A mechanism for red coloration in vertebrates.

Red coloration is a salient feature of the natural world. Many vertebrates produce red color by converting dietary yellow carotenoids into red ketocarotenoids via an unknown mechanism. Here, we show that two enzymes, cytochrome P450 2J19 (CYP2J19) and 3-hydroxybutyrate dehydrogenase 1-like (BDH1L), are sufficient to catalyze this conversion. In birds, both enzymes are expressed at the sites of ketocarotenoid biosynthesis (feather follicles and red cone photoreceptors), and genetic evidence implicates these enzymes in yellow/red color variation in feathers. In fish, the homologs of CYP2J19 and BDH1L are required for ketocarotenoid production, and we show that these enzymes are sufficient to produce ketocarotenoids in cell culture and when ectopically expressed in fish skin. Finally, we demonstrate that the red-cone-enriched tetratricopeptide repeat protein 39B (TTC39B) enhances ketocarotenoid production when co-expressed with CYP2J19 and BDH1L. The discovery of this mechanism of ketocarotenoid biosynthesis has major implications for understanding the evolution of color diversity in vertebrates.

Controlled creation and annihilation of isolated robust emergent magnetic monopole like charged vertices in square artificial spin ice.

Magnetic analogue of an isolated free electric charge, i.e., a magnet with a single north or south pole, is a long sought-after particle which remains elusive so far. In magnetically frustrated pyrochlore solids, a classical analogue of monopole was observed as a result of excitation of spin ice vertices. Direct visualization of such excitations were proposed and later confirmed in analogous artificial spin ice (ASI) systems of square as well as Kagome geometries. However, such magnetically charged vertices are randomly created as they are thermally driven and are always associated with corresponding equal and opposite emergent charges, often termed as monopole-antimonopole pairs, connected by observable strings. Here, we demonstrate a controlled stabilisation of a robust isolated emergent monopole-like magnetically charged vertices in individual square ASI systems by application of an external magnetic field. The excitation conserves the magnetic charge without the involvement of a corresponding excitation of opposite charge. Well supported by Monte Carlo simulations our experimental results enable, in absence of a true elemental magnetic monopole, creation of electron vortices and studying electrodynamics in presence of a monopole-like field in a solid state environment.

Low MSP-1 haplotype diversity in the West Palearctic population of the avian malaria parasite Plasmodium relictum.

BACKGROUND: Although avian Plasmodium species are widespread and common across the globe, limited data exist on how genetically variable their populations are. Here, the hypothesis that the avian blood parasite Plasmodium relictum exhibits very low genetic diversity in its Western Palearctic transmission area (from Morocco to Sweden in the north and Transcaucasia in the east) was tested. METHODS: The genetic diversity of Plasmodium relictum was investigated by sequencing a portion (block 14) of the fast-evolving merozoite surface protein 1 (MSP1) gene in 75 different P. relictum infections from 36 host species. Furthermore, the full-length MSP1 sequences representing the common block 14 allele was sequenced in order to investigate if additional variation could be found outside block 14. RESULTS: The majority (72 of 75) of the sequenced infections shared the same MSP1 allele. This common allele has previously been found to be the dominant allele transmitted in Europe. CONCLUSION: The results corroborate earlier findings derived from a limited dataset that the globally transmitted malaria parasite P. relictum exhibits very low genetic diversity in its Western Palearctic transmission area. This is likely the result of a recent introduction event or a selective sweep.

Molecular parallelisms between pigmentation in the avian iris and the integument of ectothermic vertebrates.

Birds exhibit striking variation in eye color that arises from interactions between specialized pigment cells named chromatophores. The types of chromatophores present in the avian iris are lacking from the integument of birds or mammals, but are remarkably similar to those found in the skin of ectothermic vertebrates. To investigate molecular mechanisms associated with eye coloration in birds, we took advantage of a Mendelian mutation found in domestic pigeons that alters the deposition of yellow pterin pigments in the iris. Using a combination of genome-wide association analysis and linkage information in pedigrees, we mapped variation in eye coloration in pigeons to a small genomic region of ~8.5kb. This interval contained a single gene, SLC2A11B, which has been previously implicated in skin pigmentation and chromatophore differentiation in fish. Loss of yellow pigmentation is likely caused by a point mutation that introduces a premature STOP codon and leads to lower expression of SLC2A11B through nonsense-mediated mRNA decay. There were no substantial changes in overall gene expression profiles between both iris types as well as in genes directly associated with pterin metabolism and/or chromatophore differentiation. Our findings demonstrate that SLC2A11B is required for the expression of pterin-based pigmentation in the avian iris. They further highlight common molecular mechanisms underlying the production of coloration in the iris of birds and skin of ectothermic vertebrates.

High-resolution multi-marker DNA metabarcoding reveals sexual dietary differentiation in a bird with minor dimorphism.

Although sexual dietary differentiation is well known in birds, it is usually linked with significant morphological dimorphism between males and females, with lower differentiation reported in sexually monomorphic or only slightly dimorphic species. However, this may be an artifact of poor taxonomic resolution achieved in most conventional dietary studies, which may be unable to detect subtle intraspecific differentiation in prey consumption. Here, we show the power of multi-marker metabarcoding to address these issues, focusing on a slightly dimorphic generalist passerine, the black wheatear Oenanthe leucura. Using markers from four genomic regions (18S, 16S, COI, and trnL), we analyzed fecal droppings collected from 93 adult black wheatears during the breeding season. We found that sexes were rather similar in bill and body features, though males had a slightly thicker bill and longer wings and tail than females. Diet was dominated in both sexes by a very wide range of arthropod species and a few fleshy fruits, but the overall diet diversity was higher for males than females, and there was a much higher frequency of occurrence of ants in female (58%) than male (29%) diets. We hypothesize that the observed sexual differentiation was likely related to females foraging closer to their offspring on abundant prey, while males consumed a wider variety of prey while foraging more widely. Overall, our results suggest that dietary sexual differentiation in birds may be more widespread than recognized at present and that multi-marker DNA metabarcoding is a particularly powerful tool to unveiling such differences.

A genetic mechanism for sexual dichromatism in birds.

Sexual dichromatism, a difference in coloration between males and females, may be due to sexual selection for ornamentation and mate choice. Here, we show that carotenoid-based dichromatism in mosaic canaries, a hybrid phenotype that arises in offspring of the sexually dichromatic red siskin and monochromatic canaries, is controlled by the gene that encodes the carotenoid-cleaving enzyme ß-carotene oxygenase 2 (BCO2). Dichromatism in mosaic canaries is explained by differential carotenoid degradation in the integument, rather than sex-specific variation in physiological functions such as pigment uptake or transport. Transcriptome analyses suggest that carotenoid degradation in the integument might be a common mechanism contributing to sexual dichromatism across finches. These results suggest that differences in ornamental coloration between sexes can evolve through simple molecular mechanisms controlled by genes of major effect.

Genetic Basis of De Novo Appearance of Carotenoid Ornamentation in Bare Parts of Canaries.

Unlike wild and domestic canaries (Serinus canaria), or any of the three dozen species of finches in genus Serinus, the domestic urucum breed of canaries exhibits bright red bills and legs. This novel trait offers a unique opportunity to understand the mechanisms of bare-part coloration in birds. To identify the mutation producing the colorful phenotype, we resequenced the genome of urucum canaries and performed a range of analyses to search for genotype-to-phenotype associations across the genome. We identified a nonsynonymous mutation in the gene BCO2 (beta-carotene oxygenase 2, also known as BCDO2), an enzyme involved in the cleavage and breakdown of full-length carotenoids into short apocarotenoids. Protein structural models and in vitro functional assays indicate that the urucum mutation abrogates the carotenoid-cleavage activity of BCO2. Consistent with the predicted loss of carotenoid-cleavage activity, urucum canaries tended to have increased levels of full-length carotenoid pigments in bill tissue and reduced levels of carotenoid-cleavage products (apocarotenoids) in retinal tissue compared with other breeds of canaries. We hypothesize that carotenoid-based bare-part coloration might be readily gained, modified, or lost through simple switches in the enzymatic activity or regulation of BCO2 and this gene may be an important mediator in the evolution of bare-part coloration among bird species.

Advancing the integration of multi-marker metabarcoding data in dietary analysis of trophic generalists.

The application of DNA metabarcoding to dietary analysis of trophic generalists requires using multiple markers in order to overcome problems of primer specificity and bias. However, limited attention has been given to the integration of information from multiple markers, particularly when they partly overlap in the taxa amplified, and vary in taxonomic resolution and biases. Here, we test the use of a mix of universal and specific markers, provide criteria to integrate multi-marker metabarcoding data and a python script to implement such criteria and produce a single list of taxa ingested per sample. We then compare the results of dietary analysis based on morphological methods, single markers, and the proposed combination of multiple markers. The study was based on the analysis of 115 faeces from a small passerine, the Black Wheatears (Oenanthe leucura). Morphological analysis detected far fewer plant taxa (12) than either a universal 18S marker (57) or the plant trnL marker (124). This may partly reflect the detection of secondary ingestion by molecular methods. Morphological identification also detected far fewer taxa (23) than when using 18S (91) or the arthropod markers IN16STK (244) and ZBJ (231), though each method missed or underestimated some prey items. Integration of multi-marker data provided far more detailed dietary information than any single marker and estimated higher frequencies of occurrence of all taxa. Overall, our results show the value of integrating data from multiple, taxonomically overlapping markers in an example dietary data set.

Intricate trophic links between threatened vertebrates confined to a small island in the Atlantic Ocean.

Trophic networks in small isolated islands are in a fragile balance, and their disturbance can easily contribute toward the extinction vortex of species. Here, we show, in a small Atlantic island (Raso) in the Cabo Verde Archipelago, using DNA metabarcoding, the extent of trophic dependence of the Endangered giant wall gecko Tarentola gigas on endemic populations of vertebrates, including one of the rarest bird species of the world, the Critically Endangered Raso lark Alauda razae. We found that the Raso lark (27%), Iago sparrow Passer iagoensis (12%), Bulwer's petrel Bulweria bulwerii (15%), and the Cabo Verde shearwater Calonectris edwardsii (10%) are the most frequent vertebrate signatures found in the feces of the giant wall gecko. This work provides the first integrative assessment of their trophic links, an important issue to be considered for the long-term conservation of these small and isolated island ecosystems.

Geographic patterns of mtDNA and Z-linked sequence variation in the Common Chiffchaff and the 'chiffchaff complex'.

The Common Chiffchaff Phylloscopus collybita is an abundant, polytypic Palearctic bird. Validity of some of its subspecies is controversial and birds from some parts of the species range remain unclassified taxonomically. The relationships among populations from different geographic areas have not been sufficiently explored with molecular data. In this study we analyzed the relationships among the four species in the 'chiffchaff complex' (Common Chiffchaff, Iberian Chiffchaff P. ibericus, Canary Islands Chiffchaff P. canariensis and Mountain Chiffchaff P. sindianus), and the patterns of intraspecific geographic variation in the mtDNA ND2 gene and intron 9 of the Z-linked aconitase gene (ACO1I9) across the Common Chiffchaff range, including a recently discovered population breeding on Mt. Hermon (Anti-Lebanon mountains). Our data supported the monophyly of the chiffchaff complex and its current systematics at the species level. Within the Common Chiffchaff, the Siberian race P. c. tristis was the most differentiated subspecies and may represent a separate or incipient species. Other Common Chiffchaff subspecies also were differentiated in their mtDNA, however, lineages of neighboring subspecies formed wide zones of introgression. The Mt. Hermon population was of mixed genetic origin but contained some birds with novel unique lineage that could not be assigned to known subspecies. All Common Chiffchaff lineages diverged at the end of the Ionian stage of Pleistocene. Lineage sorting of ACO1I9 alleles was not as complete as that of mtDNA. Chiffchaff species were mostly distinct at ACO1I9, except the Common and Canary Islands Chiffchaffs that shared multiple alleles. An AMOVA identified geographic structure in Common Chiffchaff ACO1I9 variation that was broadly consistent with that of mtDNA ND2 gene. The genetic and other data suggest the chiffchaff complex to be a group of evolutionarily young taxa that represent a paradigm of 'species evolution in action' from intergrading subspecies through to apparently complete biological speciation.

A test of the European Pleistocene refugial paradigm, using a Western Palaearctic endemic bird species.

Hewitt's paradigm for effects of Pleistocene glaciations on European populations assumes their isolation in peninsular refugia during glacial maxima, followed by re-colonization of broader Europe during interstadials. This paradigm is well supported by studies of poorly dispersing taxa, but highly dispersive birds have not been included. To test this paradigm, we use the dunnock (Prunella modularis), a Western Palaearctic endemic whose range includes all major European refugia. MtDNA gene tree, multilocus species tree and species delimitation analyses indicate the presence of three distinct lineages: one in the Iberian refugium, one in the Caucasus refugium, and one comprising the Italian and Balkan refugia and broader Europe. Our gene flow analysis suggests isolation of both the Iberian and Caucasus lineages but extensive exchange between Italy, the Balkans and broader Europe. Demographic stability could not be rejected for any refugial population, except the very recent expansion in the Caucasus. By contrast, northern European populations may have experienced two expansion periods. Iberia and Caucasus had much smaller historical populations than other populations. Although our results support the paradigm, in general, they also suggest that in highly dispersive taxa, isolation of neighbouring refugia was incomplete, resulting in large super-refugial populations.

A non-coding region near Follistatin controls head colour polymorphism in the Gouldian finch.

Discrete colour morphs coexisting within a single population are common in nature. In a broad range of organisms, sympatric colour morphs often display major differences in other traits, including morphology, physiology or behaviour. Despite the repeated occurrence of this phenomenon, our understanding of the genetics that underlie multi-trait differences and the factors that promote the long-term maintenance of phenotypic variability within a freely interbreeding population are incomplete. Here, we investigated the genetic basis of red and black head colour in the Gouldian finch (Erythrura gouldiae), a classic polymorphic system in which naturally occurring colour morphs also display differences in aggressivity and reproductive success. We show that the candidate locus is a small (approx. 70 kb) non-coding region mapping to the Z chromosome near the Follistatin (FST) gene. Unlike recent findings in other systems where phenotypic morphs are explained by large inversions containing hundreds of genes (so-called supergenes), we did not identify any structural rearrangements between the two haplotypes using linked-read sequencing technology. Nucleotide divergence between the red and black alleles was high when compared to the remainder of the Z chromosome, consistent with their maintenance as balanced polymorphisms over several million years. Our results illustrate how pleiotropic phenotypes can arise from simple genetic variation, probably regulatory in nature.

Haemosporidian parasites missed the boat during the introduction of common waxbills (Estrilda astrild) in Iberia.

Exotic species can experience fast expansion in new environments, especially if they left their pathogens behind (Enemy Release hypothesis) or brought novel pathogens to the native competitors (Novel Weapon hypothesis). Common waxbills (Estrilda astrild) are native to sub-Saharan Africa and invaded west Iberia since the 1960s. Past haemosporidian parasite surveys at four locations in Portugal showed that waxbills can be infected with parasites, though with very low prevalence. However, it is not known if this pattern generalizes across their distribution range, or if there are geographic differences in parasite prevalence. It is also not discussed if this is a case of Enemy Release, as opposed to waxbills being also little parasitized in their native range. We screened 617 waxbills in 23 sites in Portugal and detected nine parasite lineages, most of them only known to the Palearctic. Only ten individuals were parasitized, and there was no significant geographical pattern on the prevalence. Overall, this population shows very low prevalence of haemosporidians (1.6% prevalence), which contrasts with significantly higher prevalence in native grounds, as compiled from the literature. These data support Enemy Release as the most likely hypothesis, which may have been important for their success as an exotic species.

Signatures of Selection on Standing Genetic Variation Underlie Athletic and Navigational Performance in Racing Pigeons.

Racing pigeons have been selectively bred to find their way home quickly over what are often extremely long distances. This breed is of substantial commercial value and is also an excellent avian model to gain empirical insights into the evolution of traits associated with flying performance and spatial orientation. Here, we investigate the molecular basis of the superior athletic and navigational capabilities of racing pigeons using whole-genome and RNA sequencing data. We inferred multiple signatures of positive selection distributed across the genome of racing pigeons. The strongest signature overlapped the CASK gene, a gene implicated in the formation of neuromuscular junctions. However, no diagnostic alleles were found between racing pigeons and other breeds, and only a small proportion of highly differentiated variants were exclusively detected in racing pigeons. We can thus conclude that very few individual genetic changes, if any, are either strictly necessary or sufficient for superior athletics and navigation. Gene expression analysis between racing and nonracing breeds revealed modest differences in muscle (213) and brain (29). These transcripts, however, showed only slightly elevated levels of genetic differentiation between the two groups, suggesting that most differential expression is not causative but likely a consequence of alterations in regulatory networks. Our results show that the unique suite of traits that enable fast flight, long endurance, and accurate navigation in racing pigeons, do not result from few loci acting as master switches but likely from a polygenic architecture that leveraged standing genetic variation available at the onset of the breed formation.

High-density lipoprotein receptor SCARB1 is required for carotenoid coloration in birds.

Yellow, orange, and red coloration is a fundamental aspect of avian diversity and serves as an important signal in mate choice and aggressive interactions. This coloration is often produced through the deposition of diet-derived carotenoid pigments, yet the mechanisms of carotenoid uptake and transport are not well-understood. The white recessive breed of the common canary (Serinus canaria), which carries an autosomal recessive mutation that renders its plumage pure white, provides a unique opportunity to investigate mechanisms of carotenoid coloration. We carried out detailed genomic and biochemical analyses comparing the white recessive with yellow and red breeds of canaries. Biochemical analysis revealed that carotenoids are absent or at very low concentrations in feathers and several tissues of white recessive canaries, consistent with a genetic defect in carotenoid uptake. Using a combination of genetic mapping approaches, we show that the white recessive allele is due to a splice donor site mutation in the scavenger receptor B1 (SCARB1; also known as SR-B1) gene. This mutation results in abnormal splicing, with the most abundant transcript lacking exon 4. Through functional assays, we further demonstrate that wild-type SCARB1 promotes cellular uptake of carotenoids but that this function is lost in the predominant mutant isoform in white recessive canaries. Our results indicate that SCARB1 is an essential mediator of the expression of carotenoid-based coloration in birds, and suggest a potential link between visual displays and lipid metabolism.

Genetic Basis for Red Coloration in Birds.

The yellow and red feather pigmentation of many bird species [1] plays pivotal roles in social signaling and mate choice [2, 3]. To produce red pigments, birds ingest yellow carotenoids and endogenously convert them into red ketocarotenoids via an oxidation reaction catalyzed by a previously unknown ketolase [4-6]. We investigated the genetic basis for red coloration in birds using whole-genome sequencing of red siskins (Spinus cucullata), common canaries (Serinus canaria), and "red factor" canaries, which are the hybrid product of crossing red siskins with common canaries [7]. We identified two genomic regions introgressed from red siskins into red factor canaries that are required for red coloration. One of these regions contains a gene encoding a cytochrome P450 enzyme, CYP2J19. Transcriptome analysis demonstrates that CYP2J19 is significantly upregulated in the skin and liver of red factor canaries, strongly implicating CYP2J19 as the ketolase that mediates red coloration in birds. Interestingly, a second introgressed region required for red feathers resides within the epidermal differentiation complex, a cluster of genes involved in development of the integument. Lastly, we present evidence that CYP2J19 is involved in ketocarotenoid formation in the retina. The discovery of the carotenoid ketolase has important implications for understanding sensory function and signaling mediated by carotenoid pigmentation.

The Strait of Gibraltar poses an effective barrier to host-specialised but not to host-generalised lineages of avian Haemosporidia.

One of the major concerns with ongoing environmental global change is the ability of parasites to shift their distribution (both geographically and across hosts) and to increase in virulence. To understand the structure, diversity and connectivity of parasite communities across the Mediterranean Sea, we used avian haemosporidian communities associated with forest birds of northwestern Africa and northwestern Iberia as a model system. We characterised host specificity of lineages and tested whether host generalists are more likely to cross the biogeographic barrier imposed by the Strait of Gibraltar than host specialists. We sampled 321 birds of 43 species in northwestern Africa and 735 birds of 49 species in northwestern Iberia. Using a PCR-based approach to amplify Plasmodium, Haemoproteus and Leucocytozoon parasites, we retrieved 969 sequences representing 200 unique cytochrome-b lineages. Haemosporidians infected a significantly higher proportion of birds in northwestern Africa (78.5%) than in northwestern Iberia (50.5%). Relative diversity of different haemosporidian genera did not differ between our study areas, but Plasmodium was overrepresented among individual infections in northwestern Iberia. Haemoproteus and Leucocytozoon lineages were predominantly host-specialised and Plasmodium lineages were host-generalised. The number of regions occupied by lineages was significantly associated with their host specificity and abundance. These data are consistent with the positive abundance-occupancy relationship and patterns of host specificity among different haemosporidian genera observed in other studies.

Genetic and morphometric diversity of the goldcrest (Regulus regulus) populations in the Azores.

We studied the genetic diversity and phylogeography of the goldcrest Regulus regulus from the archipelago of the Azores (North Atlantic Ocean) based on sequences of two mitochondrial genes (cytochrome b and the NADH dehydrogenase subunit 2) and one nuclear gene in the Z-chromosome (intron 9 of the aconitase 1) from 69 individuals, and 41 birds from the Canary Islands and continental Europe for outgroup comparison. To understand the level of concordance between the genetic data and possible morphometric variability, 197 adult living birds from the seven Azorean islands where the species breeds were analysed in terms of eight morphometric characters. Our results are in accordance with previous studies, indicating a recent expansion of goldcrests throughout the archipelago and a low divergence in relation to continental Europe. Within the Azores, there is evidence of historical and/or recent gene flow among the island's populations, revealing a lack of current genetic structure within the archipelago. Only goldcrests from Flores Island seem to be genetically distinct and showed significantly larger body mass and tarsus length than birds on the other islands.

Does the niche breadth or trade-off hypothesis explain the abundance-occupancy relationship in avian Haemosporidia?

Two hypotheses have been proposed to explain the abundance-occupancy relationship (AOR) in parasites. The niche breadth hypothesis suggests that host generalists are more abundant and efficient at colonizing different host communities than specialists. The trade-off hypothesis argues that host specialists achieve high density across their hosts' ranges, whereas generalists incur the high cost of adaptation to diverse immuno-defence systems. We tested these hypotheses using 386 haemosporidian cytochrome-b lineages (1894 sequences) recovered from 2318 birds of 103 species sampled in NW Africa, NW Iberia, W Greater Caucasus and Transcaucasia. The number of regions occupied by lineages was associated with their frequency suggesting the presence of AOR in avian Haemosporidia. However, neither hypothesis provided a better explanation for the AOR. Although the host generalist Plasmodium SGS1 was over three times more abundant than other widespread lineages, both host specialists and generalists were successful in colonizing all study regions and achieved high overall prevalence.

Historical demographic dynamics underlying local adaptation in the presence of gene flow.

The range of a species is the result of the relative contribution of spatial tracking of environmental requirements and adaptation to ecological conditions outside the ancestral niche. The appearance of novel habitats caused by climatic oscillation can promote range expansion and accompanying demographic growth. The demographic dynamics of populations leave a signal in \ patterns. We modeled three competing scenarios pertaining to the circumstance of a range expansion by the Karoo Scrub-Robin into newly available habitat resulting from the increasing aridification of southern Africa. Genetic variation was contrasted with the theoretical expectations of a spatial range expansion, and compared with data of a putative adaptive trait. We infer that this bird likely colonized the arid zone, as a consequence of adaptive evolution in a small peripheral population, followed by an expansion with recurrent exchange of migrants with the ancestral populations.