A nuclear genome assembly of an extinct flightless bird, the little bush moa.

We present a draft genome of the little bush moa (Anomalopteryx didiformis)-one of approximately nine species of extinct flightless birds from Aotearoa, New Zealand-using ancient DNA recovered from a fossil bone from the South Island. We recover a complete mitochondrial genome at 249.9× depth of coverage and almost 900 megabases of a male moa nuclear genome at ~4 to 5× coverage, with sequence contiguity sufficient to identify more than 85% of avian universal single-copy orthologs. We describe a diverse landscape of transposable elements and satellite repeats, estimate a long-term effective population size of ~240,000, identify a diverse suite of olfactory receptor genes and an opsin repertoire with sensitivity in the ultraviolet range, show that the wingless moa phenotype is likely not attributable to gene loss or pseudogenization, and identify potential function-altering coding sequence variants in moa that could be synthesized for future functional assays. This genomic resource should support further studies of avian evolution and morphological divergence.

Global flyway evolution in red knots Calidris canutus and genetic evidence for a Nearctic refugium.

Present-day ecology and population structure are the legacies of past climate and habitat perturbations, and this is particularly true for species that are widely distributed at high latitudes. The red knot, Calidris canutus, is an arctic-breeding, long-distance migratory shorebird with six recognized subspecies defined by differences in morphology, migration behavior, and annual cycle phenology, in a global distribution thought to have arisen just since the last glacial maximum (LGM). We used nextRAD sequencing of 10,881 single-nucleotide polymorphisms (SNPs) to assess the neutral genetic structure and phylogeographic history of 172 red knots representing all known global breeding populations. Using population genetics approaches, including model-based scenario-testing in an approximate Bayesian computation (ABC) framework, we infer that red knots derive from two main lineages that diverged ca. 34,000 years ago, and thus most probably persisted at the LGM in both Palearctic and Nearctic refugia, followed by at least two instances of secondary contact and admixture. Within two Beringian subspecies (C. c. roselaari and rogersi), we detected previously unknown genetic structure among sub-populations sharing a migratory flyway, reflecting additional complexity in the phylogeographic history of the region. Conversely, we found very weak genetic differentiation between two Nearctic populations (rufa and islandica) with clearly divergent migratory phenotypes and little or no apparent contact throughout the annual cycle. Together, these results suggest that relative gene flow among migratory populations reflects a complex interplay of historical, geographical, and ecological factors.

Whole-Genome Analyses Resolve the Phylogeny of Flightless Birds (Palaeognathae) in the Presence of an Empirical Anomaly Zone.

Palaeognathae represent one of the two basal lineages in modern birds, and comprise the volant (flighted) tinamous and the flightless ratites. Resolving palaeognath phylogenetic relationships has historically proved difficult, and short internal branches separating major palaeognath lineages in previous molecular phylogenies suggest that extensive incomplete lineage sorting (ILS) might have accompanied a rapid ancient divergence. Here, we investigate palaeognath relationships using genome-wide data sets of three types of noncoding nuclear markers, together totaling 20,850 loci and over 41 million base pairs of aligned sequence data. We recover a fully resolved topology placing rheas as the sister to kiwi and emu + cassowary that is congruent across marker types for two species tree methods (MP-EST and ASTRAL-II). This topology is corroborated by patterns of insertions for 4274 CR1 retroelements identified from multispecies whole-genome screening, and is robustly supported by phylogenomic subsampling analyses, with MP-EST demonstrating particularly consistent performance across subsampling replicates as compared to ASTRAL. In contrast, analyses of concatenated data supermatrices recover rheas as the sister to all other nonostrich palaeognaths, an alternative that lacks retroelement support and shows inconsistent behavior under subsampling approaches. While statistically supporting the species tree topology, conflicting patterns of retroelement insertions also occur and imply high amounts of ILS across short successive internal branches, consistent with observed patterns of gene tree heterogeneity. Coalescent simulations and topology tests indicate that the majority of observed topological incongruence among gene trees is consistent with coalescent variation rather than arising from gene tree estimation error alone, and estimated branch lengths for short successive internodes in the inferred species tree fall within the theoretical range encompassing the anomaly zone. Distributions of empirical gene trees confirm that the most common gene tree topology for each marker type differs from the species tree, signifying the existence of an empirical anomaly zone in palaeognaths.

Convergent regulatory evolution and loss of flight in paleognathous birds.

A core question in evolutionary biology is whether convergent phenotypic evolution is driven by convergent molecular changes in proteins or regulatory regions. We combined phylogenomic, developmental, and epigenomic analysis of 11 new genomes of paleognathous birds, including an extinct moa, to show that convergent evolution of regulatory regions, more so than protein-coding genes, is prevalent among developmental pathways associated with independent losses of flight. A Bayesian analysis of 284,001 conserved noncoding elements, 60,665 of which are corroborated as enhancers by open chromatin states during development, identified 2355 independent accelerations along lineages of flightless paleognaths, with functional consequences for driving gene expression in the developing forelimb. Our results suggest that the genomic landscape associated with morphological convergence in ratites has a substantial shared regulatory component.

Natural selection shaped the rise and fall of passenger pigeon genomic diversity.

The extinct passenger pigeon was once the most abundant bird in North America, and possibly the world. Although theory predicts that large populations will be more genetically diverse, passenger pigeon genetic diversity was surprisingly low. To investigate this disconnect, we analyzed 41 mitochondrial and 4 nuclear genomes from passenger pigeons and 2 genomes from band-tailed pigeons, which are passenger pigeons' closest living relatives. Passenger pigeons' large population size appears to have allowed for faster adaptive evolution and removal of harmful mutations, driving a huge loss in their neutral genetic diversity. These results demonstrate the effect that selection can have on a vertebrate genome and contradict results that suggested that population instability contributed to this species's surprisingly rapid extinction.

Conserved Nonexonic Elements: A Novel Class of Marker for Phylogenomics.

Noncoding markers have a particular appeal as tools for phylogenomic analysis because, at least in vertebrates, they appear less subject to strong variation in GC content among lineages. Thus far, ultraconserved elements (UCEs) and introns have been the most widely used noncoding markers. Here we analyze and study the evolutionary properties of a new type of noncoding marker, conserved nonexonic elements (CNEEs), which consists of noncoding elements that are estimated to evolve slower than the neutral rate across a set of species. Although they often include UCEs, CNEEs are distinct from UCEs because they are not ultraconserved, and, most importantly, the core region alone is analyzed, rather than both the core and its flanking regions. Using a data set of 16 birds plus an alligator outgroup, and ∼3600-∼3800 loci per marker type, we found that although CNEEs were less variable than bioinformatically derived UCEs or introns and in some cases exhibited a slower approach to branch resolution as determined by phylogenomic subsampling, the quality of CNEE alignments was superior to those of the other markers, with fewer gaps and missing species. Phylogenetic resolution using coalescent approaches was comparable among the three marker types, with most nodes being fully and congruently resolved. Comparison of phylogenetic results across the three marker types indicated that one branch, the sister group to the passerine + falcon clade, was resolved differently and with moderate (>70%) bootstrap support between CNEEs and UCEs or introns. Overall, CNEEs appear to be promising as phylogenomic markers, yielding phylogenetic resolution as high as for UCEs and introns but with fewer gaps, less ambiguity in alignments and with patterns of nucleotide substitution more consistent with the assumptions of commonly used methods of phylogenetic analysis.

PHYSIOLOGIC PARAMETERS AND THEIR RESPONSE TO HANDLING STRESS IN A NEOTROPICAL MIGRATORY SHOREBIRD DURING THE NONBREEDING SEASON.

Physiologic traits are promising indicators of population health in the face of rapidly changing environments. We obtained values of diverse physiologic parameters for Two-banded Plovers (Charadrius falklandicus) in coastal sites in Patagonia, Argentina, with the objectives of determining the timeline in which these parameters become affected by the stress of capture and handling and of obtaining reference values for future monitoring of these populations. We analyzed packed cell volume, white blood cell profile, heterophil/lymphocyte ratio, bacterial agglutination titer, and total protein, glucose, triglyceride, and cholesterol levels in apparently healthy birds. Glucose, total white blood cell count, lymphocytes, and eosinophil levels showed changes with handling times >60 min after capture. The remaining parameters did not manifest significant alterations in response to capture and handling of up to 232 min (average=105.2, SD=56.7). Therefore, although researchers should attempt to obtain blood samples as soon as possible after capture, inclusion of physiologic parameters in monitoring studies of species not easily sampled in a few minutes, such as Two-banded Plovers and other shorebird species during migration, should not be discouraged. Here we provide a physiologic report for the species that can be considered as reference values during the nonbreeding season at Patagonian coastal sites.

Explosive ice age diversification of kiwi.

Molecular dating largely overturned the paradigm that global cooling during recent Pleistocene glacial cycles resulted in a burst of species diversification although some evidence exists that speciation was commonly promoted in habitats near the expanding and retracting ice sheets. Here, we used a genome-wide dataset of more than half a million base pairs of DNA to test for a glacially induced burst of diversification in kiwi, an avian family distributed within several hundred kilometers of the expanding and retracting glaciers of the Southern Alps of New Zealand. By sampling across the geographic range of the five kiwi species, we discovered many cryptic lineages, bringing the total number of kiwi taxa that currently exist to 11 and the number that existed just before human arrival to 16 or 17. We found that 80% of kiwi diversification events date to the major glacial advances of the Middle and Late Pleistocene. During this period, New Zealand was repeatedly fragmented by glaciers into a series of refugia, with the tiny geographic ranges of many kiwi lineages currently distributed in areas adjacent to these refugia. Estimates of effective population size through time show a dramatic bottleneck during the last glacial cycle in all but one kiwi lineage, as expected if kiwi were isolated in glacially induced refugia. Our results support a fivefold increase in diversification rates during key glacial periods, comparable with levels observed in classic adaptive radiations, and confirm that at least some lineages distributed near glaciated regions underwent rapid ice age diversification.

SNPs across time and space: population genomic signatures of founder events and epizootics in the House Finch (Haemorhous mexicanus).

Identifying genomic signatures of natural selection can be challenging against a background of demographic changes such as bottlenecks and population expansions. Here, we disentangle the effects of demography from selection in the House Finch (Haemorhous mexicanus) using samples collected before and after a pathogen-induced selection event. Using ddRADseq, we genotyped over 18,000 SNPs across the genome in native pre-epizootic western US birds, introduced birds from Hawaii and the eastern United States, post-epizootic eastern birds, and western birds sampled across a similar time span. We found 14% and 7% reductions in nucleotide diversity, respectively, in Hawaiian and pre-epizootic eastern birds relative to pre-epizootic western birds, as well as elevated levels of linkage disequilibrium and other signatures of founder events. Despite finding numerous significant frequency shifts (outlier loci) between pre-epizootic native and introduced populations, we found no signal of reduced genetic diversity, elevated linkage disequilibrium, or outlier loci as a result of the epizootic. Simulations demonstrate that the proportion of outliers associated with founder events could be explained by genetic drift. This rare view of genetic evolution across time in an invasive species provides direct evidence that demographic shifts like founder events have genetic consequences more widespread across the genome than natural selection.

Feather development genes and associated regulatory innovation predate the origin of Dinosauria.

The evolution of avian feathers has recently been illuminated by fossils and the identification of genes involved in feather patterning and morphogenesis. However, molecular studies have focused mainly on protein-coding genes. Using comparative genomics and more than 600,000 conserved regulatory elements, we show that patterns of genome evolution in the vicinity of feather genes are consistent with a major role for regulatory innovation in the evolution of feathers. Rates of innovation at feather regulatory elements exhibit an extended period of innovation with peaks in the ancestors of amniotes and archosaurs. We estimate that 86% of such regulatory elements and 100% of the nonkeratin feather gene set were present prior to the origin of Dinosauria. On the branch leading to modern birds, we detect a strong signal of regulatory innovation near insulin-like growth factor binding protein (IGFBP) 2 and IGFBP5, which have roles in body size reduction, and may represent a genomic signature for the miniaturization of dinosaurian body size preceding the origin of flight.

Genomic support for a moa-tinamou clade and adaptive morphological convergence in flightless ratites.

One of the most startling discoveries in avian molecular phylogenetics is that the volant tinamous are embedded in the flightless ratites, but this topology remains controversial because recent morphological phylogenies place tinamous as the closest relative of a monophyletic ratite clade. Here, we integrate new phylogenomic sequences from 1,448 nuclear DNA loci totaling almost 1 million bp from the extinct little bush moa, Chilean tinamou, and emu with available sequences from ostrich, elegant crested tinamou, four neognaths, and the green anole. Phylogenetic analysis using standard homogeneous models and heterogeneous models robust to common topological artifacts recovered compelling support for ratite paraphyly with the little bush moa closest to tinamous within ratites. Ratite paraphyly was further corroborated by eight independent CR1 retroposon insertions. Analysis of morphological characters reinterpreted on a 27-gene paleognath topology indicates that many characters are convergent in the ratites, probably as the result of adaptation to a cursorial life style.

Complete mitochondrial genomes from four subspecies of common chaffinch (Fringilla coelebs): new inferences about mitochondrial rate heterogeneity, neutral theory, and phylogenetic relationships within the order Passeriformes.

We describe whole mitochondrial genome sequences from four subspecies of the common chaffinch (Fringilla coelebs), and compare them to 31 publicly available mitochondrial genome sequences from other Passeriformes. Rates and patterns of mitochondrial gene evolution are analyzed at different taxonomic levels within this avian order, and evidence is adduced for and against the nearly neutral theory of molecular evolution and the role of positive selection in shaping genetic variation of this small but critical genome. We find evidence of mitochondrial rate heterogeneity in birds as in other vertebrates, likely due to differences in mutational pressure across the genome. Unlike in gadine fish and some of the human mitochondrial work we do not observe strong support for the nearly neutral theory of molecular evolution; instead evidence from molecular clocks, distribution of dN/dS ratios at different levels of the taxonomic hierarchy and in different lineages, McDonald-Kreitman tests within Fringillidae, and site-specific tests of selection within Passeriformes, all point to a role for positive selection, especially for the complex I NADH dehydrogenase genes. The protein-coding mitogenome phylogeny of the order Passeriformes is broadly consistent with previously-reported molecular findings, but provides support for a sister relationship between the superfamilies Muscicapoidea and Passeroidea on a short basal internode of the Passerida where relationships have been difficult to resolve. An unexpected placement of the Paridae (represented by Hume's groundpecker) within the Muscicapoidea was observed. Consistent with other molecular studies the mtDNA phylogeny reveals paraphyly within the Muscicapoidea and a sister relationship of Fringilla with Carduelis rather than Emberiza.

Characterization of MHC class I in a long-distance migrant shorebird suggests multiple transcribed genes and intergenic recombination.

The major histocompatibility complex (MHC) includes highly polymorphic gene families encoding proteins crucial to the vertebrate acquired immune system. Classical MHC class I (MHCI) genes code for molecules expressed on the surfaces of most nucleated cells and are associated with defense against intracellular pathogens, such as viruses. These genes have been studied in a few wild bird species, but have not been studied in long-distance migrating shorebirds. Red Knots Calidris canutus are medium-sized, monogamous sandpipers with migratory routes that span the globe. Understanding how such long-distance migrants protect themselves from disease has gained new relevance since the emergence of avian-borne diseases, including intracellular pathogens recognized by MHCI molecules, such as avian influenza. In this study, we characterized MHCI genes in knots and found 36 alleles in eight individuals and evidence for six putatively functional and expressed MHCI genes in a single bird. We also found evidence for recombination and for positive selection at putative peptide binding sites in exons 2 and 3. These results suggest surprisingly high MHC diversity in knots, given their demographic history. This may be a result of selection from diverse pathogens encountered by shorebirds throughout their annual migrations.

Multiple nuclear genes and retroposons support vicariance and dispersal of the palaeognaths, and an Early Cretaceous origin of modern birds.

The origin and timing of the diversification of modern birds remains controversial, primarily because phylogenetic relationships are incompletely resolved and uncertainty persists in molecular estimates of lineage ages. Here, we present a species tree for the major palaeognath lineages using 27 nuclear genes and 27 archaic retroposon insertions. We show that rheas are sister to the kiwis, emu and cassowaries, and confirm ratite paraphyly because tinamous are sister to moas. Divergence dating using 10 genes with broader taxon sampling, including emu, cassowary, ostrich, five kiwis, two rheas, three tinamous, three extinct moas and 15 neognath lineages, suggests that three vicariant events and possibly two dispersals are required to explain their historical biogeography. The age of crown group birds was estimated at 131 Ma (95% highest posterior density 122-138 Ma), similar to previous molecular estimates. Problems associated with gene tree discordance and incomplete lineage sorting in birds will require much larger gene sets to increase species tree accuracy and improve error in divergence times. The relatively rapid branching within neoaves pre-dates the extinction of dinosaurs, suggesting that the genesis of the radiation within this diverse clade of birds was not in response to the Cretaceous-Paleogene extinction event.

Eight independent nuclear genes support monophyly of the plovers: the role of mutational variance in gene trees.

Molecular phylogenies of Charadriiformes based on mtDNA genes and one to three nuclear loci do not support the traditional placement of Pluvialis in the plovers (Charadriidae), assigning it instead to oystercatchers, stilts, and avocets (Haematopodidae and Recurvirostridae). To investigate this hypothesis of plover paraphyly, the relationships among Pluvialis and closely related families were revisited by sequencing two individuals of all taxa except Peltohyas for eight independent single copy nuclear protein-coding loci selected for their informativeness at this phylogenetic depth. The species tree estimated jointly with the gene trees in the coalescent programme (*)BEAST strongly supported plover monophyly, as did Bayesian analysis of the concatenated matrix. The data sets that supported plover paraphyly in Baker et al. (2007) and Fain and Houde (2007) reflect two to four independent gene histories, and thus discordance with the plover monophyly species tree might have arisen by chance through stochastic mutational variance. For the plovers we conclude there is no conclusive evidence of coalescent variance from ancient incomplete lineage sorting across the interior branch leading to Pluvialis in the species tree. Rather, earlier studies seem have been misled by faster evolving mtDNA genes with high mutational variance, and a few nuclear genes that had low resolving power at the Pluvialis sister group level. These findings are of general relevance in avian phylogenetics, as they show that careful attention needs to be paid to the number and the phylogenetic informativeness of genes required to obtain accurate estimates of the species tree, especially where there is mutational heterogeneity in gene trees.

A trans-Amazonian screening of mtDNA reveals deep intraspecific divergence in forest birds and suggests a vast underestimation of species diversity.

The Amazonian avifauna remains severely understudied relative to that of the temperate zone, and its species richness is thought to be underestimated by current taxonomy. Recent molecular systematic studies using mtDNA sequence reveal that traditionally accepted species-level taxa often conceal genetically divergent subspecific lineages found to represent new species upon close taxonomic scrutiny, suggesting that intraspecific mtDNA variation could be useful in species discovery. Surveys of mtDNA variation in Holarctic species have revealed patterns of variation that are largely congruent with species boundaries. However, little information exists on intraspecific divergence in most Amazonian species. Here we screen intraspecific mtDNA genetic variation in 41 Amazonian forest understory species belonging to 36 genera and 17 families in 6 orders, using 758 individual samples from Ecuador and French Guiana. For 13 of these species, we also analyzed trans-Andean populations from the Ecuadorian Chocó. A consistent pattern of deep intraspecific divergence among trans-Amazonian haplogroups was found for 33 of the 41 taxa, and genetic differentiation and genetic diversity among them was highly variable, suggesting a complex range of evolutionary histories. Mean sequence divergence within families was the same as that found in North American birds (13%), yet mean intraspecific divergence in Neotropical species was an order of magnitude larger (2.13% vs. 0.23%), with mean distance between intraspecific lineages reaching 3.56%. We found no clear relationship between genetic distances and differentiation in plumage color. Our results identify numerous genetically and phenotypically divergent lineages which may result in new species-level designations upon closer taxonomic scrutiny and thorough sampling, although lineages in the tropical region could be older than those in the temperate zone without necessarily representing separate species. In-depth phylogeographic surveys are urgently needed to avoid underestimating tropical diversity, and the use of mtDNA markers can be instrumental in identifying and prioritizing taxa for species discovery.

DNA barcode detects high genetic structure within neotropical bird species.

BACKGROUND: Towards lower latitudes the number of recognized species is not only higher, but also phylogeographic subdivision within species is more pronounced. Moreover, new genetically isolated populations are often described in recent phylogenies of Neotropical birds suggesting that the number of species in the region is underestimated. Previous COI barcoding of Argentinean bird species showed more complex patterns of regional divergence in the Neotropical than in the North American avifauna. METHODS AND FINDINGS: Here we analyzed 1,431 samples from 561 different species to extend the Neotropical bird barcode survey to lower latitudes, and detected even higher geographic structure within species than reported previously. About 93% (520) of the species were identified correctly from their DNA barcodes. The remaining 41 species were not monophyletic in their COI sequences because they shared barcode sequences with closely related species (N = 21) or contained very divergent clusters suggestive of putative new species embedded within the gene tree (N = 20). Deep intraspecific divergences overlapping with among-species differences were detected in 48 species, often with samples from large geographic areas and several including multiple subspecies. This strong population genetic structure often coincided with breaks between different ecoregions or areas of endemism. CONCLUSIONS: The taxonomic uncertainty associated with the high incidence of non-monophyletic species and discovery of putative species obscures studies of historical patterns of species diversification in the Neotropical region. We showed that COI barcodes are a valuable tool to indicate which taxa would benefit from more extensive taxonomic revisions with multilocus approaches. Moreover, our results support hypotheses that the megadiversity of birds in the region is associated with multiple geographic processes starting well before the Quaternary and extending to more recent geological periods.

Genetic evaluation of the mating system in the blue-and-yellow macaw (Ara ararauna, Aves, Psittacidae) by DNA fingerprinting.

More than 90% of birds are socially monogamous, although genetic studies indicate that many are often not sexually monogamous. In the present study, DNA fingerprinting was used to estimate the genetic relationships between nestlings belonging to the same broods to evaluate the mating system in the socially monogamous macaw, Ara ararauna. We found that in 10 of 11 broods investigated, the nestlings showed genetic similarity levels congruent with values expected among full-sibs, suggesting that they shared the same parents. However, in one brood, the low genetic similarity observed between nestlings could be a result of intraspecific brood parasitism, intraspecific nest competition or extra-pair paternity. These results, along with available behavioral and life-history data, imply that the blue-and-yellow macaw is not only socially, but also genetically monogamous. However, the occurrence of eventual cases of extra-pair paternity cannot be excluded.

Characterization and locus-specific typing of MHC class I genes in the red-billed gull (Larus scopulinus) provides evidence for major, minor, and nonclassical loci.

A major challenge facing studies of major histocompatibility complex (MHC) evolution in birds is the difficulty in genotyping alleles at individual loci, and the consequent inability to investigate sequence variation and selection pressures for each gene. In this study, four MHC class I loci were isolated from the red-billed gull (Larus scopulinus), representing both the first characterized MHCI genes within Charadriiformes (shorebirds, gulls, and allies) and the first full-length MHCI sequences described outside Galloanserae (gamebirds + waterfowl). Complete multilocus genotypes were obtained for 470 individuals using a combination of reference-strand conformation analysis and direct sequencing of gene-specific amplification products, and variation of peptide-binding region (PBR) exons was surveyed for all loci. Each gene is transcribed and has conserved sequence features characteristic of antigen-presenting MHCI molecules. However, higher allelic variation, a more even allele frequency distribution, and evidence of positive selection acting on a larger number of PBR residues suggest that only one locus (Lasc-UAA) functions as a major classical MHCI gene. Lasc-UBA, with more limited variation and PBR motifs that encompass a subset of Lasc-UAA diversity, was assigned a putative minor classical function, whereas the divergent and largely invariant binding-groove motifs of Lasc-UCA and -UDA are suggestive of nonclassical loci with specialized ligand-binding roles.

Genetic evaluation of the mating system in the blue-and-yellow macaw (Ara ararauna, Aves, Psittacidae) by DNA fingerprinting

More than 90 percent of birds are socially monogamous, although genetic studies indicate that many are often not sexually monogamous. In the present study, DNA fingerprinting was used to estimate the genetic relationships between nestlings belonging to the same broods to evaluate the mating system in the socially monogamous macaw, Ara ararauna. We found that in 10 of 11 broods investigated, the nestlings showed genetic similarity levels congruent with values expected among full-sibs, suggesting that they shared the same parents. However, in one brood, the low genetic similarity observed between nestlings could be a result of intraspecific brood parasitism, intraspecific nest competition or extra-pair paternity. These results, along with available behavioral and life-history data, imply that the blue-and-yellow macaw is not only socially, but also genetically monogamous. However, the occurrence of eventual cases of extra-pair paternity cannot be excluded.