Complexity of avian evolution revealed by family-level genomes.

Despite tremendous efforts in the past decades, relationships among main avian lineages remain heavily debated without a clear resolution. Discrepancies have been attributed to diversity of species sampled, phylogenetic method and the choice of genomic regions1-3. Here we address these issues by analysing the genomes of 363 bird species4 (218 taxonomic families, 92% of total). Using intergenic regions and coalescent methods, we present a well-supported tree but also a marked degree of discordance. The tree confirms that Neoaves experienced rapid radiation at or near the Cretaceous-Palaeogene boundary. Sufficient loci rather than extensive taxon sampling were more effective in resolving difficult nodes. Remaining recalcitrant nodes involve species that are a challenge to model due to either extreme DNA composition, variable substitution rates, incomplete lineage sorting or complex evolutionary events such as ancient hybridization. Assessment of the effects of different genomic partitions showed high heterogeneity across the genome. We discovered sharp increases in effective population size, substitution rates and relative brain size following the Cretaceous-Palaeogene extinction event, supporting the hypothesis that emerging ecological opportunities catalysed the diversification of modern birds. The resulting phylogenetic estimate offers fresh insights into the rapid radiation of modern birds and provides a taxon-rich backbone tree for future comparative studies.

High MHC gene copy number maintains diversity despite homozygosity in a Critically Endangered single-island endemic bird, but no evidence of MHC-based mate choice.

Small population sizes can, over time, put species at risk due to the loss of genetic variation and the deleterious effects of inbreeding. Losing diversity in the major histocompatibility complex (MHC) could be particularly harmful, given its key role in the immune system. Here, we assess MHC class I (MHC-I) diversity and its effects on mate choice and survival in the Critically Endangered Raso lark Alauda razae, a species restricted to the 7 km2 islet of Raso, Cape Verde, since ~1460, whose population size has dropped as low as 20 pairs. Exhaustively genotyping 122 individuals, we find no effect of MHC-I genotype/diversity on mate choice or survival. However, we demonstrate that MHC-I diversity has been maintained through extreme bottlenecks by retention of a high number of gene copies (at least 14), aided by cosegregation of multiple haplotypes comprising 2-8 linked MHC-I loci. Within-locus homozygosity is high, contributing to low population-wide diversity. Conversely, each individual had comparably many alleles, 6-16 (average 11), and the large and divergent haplotypes occur at high frequency in the population, resulting in high within-individual MHC-I diversity. This functional immune gene diversity will be of critical importance for this highly threatened species' adaptive potential.

Comparing Adaptive Radiations Across Space, Time, and Taxa.

Adaptive radiation plays a fundamental role in our understanding of the evolutionary process. However, the concept has provoked strong and differing opinions concerning its definition and nature among researchers studying a wide diversity of systems. Here, we take a broad view of what constitutes an adaptive radiation, and seek to find commonalities among disparate examples, ranging from plants to invertebrate and vertebrate animals, and remote islands to lakes and continents, to better understand processes shared across adaptive radiations. We surveyed many groups to evaluate factors considered important in a large variety of species radiations. In each of these studies, ecological opportunity of some form is identified as a prerequisite for adaptive radiation. However, evolvability, which can be enhanced by hybridization between distantly related species, may play a role in seeding entire radiations. Within radiations, the processes that lead to speciation depend largely on (1) whether the primary drivers of ecological shifts are (a) external to the membership of the radiation itself (mostly divergent or disruptive ecological selection) or (b) due to competition within the radiation membership (interactions among members) subsequent to reproductive isolation in similar environments, and (2) the extent and timing of admixture. These differences translate into different patterns of species accumulation and subsequent patterns of diversity across an adaptive radiation. Adaptive radiations occur in an extraordinary diversity of different ways, and continue to provide rich data for a better understanding of the diversification of life.

The origin of the world's smallest flightless bird, the Inaccessible Island Rail Atlantisia rogersi (Aves: Rallidae).

Rails (Aves: Rallidae) are renowned for their extreme dispersal capability, which has given rise to numerous island lineages. Many insular species lost the ability to fly as a response to release from predator pressure-a feature causing rapid extinction when humans subsequently introduced mammals. The world's smallest extant flightless bird, the Inaccessible Island Rail Atlantisia rogersi, is endemic to Inaccessible Island, Tristan da Cunha archipelago, in the central South Atlantic Ocean. It is placed in a monotypic genus, but its taxonomic affinity, as well as geographic origin, are disputed. Contrary to its suggested Old World origin, we demonstrate that the Inaccessible Island Rail is nested within the mainly South American 'Laterallus clade' and that it colonized ≥3 million-year-old Inaccessible Island from South America c. 1.5 million years ago. The taxonomy of rails has traditionally been based on morphology, and convergent evolution has caused many cases of misclassification. We suggest a re-classification within the 'Laterallus clade' and call for extended coverage of taxon sampling for DNA sequencing.

Multiple instances of paraphyletic species and cryptic taxa revealed by mitochondrial and nuclear RAD data for Calandrella larks (Aves: Alaudidae).

The avian genus Calandrella (larks) was recently suggested to be non-monophyletic, and was divided into two genera, of which Calandrella sensu stricto comprises 4-5 species in Eurasia and Africa. We analysed mitochondrial cytochrome b (cytb) and nuclear Restriction-site Associated DNA (RAD) sequences from all species, and for cytb we studied 21 of the 22 recognised subspecies, with the aim to clarify the phylogenetic relationships within the genus and to compare large-scale nuclear sequence patterns with a widely used mitochondrial marker. Cytb indicated deep splits among the currently recognised species, although it failed to support the interrelationships among most of these. It also revealed unexpected deep divergences within C. brachydactyla, C. blanfordi/C. erlangeri, C. cinerea, and C. acutirostris. It also suggested that both C. brachydactyla and C. blanfordi, as presently circumscribed, are paraphyletic. In contrast, most of the many subspecies of C. brachydactyla and C. cinerea were unsupported by cytb, although two populations of C. cinerea were found to be genetically distinct. The RAD data corroborated the cytb tree (for the smaller number of taxa analysed) and recovered strongly supported interspecific relationships. However, coalescence analyses of the RAD data, analysed in SNAPP both with and without an outgroup, received equally strong support for two conflicting topologies. We suggest that the tree rooted with an outgroup - which is not recommended for SNAPP - is more trustworthy, and suggest that the reliability of analyses performed without any outgroup species should be thoroughly evaluated. We also demonstrate that degraded museum samples can be phylogenetically informative in RAD analyses following careful bioinformatic treatment. We note that the genus Calandrella is in need of taxonomic revision.

Disentangling the complex evolutionary history of the Western Palearctic blue tits (Cyanistes spp.) - phylogenomic analyses suggest radiation by multiple colonization events and subsequent isolation.

Isolated islands and their often unique biota continue to play key roles for understanding the importance of drift, genetic variation and adaptation in the process of population differentiation and speciation. One island system that has inspired and intrigued evolutionary biologists is the blue tit complex (Cyanistes spp.) in Europe and Africa, in particular the complex evolutionary history of the multiple genetically distinct taxa of the Canary Islands. Understanding Afrocanarian colonization events is of particular importance because of recent unconventional suggestions that these island populations acted as source of the widespread population in mainland Africa. We investigated the relationship between mainland and island blue tits using a combination of Sanger sequencing at a population level (20 loci; 12 500 nucleotides) and next-generation sequencing of single population representatives (>3 200 000 nucleotides), analysed in coalescence and phylogenetic frameworks. We found (i) that Afrocanarian blue tits are monophyletic and represent four major clades, (ii) that the blue tit complex has a continental origin and that the Canary Islands were colonized three times, (iii) that all island populations have low genetic variation, indicating low long-term effective population sizes and (iv) that populations on La Palma and in Libya represent relicts of an ancestral North African population. Further, demographic reconstructions revealed (v) that the Canary Islands, conforming to traditional views, hold sink populations, which have not served as source for back colonization of the African mainland. Our study demonstrates the importance of complete taxon sampling and an extensive multimarker study design to obtain robust phylogeographical inferences.

EndoS2 is a unique and conserved enzyme of serotype M49 group A Streptococcus that hydrolyses N-linked glycans on IgG and α1-acid glycoprotein.

Many bacteria have evolved ways to interact with glycosylation functions of the immune system of their hosts. Streptococcus pyogenes [GAS (group A Streptococcus)] secretes the enzyme EndoS that cleaves glycans on human IgG and impairs the effector functions of the antibody. The ndoS gene, encoding EndoS, has, until now, been thought to be conserved throughout the serotypes. However, in the present study, we identify EndoS2, an endoglycosidase in serotype M49 GAS strains. We characterized EndoS2 and the corresponding ndoS2 gene using sequencing, bioinformatics, phylogenetic analysis, recombinant expression and LC-MS analysis of glycosidic activity. This revealed that EndoS2 is present exclusively, and highly conserved, in serotype M49 of GAS and is only 37% identical with EndoS. EndoS2 showed endo-ß-N-acetylglucosaminidase activity on all N-linked glycans of IgG and on biantennary and sialylated glycans of AGP (α1-acid glycoprotein). The enzyme was found to act only on native IgG and AGP and to be specific for free biantennary glycans with or without terminal sialylation. GAS M49 expression of EndoS2 was monitored in relation to carbohydrates present in the culture medium and was linked to the presence of sucrose. We conclude that EndoS2 is a unique endoglycosidase in serotype M49 and differs from EndoS of other GAS strains by targeting both IgG and AGP. EndoS2 expands the repertoire of GAS effectors that modify key glycosylated molecules of host defence.

Avian Island Radiations Shed Light on the Dynamics of Adaptive and Nonadaptive Radiation.

Understanding the mechanisms underlying species formation and differentiation is a central goal of evolutionary biology and a formidable challenge. This understanding can provide valuable insights into the origins of the astonishing diversity of organisms living on our planet. Avian evolutionary radiations on islands have long fascinated biologists as they provide the ideal variation to study the ecological and evolutionary forces operating on the continuum between incipient lineages to complete speciation. In this review, we summarize the key insights gained from decades of research on adaptive and nonadaptive radiations of both extant and extinct insular bird species. We present a new comprehensive global list of potential avian radiations on oceanic islands, based on published island species checklists, taxonomic studies, and phylogenetic analyses. We demonstrate that our understanding of evolutionary processes is being greatly enhanced through the use of genomic tools. However, to advance the field, it is critical to complement this information with a solid understanding of the ecological and behavioral traits of both extinct and extant avian island species.

Improved haplotype resolution of highly duplicated MHC genes in a long-read genome assembly using MiSeq amplicons.

Long-read sequencing offers a great improvement in the assembly of complex genomic regions, such as the major histocompatibility complex (MHC) region, which can contain both tandemly duplicated MHC genes (paralogs) and high repeat content. The MHC genes have expanded in passerine birds, resulting in numerous MHC paralogs, with relatively high sequence similarity, making the assembly of the MHC region challenging even with long-read sequencing. In addition, MHC genes show rather high sequence divergence between alleles, making diploid-aware assemblers incorrectly classify haplotypes from the same locus as sequences originating from different genomic regions. Consequently, the number of MHC paralogs can easily be over- or underestimated in long-read assemblies. We therefore set out to verify the MHC diversity in an original and a haplotype-purged long-read assembly of one great reed warbler Acrocephalus arundinaceus individual (the focal individual) by using Illumina MiSeq amplicon sequencing. Single exons, representing MHC class I (MHC-I) and class IIB (MHC-IIB) alleles, were sequenced in the focal individual and mapped to the annotated MHC alleles in the original long-read genome assembly. Eighty-four percent of the annotated MHC-I alleles in the original long-read genome assembly were detected using 55% of the amplicon alleles and likewise, 78% of the annotated MHC-IIB alleles were detected using 61% of the amplicon alleles, indicating an incomplete annotation of MHC genes. In the haploid genome assembly, each MHC-IIB gene should be represented by one allele. The parental origin of the MHC-IIB amplicon alleles in the focal individual was determined by sequencing MHC-IIB in its parents. Two of five larger scaffolds, containing 6-19 MHC-IIB paralogs, had a maternal and paternal origin, respectively, as well as a high nucleotide similarity, which suggests that these scaffolds had been incorrectly assigned as belonging to different loci in the genome rather than as alternate haplotypes of the same locus. Therefore, the number of MHC-IIB paralogs was overestimated in the haploid genome assembly. Based on our findings we propose amplicon sequencing as a suitable complement to long-read sequencing for independent validation of the number of paralogs in general and for haplotype inference in multigene families in particular.

Evolutionary genomics of oceanic island radiations.

A recurring feature of oceanic archipelagos is the presence of adaptive radiations that generate endemic, species-rich clades that can offer outstanding insight into the links between ecology and evolution. Recent developments in evolutionary genomics have contributed towards solving long-standing questions at this interface. Using a comprehensive literature search, we identify studies spanning 19 oceanic archipelagos and 110 putative adaptive radiations, but find that most of these radiations have not yet been investigated from an evolutionary genomics perspective. Our review reveals different gaps in knowledge related to the lack of implementation of genomic approaches, as well as undersampled taxonomic and geographic areas. Filling those gaps with the required data will help to deepen our understanding of adaptation, speciation, and other evolutionary processes.

MHCtools - an R package for MHC high-throughput sequencing data: Genotyping, haplotype and supertype inference, and downstream genetic analyses in non-model organisms.

The major histocompatibility complex (MHC) plays a central role in the vertebrate adaptive immune system and has been of long-term interest in evolutionary biology. While several protocols have been developed for MHC genotyping, there is a lack of transparent and standardized tools for downstream analysis of MHC data. Here, we present the r package mhctools and demonstrate the use of its functions to (i) assist accurate MHC genotyping from high-throughput amplicon-sequencing data, (ii) infer functional MHC supertypes using bootstrapped clustering analysis, (iii) identify segregating MHC haplotypes from family data, and (iv) analyse functional and genetic distances between MHC sequences. We employed mhctools to analyse MHC class I (MHC-I) amplicon data of 559 great reed warblers (Acrocephalus arundinaceus). We identified 390 MHC-I alleles which clustered into 14 functional supertypes. A phylogenetic analysis and analyses of positive selection suggested that the MHC-I alleles belong to several distinct functional groups. We furthermore identified 107 segregating haplotypes among 116 families, and found substantial variation in diversity with 4-21 MHC-I alleles and 3-13 MHC-I supertypes per haplotype. Finally, we show that the great reed warbler haplotypes harboured combinations of MHC-I supertypes with greater functional divergence than observed in simulated populations of possible haplotypes, a result that is in accordance with the divergent allele advantage hypothesis. Our study demonstrates the power of mhctools to support genotyping and analysis of MHC in non-model species, which we hope will encourage broad implementation among researchers in MHC genetics and evolution.

A highly contiguous nuclear genome assembly of the mandarinfish Synchiropus splendidus (Syngnathiformes: Callionymidae).

The fish order Syngnathiformes has been referred to as a collection of misfit fishes, comprising commercially important fish such as red mullets as well as the highly diverse seahorses, pipefishes, and seadragons-the well-known family Syngnathidae, with their unique adaptations including male pregnancy. Another ornate member of this order is the species mandarinfish. No less than two types of chromatophores have been discovered in the spectacularly colored mandarinfish: the cyanophore (producing blue color) and the dichromatic cyano-erythrophore (producing blue and red). The phylogenetic position of mandarinfish in Syngnathiformes, and their promise of additional genetic discoveries beyond the chromatophores, made mandarinfish an appealing target for whole-genome sequencing. We used linked sequences to create synthetic long reads, producing a highly contiguous genome assembly for the mandarinfish. The genome assembly comprises 483 Mbp (longest scaffold 29 Mbp), has an N50 of 12 Mbp, and an L50 of 14 scaffolds. The assembly completeness is also high, with 92.6% complete, 4.4% fragmented, and 2.9% missing out of 4584 BUSCO genes found in ray-finned fishes. Outside the family Syngnathidae, the mandarinfish represents one of the most contiguous syngnathiform genome assemblies to date. The mandarinfish genomic resource will likely serve as a high-quality outgroup to syngnathid fish, and furthermore for research on the genomic underpinnings of the evolution of novel pigmentation.

Contrasting results from GWAS and QTL mapping on wing length in great reed warblers.

A major goal in evolutionary biology is to understand the genetic basis of adaptive traits. In migratory birds, wing morphology is such a trait. Our previous work on the great reed warbler (Acrocephalus arundinaceus) shows that wing length is highly heritable and under sexually antagonistic selection. Moreover, a quantitative trait locus (QTL) mapping analysis detected a pronounced QTL for wing length on chromosome 2, suggesting that wing morphology is partly controlled by genes with large effects. Here, we re-evaluate the genetic basis of wing length in great reed warblers using a genomewide association study (GWAS) approach based on restriction site-associated DNA sequencing (RADseq) data. We use GWAS models that account for relatedness between individuals and include covariates (sex, age and tarsus length). The resulting association landscape was flat with no peaks on chromosome 2 or elsewhere, which is in line with expectations for polygenic traits. Analysis of the distribution of p-values did not reveal biases, and the inflation factor was low. Effect sizes were however not uniformly distributed on some chromosomes, and the Z chromosome had weaker associations than autosomes. The level of linkage disequilibrium (LD) in the population decayed to background levels within c. 1 kbp. There could be several reasons to why our QTL study and GWAS gave contrasting results including differences in how associations are modelled (cosegregation in pedigree vs. LD associations), how covariates are accounted for in the models, type of marker used (multi- vs. biallelic), difference in power or a combination of these. Our study highlights that the genetic architecture even of highly heritable traits is difficult to characterize in wild populations.

Surveillance of influenza A virus in migratory waterfowl in northern Europe.

We conducted large-scale, systematic sampling of influenza type A virus in migratory waterfowl (mostly mallards [Anas platyrhynchos]) at Ottenby Bird Observatory, southeast Sweden. As with previous studies, we found a higher prevalence in fall than spring, and among juveniles compared with adults. However, in contrast to other studies, we found that prevalence in spring was sometimes high (mean 4.0%, highest 9.5%). This finding raises the possibility that ducks are capable of perpetuating influenza A virus of different subtypes and subtype combinations throughout the year and from 1 year to the next. Isolation of the H5 and H7 subtypes was common, which suggests risk for transmission to sensitive domestic animals such as poultry. We argue that wild bird screening can function as a sentinel system, and we give an example of how it could have been used to forecast a remote and deadly outbreak of influenza A in poultry.

High prevalence of influenza A virus in ducks caught during spring migration through Sweden.

As part of our ongoing screening of wild birds in Northern Europe, 358 mallards (Anas platyrhynchos) and 203 shelducks (Tadorna tadorna) were caught in southern Sweden during the spring 2003. Faecal samples were analyzed by real time RT-PCR for the presence of influenza A virus. In contrast to what has been found in North American studies; Eurasian spring migrating ducks passing through Sweden had a relatively high prevalence of influenza A virus.

Rapid advance of spring arrival dates in long-distance migratory birds.

Several bird species have advanced the timing of their spring migration in response to recent climate change. European short-distance migrants, wintering in temperate areas, have been assumed to be more affected by change in the European climate than long-distance migrants wintering in the tropics. However, we show that long-distance migrants have advanced their spring arrival in Scandinavia more than short-distance migrants. By analyzing a long-term data set from southern Italy, we show that long-distance migrants also pass through the Mediterranean region earlier. We argue that this may reflect a climate-driven evolutionary change in the timing of spring migration.