The rate of hybridization among taxa is a central consideration in any discussion of speciation, but rates of hybridization are difficult to estimate in most wild populations of animals. We used a successful citizen science dataset, eBird, to estimate the rates of hybridization for wild birds in the United States. We calculated the frequency at which hybrid individuals belonging to different species, families, and orders of birds were observed. Between 1 January 2010 and 31 December 2018, a total of 334,770,194 species records were reported to eBird within the United States. Of this total, 212,875 or 0.064% were reported as hybrids. This estimate is higher than the rate of hybridization (0.00167%) reported by Mayr based on impressions from a career studying museum specimens. However, if the 10 most influential hybrid species are removed from the eBird dataset, the rate of hybridization decreases substantially to about 0.009%. We conclude that the rate of hybridization for individuals in most bird species is extremely low, even though the potential for birds to produce fertile offspring through hybrid crosses is high. These findings indicate that there is strong prezygotic selection working in most avian species.

Carotenoid coloration is widely recognized as a signal of individual condition in various animals, but despite decades of study, the mechanisms that link carotenoid coloration to condition remain unresolved. Most birds with red feathers convert yellow dietary carotenoids to red carotenoids in an oxidation process requiring the gene encoding the putative cytochrome P450 enzyme CYP2J19. Here, we tested the hypothesis that the process of carotenoid oxidation and feather pigmentation is functionally linked to mitochondrial performance. Consistent with this hypothesis, we observed high levels of red ketolated carotenoids associated with the hepatic mitochondria of moulting wild house finches ( Haemorhous mexicanus ), and upon fractionation, we found the highest concentration of ketolated carotenoids in the inner mitochondrial membrane. We further found that the redness of growing feathers was positively related to the performance of liver mitochondria. Structural modelling of CYP2J19 supports a direct role of this protein in carotenoid ketolation that may be functionally linked to cellular respiration. These observations suggest that feather coloration serves as a signal of core functionality through inexorable links to cellular respiration in the mitochondria.

Elucidating the mechanisms of colour production in organisms is important for understanding how selection acts upon a variety of behaviours. Spiders provide many spectacular examples of colours used in courtship, predation, defence and thermoregulation, but are thought to lack many types of pigments common in other animals. Ommochromes, bilins and eumelanin have been identified in spiders, but not carotenoids or melanosomes. Here, we combined optical microscopy, refractive index matching, confocal Raman microspectroscopy and electron microscopy to investigate the basis of several types of colourful patches in spiders. We obtained four major results. First, we show that spiders use carotenoids to produce yellow, suggesting that such colours may be used for conditiondependent courtship signalling. Second, we established the Raman signature spectrum for ommochromes, facilitating the identification of ommochromes in a variety of organisms in the future. Third, we describe a potential new pigmentary-structural colour interaction that is unusual because of the use of long wavelength structural colour in combination with a slightly shorter wavelength pigment in the production of red. Finally, we present the first evidence for the presence of melanosomes in arthropods, using both scanning and transmission electron microscopy, overturning the assumption that melanosomes are a synapomorphy of vertebrates. Our research shows that spiders have a much richer colour production palette than previously thought, and this has implications for colour diversification and function in spiders and other arthropods.

16Carotenoid coloration is widely recognized as a signal of individual condition in various animals, 17 but despite decades of study, the mechanisms that link carotenoid coloration to condition remain 18 unresolved. Most birds with red feathers convert yellow dietary carotenoids to red carotenoids in 19 an oxidation process requiring the gene encoding the putative cytochrome P450 enzyme 20 CYP2J19. Here, we tested the hypothesis that the process of carotenoid oxidation and feather 21 pigmentation is functionally linked to mitochondrial performance. Consistent with this 22 30 31 Carotenoid coloration | OXPHOS | mate choice | sexual selection 32 Short Title: Mitochondrial bioenergetics and plumage color 33 34 3

We consider four key challenges related to estimating per‐individual rates of hybridization in wild birds: (1) what is the meaning of the term “hybrid”?, (2) the importance of distinguishing between shared DNA sequences and on‐going hybridization between populations, (3) the perils of focusing exclusively on known hybrid zones, and (4) the implications of very low rates of per individual hybridization. Because our focus is on using phenotype to recognize hybrids, we define a hybrid as an individual with a phenotype that is intermediate between two parental species. We emphasize the value of quantifying the rate of between‐species mating that is ongoing in current populations and distinguish this endeavor from estimates of gene flow between populations based on genomic analysis, which can reflect both current and ancient hybridization. We restate the importance of quantifying per individual rates of hybridization among all birds without prejudging which birds are presumed to engage in hybridization. And finally, we emphasize that evidence for strong prezygotic sorting is not necessarily evidence that mate choice is a driver of speciation.