patients, overt disease was recently shown to be caused by gain-of-function mutations in the erythroid-specific aminolevulinic acid synthase 2 (ALAS2/ALAS, EC 2.3.1.27) gene and named X-linked dominant protoporphyria. Diagnosis is established by finding increased levels of protoporphyrin in plasma and red blood cells, and detection of a plasma fluorescence peak at 634 nm. Investigations for hepatic involvement, ferrochelatase activity level, genetic analysis (FECH mutations, presence of the hypomorphic FECH IVS3-48C allele trans and ALAS2 mutations) and family studies are advisable. Differential diagnosis includes phototoxic drug reactions, hydroa vacciniforme, solar urticaria, contact dermatitis, angio-oedema and, in some cases, other types of porphyria. Management includes avoidance of exposure to light, reduction of protoporphyrin levels and prevention of progression of possible liver disease to liver failure. As the major risk in EPP patients is liver disease, a regular follow-up of hepatic involvement is essential. Sequential hepatic and bone marrow transplantation should be considered as a suitable treatment for most severe cases of EPP with hepatic involvement. EPP is a lifelong disorder whose prognosis depends on the evolution of the hepatic disease. However, photosensitivity may have a significant impact on quality of life of EPP patients.     

Rare extra-pair fertilizations in the semi-colonially breeding linnet Carduelis cannabina

Microsatellite analysis was used to analyse the level of extra-pair paternity (EPP) in the semi-colonially breeding linnet Carduelis cannabina in Denmark. During the breeding seasons of 1998 and 1999 EPP accounted for only 3.8% of young (4/106, 95% CL=1–13%). These extra-pair young were found in 9.1% of the broods (2/22, 95% CL=1–29%). We hypothesize that this low level of EPP is due to an efficient use of both mate guarding and frequent copulations as paternity guards. However, experimental tests with temporary detainment of males would be needed to establish whether there is a causal relationship between the low EPP level and the double paternity guard.