A new Paleogene fossil and a new dataset for waterfowl (Aves: Anseriformes) clarify phylogeny, ecological evolution, and avian evolution at the K-Pg Boundary.

Despite making up one of the most ecologically diverse groups of living birds, comprising soaring, diving and giant flightless taxa, the evolutionary relationships and ecological evolution of Anseriformes (waterfowl) remain unresolved. Although Anseriformes have a comparatively rich, global Cretaceous and Paleogene fossil record, morphological datasets for this group that include extinct taxa report conflicting relationships for all known extinct taxa. Correct placement of extinct taxa is necessary to understand whether ancestral anseriform feeding ecology was more terrestrial or one of a set of diverse aquatic ecologies and to better understand avian evolution around the K-T boundary. Here, we present a new morphological dataset for Anseriformes that includes more extant and extinct taxa than any previous anseriform-focused dataset and describe a new anseriform species from the early Eocene Green River Formation of North America. The new taxon has a mediolaterally narrow bill which is rarely found in previously described anseriform fossils. The matrix created to assess the placement of this taxon comprises 41 taxa and 719 discrete morphological characters describing skeletal morphology, musculature, syringeal morphology, ecology, and behavior. We additionally combine the morphological dataset with published sequences using Bayesian methods and perform ancestral state reconstruction for select morphological, ecological and behavioral characters. We recover the new Eocene taxon as the sister taxon to (Anseranatidae+Anatidae) across all analyses, and find that the new taxon represents a novel ecology within known Anseriformes and the Green River taxa. Results provide insight into avian evolution during and following the K-Pg mass extinction and indicate that Anseriformes were likely ancestrally aquatic herbivores with rhamphothecal lamellae..

A high-quality chromosomal-level genome assembly of Greater Scaup (Aythya marila).

Aythya marila is one of the few species of Anatidae, and the only Aythya to live in the circumpolar. However, there is a relative lack of research on genetics of this species. In this study, we reported and assembled the first high-quality chromosome-level genome assembly of A. marila. This genome was assembled using Nanopore long reads, and errors corrected using Illumina short reads, with a final genome size of 1.14 Gb, scaffold N50 of 85.44 Mb, and contig N50 of 32.46 Mb. 106 contigs were clustered and ordered onto 35 chromosomes based on Hi-C data, covering approximately 98.28% of the genome. BUSCO assessment showed that 97.0% of the highly conserved genes in aves_odb10 were present intact in the genome assembly. In addition, a total of 154.94 Mb of repetitive sequences were identified. 15,953 protein-coding genes were predicted in the genome, and 98.96% of genes were functionally annotated. This genome will be a valuable resource for future genetic diversity and genomics studies of A. marila.

Bacterial composition along the digestive tract of the Horned Screamer (Anhima cornuta), a tropical herbivorous bird.

Background: The Horned Screamer (Anhima cornuta) is an herbivorous bird that inhabits wetlands of the South American tropical region. We hypothesize that due to its herbivorous niche, its digestive tract compartments may have bacteria specialized in fermenting complex plant carbohydrates. To test this hypothesis, we compared the bacterial communities along the gastrointestinal tract (GIT) of a Horned Screamer captured in Venezuela. Methods: Samples were taken from tissues and content of the proventriculus and the small intestine (considered for this study as upper GIT), and the large intestine and cecum (lower GIT). The bacterial community was characterized by sequencing the V4 region of the 16S rRNA gene. Bioinformatic analysis was performed using QIIME, QIITA and Microbiome Analyst. The association between microbial taxonomy and function was analyzed using their Greengenes OTU IDs and a custom KEGG BRITE hierarchical tree and visualized with BURRITO. Results: The Screamer's gastrointestinal microbiota was composed by seven phyla being Firmicutes and Bacteroidetes the most predominant. The dominant taxa in the upper GIT were Helicobacter, Vibrio, Enterobacter, Acinetobacter and Staphylococcus. The dominant taxa in the lower GIT were Oribacterium, Blautia, Roseburia, Ruminococcus, Desulfovibrio, Intestinimonas, Marvinbryantia and Parabacteroides. Complete degradation of cellulose to the end-products acetate, propanoate, butanoate and acetoacetate was found in the upper and lower GIT without significant differences. Conclusion: Our study confirmed changes in bacterial community composition throughout the GIT of the Horned Screamer primarily associated with the production of metabolic end-products of carbohydrate digestion essential for the fermentation of the herbivorous diet.

Health monitoring in birds using bio-loggers and whole blood transcriptomics.

Monitoring and early detection of emerging infectious diseases in wild animals is of crucial global importance, yet reliable ways to measure immune status and responses are lacking for animals in the wild. Here we assess the usefulness of bio-loggers for detecting disease outbreaks in free-living birds and confirm detailed responses using leukocyte composition and large-scale transcriptomics. We simulated natural infections by viral and bacterial pathogens in captive mallards (Anas platyrhynchos), an important natural vector for avian influenza virus. We show that body temperature, heart rate and leukocyte composition change reliably during an acute phase immune response. Using genome-wide gene expression profiling of whole blood across time points we confirm that immunostimulants activate pathogen-specific gene regulatory networks. By reporting immune response related changes in physiological and behavioural traits that can be studied in free-ranging populations, we provide baseline information with importance to the global monitoring of zoonotic diseases.

Molecular cytogenetic characterization of repetitive sequences comprising centromeric heterochromatin in three Anseriformes species.

The highly repetitive DNA sequence of centromeric heterochromatin is an effective molecular cytogenetic marker for investigating genomic compartmentalization between macrochromosomes and microchromosomes in birds. We isolated four repetitive sequence families of centromeric heterochromatin from three Anseriformes species, viz., domestic duck (Anas platyrhynchos, APL), bean goose (Anser fabalis, AFA), and whooper swan (Cygnus cygnus, CCY), and characterized the sequences by molecular cytogenetic approach. The 190-bp APL-HaeIII and 101-bp AFA-HinfI-S sequences were localized in almost all chromosomes of A. platyrhynchos and A. fabalis, respectively. However, the 192-bp AFA-HinfI-L and 290-bp CCY-ApaI sequences were distributed in almost all microchromosomes of A. fabalis and in approximately 10 microchromosomes of C. cygnus, respectively. APL-HaeIII, AFA-HinfI-L, and CCY-ApaI showed partial sequence homology with the chicken nuclear-membrane-associated (CNM) repeat families, which were localized primarily to the centromeric regions of microchromosomes in Galliformes, suggesting that ancestral sequences of the CNM repeat families are observed in the common ancestors of Anseriformes and Galliformes. These results collectively provide the possibility that homogenization of centromeric heterochromatin occurred between microchromosomes in Anseriformes and Galliformes; however, homogenization between macrochromosomes and microchromosomes also occurred in some centromeric repetitive sequences.

Comparative proteomic analysis revealed complex responses to classical/novel duck reovirus infections in Cairna moschata.

Duck reovirus (DRV) is an typical aquatic bird pathogen belonging to the Orthoreovirus genus of the Reoviridae family. Reovirus causes huge economic losses to the duck industry. Although DRV has been identified and isolated long ago, the responses of Cairna moschata to classical/novel duck reovirus (CDRV/NDRV) infections are largely unknown. To investigate the relationship of pathogenesis and immune response, proteomes of C. moschata liver cells under the C/NDRV infections were analyzed, respectively. In total, 5571 proteins were identified, among which 5015 proteins were quantified. The differential expressed proteins (DEPs) between the control and infected liver cells displayed diverse biological functions and subcellular localizations. Among the DEPs, most of the metabolism-related proteins were down-regulated, suggesting a decrease in the basal metabolisms under C/NDRV infections. Several important factors in the complement, coagulation and fibrinolytic systems were significantly up-regulated by the C/NDRV infections, indicating that the serine protease-mediated innate immune system might play roles in the responses to the C/NDRV infections. Moreover, a number of molecular chaperones were identified, and no significantly changes in their abundances were observed in the liver cells. Our data may give a comprehensive resource for investigating the regulation mechanism involved in the responses of C. moschata to the C/NDRV infections.

Influenza A virus recovery, diversity, and intercontinental exchange: A multi-year assessment of wild bird sampling at Izembek National Wildlife Refuge, Alaska.

Western Alaska is a potential point-of-entry for foreign-origin influenza A viruses (IAVs) into North America via migratory birds. We sampled waterfowl and gulls for IAVs at Izembek National Wildlife Refuge (NWR) in western Alaska, USA, during late summer and autumn months of 2011-2015, to evaluate the abundance and diversity of viruses at this site. We collected 4842 samples across five years from 25 species of wild birds resulting in the recovery, isolation, and sequencing of 172 IAVs. With the intent of optimizing sampling efficiencies, we used information derived from this multi-year effort to: 1) evaluate from which species we consistently recover viruses, 2) describe viral subtypes of isolates by host species and year, 3) characterize viral gene segment sequence diversity with respect to host species, and assess potential differences in the viral lineages among the host groups, and 4) examine how evidence of intercontinental exchange of IAVs relates to host species. We consistently recovered viruses from dabbling ducks (Anas spp.), emperor geese (Chen canagica) and glaucous-winged gulls (Larus glaucescens). There was little evidence for differences in viral subtypes and diversity from different waterfowl hosts, however subtypes and viral diversity varied between waterfowl host groups and glaucous-winged gulls. Furthermore, higher proportions of viral sequences from northern pintails (Anas acuta), emperor geese and glaucous-winged gulls were grouped in phylogenetic clades that included IAV sequences originating from wild birds sampled in Asia as compared to non-pintail dabbling ducks, a difference that may be related to intercontinental migratory tendencies of host species. Our summary of research and surveillance efforts at Izembek NWR will assist in future prioritization of which hosts to sample and swab types to collect in Alaska and elsewhere in order to maximize isolate recovery, subtype and sequence diversity for resultant viruses, and detection of evidence for intercontinental viral exchange.

Rapid and recent diversification patterns in Anseriformes birds: Inferred from molecular phylogeny and diversification analyses.

The Anseriformes is a well-known and widely distributed bird order, with more than 150 species in the world. This paper aims to revise the classification, determine the phylogenetic relationships and diversification patterns in Anseriformes by exploring the Cyt b, ND2, COI genes and the complete mitochondrial genomes (mito-genomes). Molecular phylogeny and genetic distance analyses suggest that the Dendrocygna species should be considered as an independent family, Dendrocygnidae, rather than a member of Anatidae. Molecular timescale analyses suggests that the ancestral diversification occurred during the Early Eocene Climatic Optimum (58 ~ 50 Ma). Furthermore, diversification analyses showed that, after a long period of constant diversification, the median initial speciation rate was accelerated three times, and finally increased to approximately 0.3 sp/My. In the present study, both molecular phylogeny and diversification analyses results support that Anseriformes birds underwent rapid and recent diversification in their evolutionary history, especially in modern ducks, which show extreme diversification during the Plio-Pleistocene (~ 5.3 Ma). Therefore, our study support that the Plio-Pleistocene climate fluctuations are likely to have played a significant role in promoting the recent diversification for Anseriformes.

Characterising the interspecific variations and convergence of gut microbiota in Anseriformes herbivores at wintering areas.

Microorganisms in vertebrate guts have been recognized as important symbionts influencing host life. However, it remains unclear about the gut microbiota in long-distance migratory Anseriformes herbivores, which could be functionally important for these wetland-dependent animals. We collected faeces of the greater white-fronted goose (GWFG), bean goose (BG) and swan goose (SG) from Shengjin Lake (SJL) and Poyang Lake (PYL) in the Yangtze River Floodplain, China. High-throughput sequencing of 16S rRNA V4 region was employed to depict the composition and structure of geese gut microbiota during wintering period. The dominant bacterial phyla across all samples were Firmicutes, Proteobacteria and Actinobacteria, but significant variations were detected among different goose species and sampling sites, in terms of α diversity, community structures and microbial interactions. We found a significant correlation between diet and the microbial community structure in GWFG-SJL samples. These results demonstrated that host species and diet are potential drivers of goose gut microbiota assemblies. Despite these variations, functions of geese gut microbiota were similar, with great abundances of potential genes involved in nutrient metabolism. This preliminary study would be valuable for future, exhaustive investigations of geese gut microbiota and their interactions with host.

DNA double-strand breaks in incubating female common eiders (Somateria mollissima): Comparison between a low and a high polluted area.

Alterations in the genetic material may have severe consequences for individuals and populations. Hence, genotoxic effects of environmental exposure to pollutants are of great concern. We assessed the impact of blood concentrations of persistent organic pollutants (POPs) and mercury (Hg) on DNA double-strand break (DSB) frequency, in blood cells of a high-exposed Baltic, and lower exposed Arctic population of common eiders (Somateria mollissima). Furthermore, we examined whether the genotoxic response was influenced by antioxidant concentration (plasma total glutathione (tGSH) and total antioxidant capacity) and female body mass. The DNA DSB frequency did not differ between the two populations. We found significant positive relationships between Hg and DNA DSB frequency in Baltic, but not in Arctic eiders. Although both p,p'-DDE and PCB 118 had a lesser effect than Hg, they exhibited a positive association with DNA DSB frequency in Baltic eiders. Antioxidant levels were not important for the genotoxic effect, suggesting alternative mechanisms other than GSH depletion for the relationship between Hg and DNA DSBs. Hence, the Baltic population, which is considered to be endangered and is under the influence of several environmental stressors, may be more susceptible to genotoxic effects of environmental exposure to Hg than the Arctic population.

The Evolution of Innate Immune Genes: Purifying and Balancing Selection on ß-Defensins in Waterfowl.

In disease dynamics, high immune gene diversity can confer a selective advantage to hosts in the face of a rapidly evolving and diverse pathogen fauna. This is supported empirically for genes involved in pathogen recognition and signalling. In contrast, effector genes involved in pathogen clearance may be more constrained. ß-Defensins are innate immune effector genes; their main mode of action is via disruption of microbial membranes. Here, five ß-defensin genes were characterized in mallards (Anas platyrhynchos) and other waterfowl; key reservoir species for many zoonotic diseases. All five genes showed remarkably low diversity at the individual-, population-, and species-level. Furthermore, there was widespread sharing of identical alleles across species divides. Thus, specific ß-defensin alleles were maintained not only spatially but also over long temporal scales, with many amino acid residues being fixed across all species investigated. Purifying selection to maintain individual, highly efficacious alleles was the primary evolutionary driver of these genes in waterfowl. However, we also found evidence for balancing selection acting on the most recently duplicated ß-defensin gene (AvBD3b). For this gene, we found that amino acid replacements were more likely to be radical changes, suggesting that duplication of ß-defensin genes allows exploration of wider functional space. Structural conservation to maintain function appears to be crucial for avian ß-defensin effector molecules, resulting in low tolerance for new allelic variants. This contrasts with other types of innate immune genes, such as receptor and signalling molecules, where balancing selection to maintain allelic diversity has been shown to be a strong evolutionary force.

Waterbird Susceptibility to Avian Cholera at Hayward Marsh, California, USA.

We characterized past avian cholera outbreaks in waterbirds at Hayward Marsh, California, US. In 2013, we surveyed populations and determined the presence of disease using several diagnostic methods, including behavioral and physical observations, field necropsy, and bacterial culture. We compiled this information with data from previous outbreaks from 1990-2012 to compare waterbird abundance to various measures of mortality, including percentage of mortality and percentage of difference between abundance and mortality by species. We suggest that Ruddy Duck ( Oxyura jamaicensis ) have consistently suffered greater mortality from this disease than have other species at this site.

The complete mitochondrial genome of Anser indicus (Aves, Anseriformes, Anatidae).

The bar-headed goose is known one of the world's highest-flying birds. In this study, the complete mitochondrial genome of Anser indicus (16,728 bp in length) was sequenced. Similar to the typical mtDNA of other vertebrates, goose mtDNA contained 37 genes (13 protein-coding genes, 2 rRNA genes and 22 tRNA genes) and a non-coding region (D-loop). The characteristics of the mitochondrial genome were analyzed in detail. We deduce that ND5 may be a major gene required for adaptation to high-altitude flight. The complete mitochondrial genome sequence of A. indicus obtained will be useful for phylogenetics, and biological characteristics.

Structure and variation of the Anseriformes mitochondrial DNA control region.

The control region is the major non-coding segment of animal mitochondrial DNA. To infer the structure and variation of Anseriformes mitochondrial DNA control region, the control region sequences of 52 species were analyzed. The length of the control region sequences ranged from 968 bp (Chenonetta jubata) to 1335 bp (Anseranas semipalmata) and can be separated into three domains. There is a deletion of 100-130 bp in Anatinae, compared to other groups of Anserinae. The average genetic distances among the species within the genera varied from 4.14% (Anser) to 10.58% (Cygnus). The average genetic distances showed insignificantly negative correlation with ts/tv. Domain I is the most variable among the three domains among all the genera. Five conserved sequence boxes in the domain II of Anseriformes sequences were identified. The alignment of the Anseriformes five boxes sequences showed considerable sequence variation. CSB-1, -2 and 3 were not found in the Anseriformes. Maximum-likelihood method was used to construct a phylogenetic tree, which grouped all of the genera into four divergent clades. Anseranas + Chauna and Dendrocygna were identified as early offshoots of the Anatidae. All the remaining taxa fell into two clades that correspond to the two subfamilies Anserinae and Anatiane.

The complete mitochondrial genome of Aythya fuligula (Anatidae: Aythya).

Aythya fuligula belongs to the genus of Aythya (Anseriformes, Anatidae), which only has 12 species worldwide. The complete mitochondrial genome sequence of A. fuligula was implemented. A circular molecule of 16,616 bp in length, containing 13 protein-coding genes, 2 rRNA genes, 22 tRNAs and a non-coding AT-rich region. Its gene arrangement patterns were identical to the most other species of Anseriformes.

Complete mitochondrial genome of Tundra swan Cygnus columbianus jankowskii (Anseriformes: Anatidae).

Cygnus columbianus jankowskii is a subspecies of Tundra swan, which breeds in eastern Russia and northeast China, wintering in the middle and lower reaches of the Yangtze River. In this study, we used PCR-based method to obtain the complete mtDNA of this subspecies. The arrangement pattern of the complete mtDNA is identical with typical bird species, which is the shortest (16,723 bp) in three subspecies. The length of the PCGs is same except ND2, ND3 and ND6, and the initiation/termination codons are all same with other subspecies. The length of 12S rRNA and 16S rRNA is same with C. c. columbianus, but different from C. c. bewickii. The length and structure of all tRNAs are the same with other subspecies except for tRNA(Ile), tRNA(leu(CUN)) and tRNA(ser(AGY)). The control region is located between tRNA(Phe) and tRNA(Glu), but the length is different with others.

Complete mitochondrial genome of Swan goose Anser cygnoides (Anseriformes: Anatidae).

The Swan goose Anser cygnoides is a large threatened goose species in the IUCN Red List, with a natural breeding range in East Asia, migratory and wintering mainly in central and eastern China. In this study, we used PCR-based method to determine the complete mitochondrial genome (mtDNA) of this Anatidae species. The complete mtDNA is a 16,740 bp circular molecule containing 37 typical genes. The gene order is identical with that of the standard avian gene order. All protein-coding genes start with a typical ATG codon except ND5, COI and COII. The termination codon is usually the standard TAA. The non-coding region contains some inter-genic spacers and a control region.

Complete mitochondrial genome of Cygnus olor (Aves, Anseriformes, Anatidae).

The complete mitochondrial genome of Cygnus olor (Aves, Anseriformes, Anatidae) was revealed in this study. Total 16 739 base pairs (bp) of this mitogenome encoded genes for 13 protein coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs) and a D-loop (control region). The 12S rRNA and 16S rRNA genes are located between tRNA-Phe and tRNA-Leu (UUR) and segmentalized by the tRNA-Val. D-loop is located between tRNA-Glu and tRNA-Phe. The overall base composition of C. olor is G + C: 47.8%, A + T: 52.2%, apparently with a slight AT bias. Following the phylogenetic analysis, the C. olor was closed to Anser cygnoides.

Complete mitochondrial genome of Cygnus cygnus (Aves, Anseriformes, Anatidae).

In this study, the complete mitochondrial genome of Cygnus cygnus (Aves, Anseriformes, Anatidae) was sequenced. The genome, consisting of 16 724 base pairs (bp), encoded 13 protein coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and a control region (CR). Two rRNA genes for 12S rRNA (991 bases) and 16S rRNA (1608 bases) are located between tRNA-Phe and tRNA-Leu (UUR) and divided by the tRNA-Val. The CR, of 1156 bp in length, is located between tRNA-Glu and tRNA-Phe. The overall base composition of C. cygnus is G + C: 47.2%, A + T: 52.8%, apparently with a slight AT bias. Phylogenetic analysis showed that the C. cygnus was closed to Cygnus columbianus.

Phylogenetic studies of two Anas platyrhynchos (Anatini: Anatinae) in Hunan province of China based on complete mitochondrial DNA sequences.

In this study, we cloned and sequenced the complete mitochondrial DNAs of Chinese duck, Anas platyrhynchos, population from two different areas of Hunan province in China. The Anas platyrhynchos breed Linwu duck (LW) sample was taken from the Linwu county of Chenzhou city, and the Anas platyrhynchos breed Youxian duck (YX) sample was taken from the Youxian county of Zhuzhou city. The lengths of their complete mitochondrial genome were 16,604 bp (LW) and 16,606 bp (YX), respectively. The organization of the two Anas platyrhynchos breed mitochondrial genomes was similar to those reported from other duck mitochondrial genomes. Phylogenetic analyses using N-J computational algorithms showed that the analyzed species are divided into four major clades: Anatinae, Anserinae, Dendrocygninae and Anseranatidae. Also, the Linwu duck and Youxian duck have highly similar phylogenetic relationship.