Diversification of light capture ability was accompanied by the evolution of phycobiliproteins in cryptophyte algae.

Evolutionary biologists have long sought to identify phenotypic traits whose evolution enhances an organism's performance in its environment. Diversification of traits related to resource acquisition can occur owing to spatial or temporal resource heterogeneity. We examined the ability to capture light in the Cryptophyta, a phylum of single-celled eukaryotic algae with diverse photosynthetic pigments, to better understand how acquisition of an abiotic resource may be associated with diversification. Cryptophytes originated through secondary endosymbiosis between an unknown eukaryotic host and a red algal symbiont. This merger resulted in distinctive pigment-protein complexes, the cryptophyte phycobiliproteins, which are the products of genes from both ancestors. These novel complexes may have facilitated diversification across environments where the spectrum of light available for photosynthesis varies widely. We measured light capture and pigments under controlled conditions in a phenotypically and phylogenetically diverse collection of cryptophytes. Using phylogenetic comparative methods, we found that phycobiliprotein characteristics were evolutionarily associated with diversification of light capture in cryptophytes, while non-phycobiliprotein pigments were not. Furthermore, phycobiliproteins were evolutionarily labile with repeated transitions and reversals. Thus, the endosymbiotic origin of cryptophyte phycobiliproteins provided an evolutionary spark that drove diversification of light capture, the resource that is the foundation of photosynthesis.

Light capture and pigment diversity in marine and freshwater cryptophytes.

Phenotypic traits associated with light capture and phylogenetic relationships were characterized in 34 strains of diversely pigmented marine and freshwater cryptophytes. Nuclear SSU and partial LSU rDNA sequence data from 33 of these strains plus an additional 66 strains produced a concatenated rooted maximum likelihood tree that classified the strains into 7 distinct clades. Molecular and phenotypic data together support: (i) the reclassification of Cryptomonas irregularis NIES 698 to the genus Rhodomonas, (ii) revision of phycobiliprotein (PBP) diversity within the genus Hemiselmis to include cryptophyte phycocyanin (Cr-PC) 569, (iii) the inclusion of previously unidentified strain CCMP 2293 into the genus Falcomonas, even though it contains cryptophyte phycoerythrin 545 (Cr-PE 545), and (iv) the inclusion of previously unidentified strain CCMP 3175, which contains Cr-PE 545, in a clade with PC-containing Chroomonas species. A discriminant analysis-based model of group membership correctly predicted 70.6% of the clades using three traits: PBP concentration · cell-1 , the wavelength of PBP maximal absorption, and habitat. Non-PBP pigments (alloxanthin, chl-a, chl-c2 , α-carotene) did not contribute significantly to group classification, indicating the potential plasticity of these pigments and the evolutionary conservation of the PBPs. Pigment data showed evidence of trade-offs in investments in PBPs vs. chlorophylls (a +c2 ).

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.

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.

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

BACKGROUND: Vertebrate skin appendages are constructed of keratins produced by multigene families. Alpha (α) keratins are found in all vertebrates, while beta (ß) 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. RESULTS: We 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 ß-keratins, however, demonstrate a dynamic evolution associated with avian lifestyle. The avian specific feather ß-keratins comprise a large majority of the total number of ß-keratins, but independently derived lineages of aquatic and predatory birds have smaller proportions of feather ß-keratin genes and larger proportions of keratinocyte ß-keratin genes. Additionally, birds of prey have a larger proportion of claw ß-keratins. Analysis of α- and ß-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 ß-keratin genes far exceeds that of α-keratins. CONCLUSIONS: These results support the view that the number of α- and ß-keratin genes expressed, the proportion of the ß-keratin subfamily genes expressed and the diversification of the ß-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 ß-keratins: diversification and expansion of archosaurian ß-keratins and the origin of feather ß-keratins.

The archosauria consist of two living groups, crocodilians, and birds. Here we compare the structure, expression, and phylogeny of the beta (ß)-keratins in two crocodilian genomes and two avian genomes to gain a better understanding of the evolutionary origin of the feather ß-keratins. Unlike squamates such as the green anole with 40 ß-keratins in its genome, the chicken and zebra finch genomes have over 100 ß-keratin genes in their genomes, while the American alligator has 20 ß-keratin genes, and the saltwater crocodile has 21 ß-keratin genes. The crocodilian ß-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 ß-sheets, which form the 2-3 nm filament. Overall the expression of alligator ß-keratin genes in the integument increases during development. Phylogenetic analysis demonstrates that a crocodilian ß-keratin clade forms a monophyletic group with the avian scale and feather ß-keratins, suggesting that avian scale and feather ß-keratins along with a subset of crocodilian ß-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 ß-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 ß-keratins. The expansion of the avian specific feather ß-keratin genes accompanied the diversification of birds and the evolution of feathers.

A three-genome ultraconserved element phylogeny of cryptophytes.

Cryptophytes are single celled protists found in all aquatic environments. They are composed of a heterotrophic genus, Goniomonas, and a largely autotrophic group comprising many genera. Cryptophytes evolved through secondary endosymbiosis between a host eukaryotic heterotroph and a symbiont red alga. This merger resulted in a four-genome system that includes the nuclear and mitochondrial genomes from the host and a second nuclear genome (nucleomorph) and plastid genome inherited from the symbiont. Here, we make use of different genomes (with potentially distinct evolutionary histories) to perform a phylogenomic study of the early history of cryptophytes. Using ultraconserved elements from the host nuclear genome and symbiont nucleomorph and plastid genomes, we produce a three-genome phylogeny of 91 strains of cryptophytes. Our phylogenetic analyses find that that there are three major cryptophyte clades: Clade 1 comprises Chroomonas and Hemiselmis species, Clade 2, a taxonomically rich clade, comprises at least twelve genera, and Clade 3, comprises the heterotrophic Goniomonas species. Each of these major clades include both freshwater and marine species, but subclades within these clades differ in degrees of niche conservatism. Finally, we discuss priorities for taxonomic revision to Cryptophyceae based on previous studies and in light of these phylogenomic analyses.

Linking the molecular evolution of avian beta (ß) keratins to the evolution of feathers.

Feathers of today's birds are constructed of beta (ß)-keratins, structural proteins of the epidermis that are found solely in reptiles and birds. Discoveries of "feathered dinosaurs" continue to stimulate interest in the evolutionary origin of feathers, but few studies have attempted to link the molecular evolution of their major structural proteins (ß-keratins) to the appearance of feathers in the fossil record. Using molecular dating methods, we show that before the appearance of Anchiornis (∼155 Million years ago (Ma)) the basal ß-keratins of birds began diverging from their archosaurian ancestor ∼216 Ma. However, the subfamily of feather ß-keratins, as found in living birds, did not begin diverging until ∼143 Ma. Thus, the pennaceous feathers on Anchiornis, while being constructed of avian ß-keratins, most likely did not contain the feather ß-keratins found in the feathers of modern birds. Our results demonstrate that the evolutionary origin of feathers does not coincide with the molecular evolution of the feather ß-keratins found in modern birds. More likely, during the Late Jurassic, the epidermal structures that appeared on organisms in the lineage leading to birds, including early forms of feathers, were constructed of avian ß-keratins other than those found in the feathers of modern birds. Recent biophysical studies of the ß-keratins in feathers support the view that the appearance of the subfamily of feather ß-keratins altered the biophysical nature of the feather establishing its role in powered flight.

Evolution of an Epidermal Differentiation Complex (EDC) Gene Family in Birds.

The transition of amniotes to a fully terrestrial lifestyle involved the adaptation of major molecular innovations to the epidermis, often in the form of epidermal appendages such as hair, scales and feathers. Feathers are diverse epidermal structures of birds, and their evolution has played a key role in the expansion of avian species to a wide range of lifestyles and habitats. As with other epidermal appendages, feather development is a complex process which involves many different genetic and protein elements. In mammals, many of the genetic elements involved in epidermal development are located at a specific genetic locus known as the epidermal differentiation complex (EDC). Studies have identified a homologous EDC locus in birds, which contains several genes expressed throughout epidermal and feather development. A family of avian EDC genes rich in aromatic amino acids that also contain MTF amino acid motifs (EDAAs/EDMTFs), that includes the previously reported histidine-rich or fast-protein (HRP/fp), an important marker in feather development, has expanded significantly in birds. Here, we characterize the EDAA gene family in birds and investigate the evolutionary history and possible functions of EDAA genes using phylogenetic and sequence analyses. We provide evidence that the EDAA gene family originated in an early archosaur ancestor, and has since expanded in birds, crocodiles and turtles, respectively. Furthermore, this study shows that the respective amino acid compositions of avian EDAAs are characteristic of structural functions associated with EDC genes and feather development. Finally, these results support the hypothesis that the genes of the EDC have evolved through tandem duplication and diversification, which has contributed to the evolution of the intricate avian epidermis and epidermal appendages.

Genomic organization and molecular phylogenies of the beta (beta) keratin multigene family in the chicken (Gallus gallus) and zebra finch (Taeniopygia guttata): implications for feather evolution.

BACKGROUND: The epidermal appendages of reptiles and birds are constructed of beta (beta) keratins. The molecular phylogeny of these keratins is important to understanding the evolutionary origin of these appendages, especially feathers. Knowing that the crocodilian beta-keratin genes are closely related to those of birds, the published genomes of the chicken and zebra finch provide an opportunity not only to compare the genomic organization of their beta-keratins, but to study their molecular evolution in archosaurians. RESULTS: The subfamilies (claw, feather, feather-like, and scale) of beta-keratin genes are clustered in the same 5' to 3' order on microchromosome 25 in chicken and zebra finch, although the number of claw and feather genes differs between the species. Molecular phylogenies show that the monophyletic scale genes are the basal group within birds and that the monophyletic avian claw genes form the basal group to all feather and feather-like genes. Both species have a number of feather clades on microchromosome 27 that form monophyletic groups. An additional monophyletic cluster of feather genes exist on macrochromosome 2 for each species. Expression sequence tag analysis for the chicken demonstrates that all feather beta-keratin clades are expressed. CONCLUSIONS: Similarity in the overall genomic organization of beta-keratins in Galliformes and Passeriformes suggests similar organization in all Neognathae birds, and perhaps in the ancestral lineages leading to modern birds, such as the paravian Anchiornis huxleyi. Phylogenetic analyses demonstrate that evolution of archosaurian epidermal appendages in the lineage leading to birds was accompanied by duplication and divergence of an ancestral beta-keratin gene cluster. As morphological diversification of epidermal appendages occurred and the beta-keratin multigene family expanded, novel beta-keratin genes were selected for novel functions within appendages such as feathers.

Complex Gene Loss and Duplication Events Have Facilitated the Evolution of Multiple Loricrin Genes in Diverse Bird Species.

The evolution of a mechanically resilient epidermis was a key adaptation in the transition of amniotes to a fully terrestrial lifestyle. Skin appendages usually form via a specialized type of programmed cell death known as cornification which is characterized by the formation of an insoluble cornified envelope (CE). Many of the substrates of cornification are encoded by linked genes located at a conserved genetic locus known as the epidermal differentiation complex (EDC). Loricrin is the main protein component of the mammalian CE and is encoded for by a gene located within the EDC. Recently, genes resembling mammalian loricrin, along with several other proteins most likely involved in CE formation, have been identified within the EDC of birds and several reptiles. To better understand the evolution and function of loricrin in birds, we screened the genomes of 50 avian species and 3 crocodilians to characterize their EDC regions. We found that loricrin is present within the EDC of all species investigated, and that three loricrin genes were present in birds. Phylogenetic and molecular evolution analyses found evidence that gene deletions and duplications as well as concerted evolution has shaped the evolution of avian loricrins. Our results suggest a complex evolutionary history of avian loricrins which has accompanied the evolution of bird species with diverse morphologies and lifestyles.

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 ß-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.

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

BACKGROUND: Penguins 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]. RESULTS: Phylogenetic 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. CONCLUSIONS: Our 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.

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.