The genomic architecture of continuous plumage colour variation in the European barn owl (Tyto alba).

The maintenance of colour variation in wild populations has long fascinated evolutionary biologists, although most studies have focused on discrete traits exhibiting rather simple inheritance patterns and genetic architectures. However, the study of continuous colour traits and their potentially oligo- or polygenic genetic bases remains rare in wild populations. We studied the genetics of the continuously varying white-to-rufous plumage coloration of the European barn owl (Tyto alba) using a genome-wide association approach on the whole-genome data of 75 individuals. We confirmed a mutation at the melanocortin-1-receptor gene (MC1R) is involved in the coloration and identified two new regions, located in super-scaffolds 9 and 42. The combination of the three regions explains most of the colour variation (80.37%, 95% credible interval 58.45-100%). One discovered region, located in the sex chromosome, differs between the most extreme colorations in owls sharing a specific MC1R genotype. This region may play a role in the colour sex dimorphism of this species, possibly in interaction with the autosomal MC1R. We thus provide insights into the genetic architecture of continuous colour variation, pointing to an oligogenic basis with potential epistatic effects among loci that should aid future studies understanding how continuous colour variation is maintained in nature.

Characterization of the diversity of barn owl's mitochondrial genome reveals high copy number variations in the control region.

Mitochondria are known to play an essential role in the cell. These organelles contain their own DNA, which is divided in a coding and non-coding region (NCR). While much of the NCR's function is unknown, tandem repeats have been observed in several vertebrates, with extreme intra-individual, intraspecific and interspecific variation. Taking advantage of a new complete reference for the mitochondrial genome of the Afro-European Barn Owl (Tyto alba), as well as 172 whole genome-resequencing; we (i) describe the reference mitochondrial genome with a special focus on the repeats in the NCR, (ii) quantify the variation in number of copies between individuals, and (iii) explore the possible factors associated with the variation in the number of repetitions. The reference mitochondrial genome revealed a long (256bp) and a short (80bp) tandem repeat in the NCR region. The re-sequenced genomes showed a great variation in number of copies between individuals, with 4 to 38 copies of the Long and 6 to 135 copies of the short repeat. Among the factors associated with this variation between individuals, the tissue used for extraction was the most significant. The exact mechanisms of the formations of these repeats are still to be discovered and understanding them will help explain the maintenance of the polymorphism in the number of copies, as well as their interactions with the metabolism, the aging and health of the individuals.

Genomic basis of melanin-associated phenotypes suggests colour-specific environmental adaptations in tawny owls.

Feathers comprise a series of evolutionary innovations but also harbour colour, a key biological trait known to co-vary with life history or complex traits. Those relationships are particularly true in melanin-based pigmentation species due to known pleiotropic effects of the melanocortin pathway - originating from melanin-associated phenotypes. Here, we explore the molecular basis of melanin colouration and expected co-variation at the molecular level in the melanin-based, colour polymorphic system of the tawny owl (Strix aluco). An extensive body of literature has revealed that grey and brown tawny owl colour morphs differ in a series of life history and behavioural traits. Thus, it is plausible to expect co-variation also at molecular level between colour morphs. To investigate this possibility, we assembled the first draft genome of the species against which we mapped ddRADseq reads from 220 grey and 150 brown morphs - representing 10 years of pedigree data from a population in Southern Finland - and explored genome-wide associations with colour phenotype. Our results revealed putative molecular signatures of cold adaptation strongly associated with the grey phenotype, namely, a non-synonymous substitution in MCHR1, plus 2 substitutions in non-coding regions of FTCD and FAM135A whose genotype combinations obtained a predictive power of up to 100% (predicting grey colour). These suggest a molecular basis of cold environment adaptations predicted to be grey-morph specific. Our results potentially reveal part of the molecular machinery of melanin-associated phenotypes and provide novel insights towards understanding the functional genomics of colour polymorphism in melanin-based pigmented species.

Range-wide phylogenomics of the Great Horned Owl (Bubo virginianus) reveals deep north-south divergence in northern Peru.

The Great Horned Owl (Bubo virginianus) inhabits myriad habitats throughout the Americas and shows complex patterns of individual and geographic morphological variation. The owl family Strigidae is known to follow ecogeographic rules, such as Gloger's rule. Although untested at the species level, these ecogeographic rules may affect B. virginianus plumage coloration and body size. Previous studies have indicated that, despite this species' morphological variability, little genetic differentiation exists across parts of their range. This study uses reduced representation genome-wide nuclear and complete mitochondrial DNA sequence data to assess range-wide relationships among B. virginianus populations and the disputed species status of B. v. magellanicus (Magellanic or Lesser Horned Owl) of the central and southern Andes. We found shallow phylogenetic relationships generally structured latitudinally to the north of the central Andes, and a deep divergence between a southern and northern clade close to the Marañón Valley in the central Andes, a common biogeographic barrier. We identify evidence of gene flow between B. v. magellanicus and other subspecies based on mitonuclear discordance and F-branch statistics. Overall differences in morphology, plumage coloration, voice, and genomic divergence support species status for B. v. magellanicus.

Description of Amblyomma monteiroae n. sp. (Acari: Ixodidae), a parasite of the great horned owl (Strigiformes: Strigidae) in southern Brazil.

In 2020, adult hard ticks (males and females) were collected from great horned owls [Bubo virginianus (Gmelin, 1788)] in the coastal region in southern Brazil. The engorged females were allowed to oviposit in the laboratory and hatched larvae could be obtained. Analyses of the external morphology of the adult ticks revealed that they represent a new species, which was named Amblyomma monteiroae n. sp. Partial sequences of the mitochondrial 16S rRNA gene and the nuclear second internal transcribed spacer (ITS2) were generated from a male and a female. Their 16S rRNA haplotypes were identical to each other and closest (96% identity) to corresponding sequences of Amblyomma parvitarsum Neumann, 1901, and 90% identical to Amblyomma neumanni Ribaga, 1902. Their ITS2 haplotypes were 95.8 to 96.0 identical to the single ITS-2 partial sequence of A. parvitarsum available in GenBank. In the phylogenetic trees inferred by both 16S rRNA and ITS2 partial sequences, A. monteiroae n. sp. formed a clade with A. parvitarsum, with A. neumanni branching sister to this clade. Amblyomma monteiroae n. sp. is genetically and morphologically related to A. parvitarsum. Both tick species are unique in combining the following morphological characters: scutum extensively ornate; eyes rounded and bulging; coxa I with two moderate pointed spurs, the external longer than the internal; a single triangular short spur on coxae II-III; presence of two spines on the tibia of legs II-IV; hypostomal dentition 3/3, trochanters without spurs. However, the males of the two species can be separated by specific features in palps and festoons, whereas the females differ in specific features of the coxal spurs. The larva of A. monteiroae n. sp. can be morphologically distinguished from A. parvitarsum only by morphometry, with the former species being slightly smaller. Currently, A. monteiroae n. sp. is restricted to southern Brazil, and the only known host is B. virginianus (Strigiformes: Strigidae). The present study increases the Amblyomma Brazilian fauna to 34 species.

Genomic basis of insularity and ecological divergence in barn owls (Tyto alba) of the Canary Islands.

Islands, and the particular organisms that populate them, have long fascinated biologists. Due to their isolation, islands offer unique opportunities to study the effect of neutral and adaptive mechanisms in determining genomic and phenotypical divergence. In the Canary Islands, an archipelago rich in endemics, the barn owl (Tyto alba), present in all the islands, is thought to have diverged into a subspecies (T. a. gracilirostris) on the eastern ones, Fuerteventura and Lanzarote. Taking advantage of 40 whole-genomes and modern population genomics tools, we provide the first look at the origin and genetic makeup of barn owls of this archipelago. We show that the Canaries hold diverse, long-standing and monophyletic populations with a neat distinction of gene pools from the different islands. Using a new method, less sensitive to structure than classical FST, to detect regions involved in local adaptation to insular environments, we identified a haplotype-like region likely under selection in all Canaries individuals and genes in this region suggest morphological adaptations to insularity. In the eastern islands, where the subspecies is present, genomic traces of selection pinpoint signs of adapted body proportions and blood pressure, consistent with the smaller size of this population living in a hot arid climate. In turn, genomic regions under selection in the western barn owls from Tenerife showed an enrichment in genes linked to hypoxia, a potential response to inhabiting a small island with a marked altitudinal gradient. Our results illustrate the interplay of neutral and adaptive forces in shaping divergence and early onset speciation.

Density-dependent selection and the maintenance of colour polymorphism in barn owls.

The capacity of natural selection to generate adaptive changes is (according to the fundamental theorem of natural selection) proportional to the additive genetic variance in fitness. In spite of its importance for development of new adaptations to a changing environment, processes affecting the magnitude of the genetic variance in fitness-related traits are poorly understood. Here, we show that the red-white colour polymorphism in female barn owls is subject to density-dependent selection at the phenotypic and genotypic level. The diallelic melanocortin-1 receptor gene explained a large amount of the phenotypic variance in reddish coloration in the females ([Formula: see text]). Red individuals (RR genotype) were selected for at low densities, while white individuals (WW genotype) were favoured at high densities and were less sensitive to changes in density. We show that this density-dependent selection favours white individuals and predicts fixation of the white allele in this population at longer time scales without immigration or other selective forces. Still, fluctuating population density will cause selection to fluctuate and periodically favour red individuals. These results suggest how balancing selection caused by fluctuations in population density can be a general mechanism affecting the level of additive genetic variance in natural populations.

Genomic signatures of the evolution of a diurnal lifestyle in Strigiformes.

Understanding the targets of selection associated with changes in behavioral traits represents an important challenge of current evolutionary research. Owls (Strigiformes) are a diverse group of birds, most of which are considered nocturnal raptors. However, a few owl species independently adopted a diurnal lifestyle in their recent evolutionary history. We searched for signals of accelerated rates of evolution associated with a diurnal lifestyle using a genome-wide comparative approach. We estimated substitution rates in coding and noncoding conserved regions of the genome of seven owl species, including three diurnal species. Substitution rates of the noncoding elements were more accelerated than those of protein-coding genes. We identified new, owl-specific conserved noncoding elements as candidates of parallel evolution during the emergence of diurnality in owls. Our results shed light on the molecular basis of adaptation to a new niche and highlight the importance of regulatory elements for evolutionary changes in behavior. These elements were often involved in the neuronal development of the brain.

Unexpected post-glacial colonisation route explains the white colour of barn owls (Tyto alba) from the British Isles.

The climate fluctuations of the Quaternary shaped the movement of species in and out of glacial refugia. In Europe, the majority of species followed one of the described traditional postglacial recolonization routes from the southern peninsulas towards the north. Like most organisms, barn owls are assumed to have colonized the British Isles by crossing over Doggerland, a land bridge that connected Britain to northern Europe. However, while they are dark rufous in northern Europe, barn owls in the British Isles are conspicuously white, a contrast that could suggest selective forces are at play on the islands. Yet, our analysis of known candidate genes involved in coloration found no signature of selection. Instead, using whole genome sequences and species distribution modelling, we found that owls colonised the British Isles soon after the last glaciation, directly from a white coloured refugium in the Iberian Peninsula, before colonising northern Europe. They would have followed a hitherto unknown post-glacial colonization route to the Isles over a westwards path of suitable habitat in now submerged land in the Bay of Biscay, thus not crossing Doggerland. As such, they inherited the white colour of their Iberian founders and maintained it through low gene flow with the mainland that prevents the import of rufous alleles. Thus, we contend that neutral processes probably explain this contrasting white colour compared to continental owls. With the barn owl being a top predator, we expect future research will show this unanticipated route was used by other species from its paleo community.

Landscape and Climatic Variations Shaped Secondary Contacts amid Barn Owls of the Western Palearctic.

The combined actions of climatic variations and landscape barriers shape the history of natural populations. When organisms follow their shifting niches, obstacles in the landscape can lead to the splitting of populations, on which evolution will then act independently. When two such populations are reunited, secondary contact occurs in a broad range of admixture patterns, from narrow hybrid zones to the complete dissolution of lineages. A previous study suggested that barn owls colonized the Western Palearctic after the last glaciation in a ring-like fashion around the Mediterranean Sea, and conjectured an admixture zone in the Balkans. Here, we take advantage of whole-genome sequences of 94 individuals across the Western Palearctic to reveal the complex history of the species in the region using observational and modeling approaches. Even though our results confirm that two distinct lineages colonized the region, one in Europe and one in the Levant, they suggest that it predates the last glaciation and identify a secondary contact zone between the two in Anatolia. We also show that barn owls recolonized Europe after the glaciation from two distinct glacial refugia: a previously identified western one in Iberia and a new eastern one in Italy. Both glacial lineages now communicate via eastern Europe, in a wide and permeable contact zone. This complex history of populations enlightens the taxonomy of Tyto alba in the region, highlights the key role played by mountain ranges and large water bodies as barriers and illustrates the power of population genomics in uncovering intricate demographic patterns.

Genomic consequences of colonisation, migration and genetic drift in barn owl insular populations of the eastern Mediterranean.

The study of insular populations was key in the development of evolutionary theory. The successful colonisation of an island depends on the geographic context, and specific characteristics of the organism and the island, but also on stochastic processes. As a result, apparently identical islands may harbour populations with contrasting histories. Here, we use whole genome sequences of 65 barn owls to investigate the patterns of inbreeding and genetic diversity of insular populations in the eastern Mediterranean Sea. We focus on Crete and Cyprus, islands with similar size, climate and distance to mainland, that provide natural replicates for a comparative analysis of the impacts of microevolutionary processes on isolated populations. We show that barn owl populations from each island have a separate origin, Crete being genetically more similar to other Greek islands and mainland Greece, and Cyprus more similar to the Levant. Further, our data show that their respective demographic histories following colonisation were also distinct. On the one hand, Crete harbours a small population and maintains very low levels of gene flow with neighbouring populations. This has resulted in low genetic diversity, strong genetic drift, increased relatedness in the population and remote inbreeding. Cyprus, on the other hand, appears to maintain enough gene flow with the mainland to avoid such an outcome. Our study provides a comparative population genomic analysis of the effects of neutral processes on a classical island-mainland model system. It provides empirical evidence for the role of stochastic processes in determining the fate of diverging isolated populations.

Cross-Amplification in Strigiformes: A New STR Panel for Forensic Purposes.

Strigiformes are affected by a substantial decline mainly caused by habitat loss and destruction, poaching, and trapping. Moreover, the increasing trend in bird trade and the growing interest in wild-caught rather than captive-bred birds are expected to encourage illegal trade. The biomolecular investigation represents a valuable tool to track illegal trade and to explore the genetic variability to preserving biodiversity. Microsatellite loci (STRs) are the most used markers to study genetic variability. Despite the availability of species-specific microsatellite loci in Strigiformes, a unique panel permitting the description of the genetic variability across species has not been identified yet. We tested 32 highly polymorphic microsatellite markers to evaluate the reliability of a unique microsatellite panel in different species of Strigiformes and its use for conservation and forensic purposes. We included in the study 84 individuals belonging to 28 parental groups and 11 species of Strigiformes. After screening polymorphic microsatellite loci, the description of genetic variability, and the kinship assessment, we characterized a final panel of 12 microsatellite loci able to identify individuals in 9 Strigiformes species. This STR panel might support the authorities in the forensic investigation for suspected smugglers and false parental claims; moreover, it can be useful to evaluate relatedness among individuals in captive-bred populations and to implement research projects finalized to the description of the genetic variability in wild populations.

Genomic Variation and Recent Population Histories of Spotted (Strix occidentalis) and Barred (Strix varia) Owls.

Spotted owls (SOs, Strix occidentalis) are a flagship species inhabiting old-growth forests in western North America. In recent decades, their populations have declined due to ongoing reductions in suitable habitat caused by logging, wildfires, and competition with the congeneric barred owl (BO, Strix varia). The northern spotted owl (S. o. caurina) has been listed as "threatened" under the Endangered Species Act since 1990. Here, we use an updated SO genome assembly along with 51 high-coverage whole-genome sequences to examine population structure, hybridization, and recent changes in population size in SO and BO. We found that potential hybrids identified from intermediate plumage morphology were a mixture of pure BO, F1 hybrids, and F1 × BO backcrosses. Also, although SO underwent a population bottleneck around the time of the Pleistocene-Holocene transition, their population sizes rebounded and show no evidence of any historical (i.e., 100-10,000 years ago) population decline. This suggests that the current decrease in SO abundance is due to events in the past century. Finally, we estimate that western and eastern BOs have been genetically separated for thousands of years, instead of the previously assumed recent (i.e., <150 years) divergence. Although this result is surprising, it is unclear where the ancestors of western BO lived after the separation. In particular, although BO may have colonized western North America much earlier than the first recorded observations, it is also possible that the estimated divergence time reflects unsampled BO population structure within central or eastern North America.

Fledging Mass Is Color Morph Specific and Affects Local Recruitment in a Wild Bird.

AbstractEarly-life conditions may have long-lasting effects on life history. In color polymorphic species, morph-specific sensitivity to environmental conditions may lead to differential fitness. In tawny owls (Strix aluco), pheomelanin-based color polymorphism is expected to be maintained because the brown morph has higher adult fitness in warmer environments, while selection favors the gray morph under colder conditions. Here we investigate body mass at fledging and its consequences until adulthood in a population at the species' cold range margin. Using 40 years of data (1979-2017), we show that brown pairs, which mainly produce brown offspring consistent with a one-locus-two-alleles inheritance model, consistently raised heavier offspring than mixed (gray-brown) pairs and gray pairs. Offspring mass declined seasonally, except among offspring raised by brown pairs. Brown offspring could be heavier because of morph-specific parental care and/or offspring growth. Furthermore, mass at fledging is associated with fitness: the probability of local recruitment into the breeding population increased with higher mass at fledging, especially in mild winters and with favorable food conditions, although recruitment is not morph specific. Fledgling mass thus provides a fitness benefit in terms of recruitment probability that is modulated by environmental factors, which appear to level off any direct morph-specific recruitment benefits.

Genomic Evidence for Sensorial Adaptations to a Nocturnal Predatory Lifestyle in Owls.

Owls (Strigiformes) evolved specific adaptations to their nocturnal predatory lifestyle, such as asymmetrical ears, a facial disk, and a feather structure allowing silent flight. Owls also share some traits with diurnal raptors and other nocturnal birds, such as cryptic plumage patterns, reversed sexual size dimorphism, and acute vision and hearing. The genetic basis of some of these adaptations to a nocturnal predatory lifestyle has been studied by candidate gene approaches but rarely with genome-wide scans. Here, we used a genome-wide comparative analysis to test for selection in the early history of the owls. We estimated the substitution rates in the coding regions of 20 bird genomes, including 11 owls of which five were newly sequenced. Then, we tested for functional overrepresentation across the genes that showed signals of selection. In the ancestral branch of the owls, we found traces of positive selection in the evolution of genes functionally related to visual perception, especially to phototransduction, and to chromosome packaging. Several genes that have been previously linked to acoustic perception, circadian rhythm, and feather structure also showed signals of an accelerated evolution in the origin of the owls. We discuss the functions of the genes under positive selection and their putative association with the adaptation to the nocturnal predatory lifestyle of the owls.

Cranial evolution in the extinct Rodrigues Island owl Otus murivorus (Strigidae), associated with unexpected ecological adaptations.

Island birds that were victims of anthropic extinctions were often more specialist species, having evolved their most distinctive features in isolation, making the study of fossil insular birds most interesting. Here we studied a fossil cranium of the 'giant' extinct scops owl Otus murivorus from Rodrigues Island (Mascarene Islands, southwestern Indian Ocean), to determine any potential unique characters. The fossil and extant strigids were imaged through X-ray microtomography, providing 3D views of external and internal (endocast, inner ear) cranial structures. Geometric morphometrics and analyses of traditional measurements yielded new information about the Rodrigues owl's evolution and ecology. Otus murivorus exhibits a 2-tier "lag behind" phenomenon for cranium and brain evolution, both being proportionately small relative to increased body size. It also had a much more developed olfactory bulb than congeners, indicating an unexpectedly developed olfactory sense, suggesting a partial food scavenging habit. In addition, O. murivorus had the eyes placed more laterally than O. sunia, the species from which it was derived, probably a side effect of a small brain; rather terrestrial habits; probably relatively fearless behavior; and a less vertical posture (head less upright) than other owls (this in part an allometric effect of size increase). These evolutionary features, added to gigantism and wing reduction, make the extinct Rodrigues owl's evolution remarkable, and with multiple causes.

Molecular evolution of the proopiomelanocortin system in Barn owl species.

Examination of genetic polymorphisms in outbred wild-living species provides insights into the evolution of complex systems. In higher vertebrates, the proopiomelanocortin (POMC) precursor gives rise to α-, ß-, and γ-melanocyte-stimulating hormones (MSH), which are involved in numerous physiological aspects. Genetic defects in POMC are linked to metabolic disorders in humans and animals. In the present study, we undertook an evolutionary genetic approach complemented with biochemistry to investigate the functional consequences of genetic polymorphisms in the POMC system of free-living outbred barn owl species (family Tytonidae) at the molecular level. Our phylogenetic studies revealed a striking correlation between a loss-of-function H9P mutation in the ß-MSH receptor-binding motif and an extension of a poly-serine stretch in γ3-MSH to ≥7 residues that arose in the barn owl group 6-8 MYA ago. We found that extension of the poly-serine stretches in the γ-MSH locus affects POMC precursor processing, increasing γ3-MSH production at the expense of γ2-MSH and resulting in an overall reduction of γ-MSH signaling, which may be part of a negative feedback mechanism. Extension of the γ3-MSH poly-serine stretches ≥7 further markedly increases peptide hormone stability in plasma, which is conserved in humans, and is likely relevant to its endocrine function. In sum, our phylogenetic analysis of POMC in wild living owls uncovered a H9P ß-MSH mutation subsequent to serine extension in γ3-MSH to 7 residues, which was then followed by further serine extension. The linked MSH mutations highlight the genetic plasticity enabled by the modular design of the POMC gene.

Identification of two novel adenoviruses in smooth-billed ani and tropical screech owl.

Avian adenoviruses (AdVs) are a very diverse group of pathogens causing diseases in poultry and wild birds. Wild birds, endangered by habitat loss and habitat fragmentation in the tropical forests, are recognised to play a role in the transmission of various AdVs. In this study, two novel, hitherto unknown AdVs were described from faecal samples of smooth-billed ani and tropical screech owl. The former was classified into genus Aviadenovirus, the latter into genus Atadenovirus, and both viruses most probably represent new AdV species as well. These results show that there is very limited information about the biodiversity of AdVs in tropical wild birds, though viruses might have a major effect on the population of their hosts or endanger even domesticated animals. Surveys like this provide new insights into the diversity, evolution, host variety, and distribution of avian AdVs.

Mitogenome of the little owl Athene noctua and phylogenetic analysis of Strigidae.

New advances in molecular approaches for DNA analysis have enhanced our understanding of the phylogenetic relationship of birds. The Little Owl (Athene noctua) is of great significance for the integrated management of forest diseases and control of regional pests. Here, we sequenced and annotated the 17,772 bp complete mitogenome of A. noctua. The mitogenome encoded 37 typical mitochondrial genes: 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA, and one non-coding control region (D-loop). The organization and location of genes in the A. noctua mitogenome were consistent with those reported for other Strigidae birds. Phylogenetic relationships based on Bayesian inference and Maximum likelihood methods showed that A. noctua has close relationships with Athene brama and Glaucidium cuculoides, confirming that A. noctua belongs to the Strigidae family. The phylogenetic relationships among seven genera of the Strigidae family used in this study were: Ninox and the other six genera were far apart, Otus and the clade ((Bubo + Strix) + Asio) were clustered into one branch, and Athene and Glaucidium were clustered into one branch. This phylogenetic classification is consistent with prior taxonomic studies on the Strigidae family. Our results provide new mitogenomic data to support further phylogenetic and taxonomic studies of Strigidae.

Lack of statistical power as a major limitation in understanding MHC-mediated immunocompetence in wild vertebrate populations.

Disentangling the sources of variation in developing an effective immune response against pathogens is of major interest to immunoecology and evolutionary biology. To date, the link between immunocompetence and genetic variation at the major histocompatibility complex (MHC) has received little attention in wild animals, despite the key role of MHC genes in activating the adaptive immune system. Although several studies point to a link between MHC and immunocompetence, negative findings have also been reported. Such disparate findings suggest that limited statistical power might be affecting studies on this topic, owing to insufficient sample sizes and/or a generally small effect of MHC on the immunocompetence of wild vertebrates. To clarify this issue, we investigated the link between MHC variation and seven immunocompetence proxies in a large sample of barn owls and estimated the effect sizes and statistical power of this and published studies on this topic. We found that MHC poorly explained variation in immunocompetence of barn owls, with small-to-moderate associations between MHC and immunocompetence in owls (effect size: .1 ≥ r ≤ .3) similar to other vertebrates studied to date. Such small-to-moderate effects were largely associated with insufficient power, which was only sufficient (>0.8) to detect moderate-to-large effect sizes (r ≥ .3). Thus, studies linking MHC variation with immunocompetence in wild populations are underpowered to detect MHC effects, which are likely to be of generally small magnitude. Larger sample sizes (>200) will be required to achieve sufficient power in future studies aiming to robustly test for a link between MHC variation and immunocompetence.