Matthew J. Greenwold

Comparative genomics reveals insights into avian genome evolution and adaptation

Birds are the most species-rich class of tetrapod vertebrates and have wide relevance across many research fields. We explored bird macroevolution using full genomes from 48 avian species representing all major extant clades. The avian genome is principally characterized by its constrained size, which predominantly arose because of lineage-specific erosion of repetitive elements, large segmental deletions, and gene loss. Avian genomes furthermore show a remarkably high degree of evolutionary stasis at the levels of nucleotide sequence, gene synteny, and chromosomal structure. Despite this pattern of conservation, we detected many non-neutral evolutionary changes in protein-coding genes and noncoding regions. These analyses reveal that pan-avian genomic diversity covaries with adaptations to different lifestyles and convergent evolution of traits.

Sequencing three crocodilian genomes to illuminate the evolution of archosaurs and amniotes

The International Crocodilian Genomes Working Group (ICGWG) will sequence and assemble the American alligator (Alligator mississippiensis), saltwater crocodile (Crocodylus porosus) and Indian gharial (Gavialis gangeticus) genomes. The status of these projects and our planned analyses are described.

Two Antarctic penguin genomes reveal insights into their evolutionary history and molecular changes related to the Antarctic environment.

BackgroundPenguins are flightless aquatic birds widely distributed in the Southern Hemisphere. The distinctive morphological and physiological features of penguins allow them to live an aquatic life, and some of them have successfully adapted to the hostile environments in Antarctica. To study the phylogenetic and population history of penguins and the molecular basis of their adaptations to Antarctica, we sequenced the genomes of the two Antarctic dwelling penguin species, the Adélie penguin [Pygoscelis adeliae] and emperor penguin [Aptenodytes forsteri].ResultsPhylogenetic dating suggests that early penguins arose ~60 million years ago, coinciding with a period of global warming. Analysis of effective population sizes reveals that the two penguin species experienced population expansions from ~1 million years ago to ~100 thousand years ago, but responded differently to the climatic cooling of the last glacial period. Comparative genomic analyses with other available avian genomes identified molecular changes in genes related to epidermal structure, phototransduction, lipid metabolism, and forelimb morphology.ConclusionsOur sequencing and initial analyses of the first two penguin genomes provide insights into the timing of penguin origin, fluctuations in effective population sizes of the two penguin species over the past 10 million years, and the potential associations between these biological patterns and global climate change. The molecular changes compared with other avian genomes reflect both shared and diverse adaptations of the two penguin species to the Antarctic environment.

Dynamic evolution of the alpha (α) and beta (β) keratins has accompanied integument diversification and the adaptation of birds into novel lifestyles.

BackgroundVertebrate skin appendages are constructed of keratins produced by multigene families. Alpha (α) keratins are found in all vertebrates, while beta (β) keratins are found exclusively in reptiles and birds. We have studied the molecular evolution of these gene families in the genomes of 48 phylogenetically diverse birds and their expression in the scales and feathers of the chicken.ResultsWe found that the total number of α-keratins is lower in birds than mammals and non-avian reptiles, yet two α-keratin genes (KRT42 and KRT75) have expanded in birds. The β-keratins, however, demonstrate a dynamic evolution associated with avian lifestyle. The avian specific feather β-keratins comprise a large majority of the total number of β-keratins, but independently derived lineages of aquatic and predatory birds have smaller proportions of feather β-keratin genes and larger proportions of keratinocyte β-keratin genes. Additionally, birds of prey have a larger proportion of claw β-keratins. Analysis of α- and β-keratin expression during development of chicken scales and feathers demonstrates that while α-keratins are expressed in these tissues, the number and magnitude of expressed β-keratin genes far exceeds that of α-keratins.ConclusionsThese results support the view that the number of α- and β-keratin genes expressed, the proportion of the β-keratin subfamily genes expressed and the diversification of the β-keratin genes have been important for the evolution of the feather and the adaptation of birds into multiple ecological niches.

Molecular evolution and expression of archosaurian β-keratins: Diversification and expansion of archosaurian β-keratins and the origin of feather β-keratins

The archosauria consist of two living groups, crocodilians, and birds. Here we compare the structure, expression, and phylogeny of the beta (β)-keratins in two crocodilian genomes and two avian genomes to gain a better understanding of the evolutionary origin of the feather β-keratins. Unlike squamates such as the green anole with 40 β-keratins in its genome, the chicken and zebra finch genomes have over 100 β-keratin genes in their genomes, while the American alligator has 20 β-keratin genes, and the saltwater crocodile has 21 β-keratin genes. The crocodilian β-keratins are similar to those of birds and these structural proteins have a central filament domain and N- and C-termini, which contribute to the matrix material between the twisted β-sheets, which form the 2-3 nm filament. Overall the expression of alligator β-keratin genes in the integument increases during development. Phylogenetic analysis demonstrates that a crocodilian β-keratin clade forms a monophyletic group with the avian scale and feather β-keratins, suggesting that avian scale and feather β-keratins along with a subset of crocodilian β-keratins evolved from a common ancestral gene/s. Overall, our analyses support the view that the epidermal appendages of basal archosaurs used a diverse array of β-keratins, which evolved into crocodilian and avian specific clades. In birds, the scale and feather subfamilies appear to have evolved independently in the avian lineage from a subset of archosaurian claw β-keratins. The expansion of the avian specific feather β-keratin genes accompanied the diversification of birds and the evolution of feathers.

Expressed miRNAs target feather related mRNAs involved in cell signaling, cell adhesion and structure during chicken epidermal development.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Previous studies have shown that miRNA regulation contributes to a diverse set of processes including cellular differentiation and morphogenesis which leads to the creation of different cell types in multicellular organisms and is thus key to animal development. Feathers are one of the most distinctive features of extant birds and are important for multiple functions including flight, thermal regulation, and sexual selection. However, the role of miRNAs in feather development has been woefully understudied despite the identification of cell signaling pathways, cell adhesion molecules and structural genes involved in feather development. In this study, we performed a microarray experiment comparing the expression of miRNAs and mRNAs among three embryonic stages of development and two tissues (scutate scale and feather) of the chicken. We combined this expression data with miRNA target prediction tools and a curated list of feather related genes to produce a set of 19 miRNA-mRNA duplexes. These targeted mRNAs have been previously identified as important cell signaling and cell adhesion genes as well as structural genes involved in feather and scale morphogenesis. Interestingly, the miRNA target site of the cell signaling pathway gene, Aldehyde Dehydrogenase 1 Family, Member A3 (ALDH1A3), is unique to birds indicating a novel role in Aves. The identified miRNA target site of the cell adhesion gene, Tenascin C (TNC), is only found in specific chicken TNC splice variants that are differentially expressed in developing scutate scale and feather tissue indicating an important role of miRNA regulation in epidermal differentiation. Additionally, we found that β-keratins, a major structural component of avian and reptilian epidermal appendages, are targeted by multiple miRNA genes. In conclusion, our work provides quantitative expression data on miRNAs and mRNAs during feather and scale development and has produced a highly diverse, but manageable list of miRNA-mRNA duplexes for future validation experiments.

Ancient and Recent Duplications Support Functional Diversity of Daphnia Opsins

Daphnia pulex has the largest known family of opsins, genes critical for photoreception and vision in animals. This diversity may be functionally redundant, arising from recent processes, or ancient duplications may have been preserved due to distinct functions and independent contributions to fitness. We analyzed opsins in D. pulex and its distant congener Daphnia magna. We identified 48 opsins in the D. pulex genome and 32 in D. magna. We inferred the complement of opsins in the last common ancestor of all Daphnia and evaluated the history of opsin duplication and loss. We further analyzed sequence variation to assess possible functional diversification among Daphnia opsins. Much of the opsin expansion occurred before the D. pulex-D. magna split more than 145 Mya, and both Daphnia lineages preserved most ancient opsins. More recent expansion occurred in pteropsins and long-wavelength visual opsins in both species, particularly D. pulex. Recent duplications were not random: the same ancestral genes duplicated independently in each modern species. Most ancient and some recent duplications involved differentiation at residues known to influence spectral tuning of visual opsins. Arthropsins show evidence of gene conversion between tandemly arrayed paralogs in functionally important domains. Intron–exon gene structure was generally conserved within clades inferred from sequences, although pteropsins showed substantial intron size variation. Overall, our analyses support the hypotheses that diverse opsins are maintained due to diverse functional roles in photoreception and vision, that functional diversification is both ancient and recent, and that multiple evolutionary processes have influenced different types of opsins.

Using scale and feather traits for module construction provides a functional approach to chicken epidermal development

Gene co-expression network analysis has been a research method widely used in systematically exploring gene function and interaction. Using the Weighted Gene Co-expression Network Analysis (WGCNA) approach to construct a gene co-expression network using data from a customized 44K microarray transcriptome of chicken epidermal embryogenesis, we have identified two distinct modules that are highly correlated with scale or feather development traits. Signaling pathways related to feather development were enriched in the traditional KEGG pathway analysis and functional terms relating specifically to embryonic epidermal development were also enriched in the Gene Ontology analysis. Significant enrichment annotations were discovered from customized enrichment tools such as Modular Single-Set Enrichment Test (MSET) and Medical Subject Headings (MeSH). Hub genes in both trait-correlated modules showed strong specific functional enrichment toward epidermal development. Also, regulatory elements, such as transcription factors and miRNAs, were targeted in the significant enrichment result. This work highlights the advantage of this methodology for functional prediction of genes not previously associated with scale- and feather trait-related modules.