Email updates

Keep up to date with the latest news and content from BMC Evolutionary Biology and BioMed Central.

This article is part of the series Zebra finch: the companion papers for the publication of the zebra finch genome sequence.

Open Access Highly Accessed Research article

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

Matthew J Greenwold and Roger H Sawyer*

Author affiliations

Department of Biological Sciences, University of South Carolina, Columbia, SC 29205, USA

For all author emails, please log on.

Citation and License

BMC Evolutionary Biology 2010, 10:148  doi:10.1186/1471-2148-10-148


The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2148/10/148


Received:26 October 2009
Accepted:18 May 2010
Published:18 May 2010

© 2010 Greenwold and Sawyer; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The epidermal appendages of reptiles and birds are constructed of beta (β) keratins. The molecular phylogeny of these keratins is important to understanding the evolutionary origin of these appendages, especially feathers. Knowing that the crocodilian β-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 β-keratins, but to study their molecular evolution in archosaurians.

Results

The subfamilies (claw, feather, feather-like, and scale) of β-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 β-keratin clades are expressed.

Conclusions

Similarity in the overall genomic organization of β-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 β-keratin gene cluster. As morphological diversification of epidermal appendages occurred and the β-keratin multigene family expanded, novel β-keratin genes were selected for novel functions within appendages such as feathers.

Background

The skin of terrestrial vertebrates evolved to prevent water loss and to provide a barrier between the organism and its environment [1]. In reptiles and birds, skin appendages such as claws, scales, beaks and feathers develop, and provide novel functions. These diverse epidermal structures are composed of beta (β) keratins, whose genes have been isolated from all major groups of reptiles including squamates, crocodilians, and chelonians [2-6]. To date β-keratin sequences are known for three crocodilian species, which are highly similar to avian β-keratins [4,6]. Initial analysis of the chicken genome demonstrated that there are ~150 avian β-keratins with a tandem array of 30 being located on microchromosome 27 [7]. Recently, the feather subfamily of β-keratins has been located on multiple chromosomes in the chicken genome, yet the claw, feather-like, and scale β-keratins are restricted to microchromosome 25 [8]. Glenn et al. [9] isolated multiple copies of feather β-keratins from eight orders of the class aves, but they were unable to amplify sequences in the Passeriforme order, which makes up over fifty percent of all living birds.

Phenotypically the chicken has a larger body size and longer life span than the zebra finch. Furthermore altricial birds like the zebra finch, hatch naked, blind, and nearly helpless and are dependent on their parents for survival. In contrast precocial birds, such as the chicken are generally well-developed, fully feathered and need little parental care [10].

Expression of Chicken β-keratins

In the chicken four subfamilies (claw, feather, feather-like and scale) of β-keratin genes have been named in accordance with tissue specific expression and sequence heterogeneity [11-13]. However, during development of the epidermis and its appendages more than one subfamily may be expressed in a specific tissue (Table 1). For example, the feather-like gene is not only expressed in feathers, but also in embryonic scales, and claw genes are expressed in embryonic feathers [12,13]. Furthermore the scaleless (sc/sc) mutant chicken, which does not undergo scale and feather development, expresses β-keratins from all four subfamilies in its embryonic epidermis [14]. This embryonic epidermis is generated by the initial stem cell population of the embryonic ectoderm [15].

Table 1. Expression of β-keratin Sequences in Chicken Tissue:

As appendage morphogenesis and epidermal differentiation progress in normal birds, new epidermal stem cell lineages (germinative basal cell populations) differentiate and the expression of the β-keratin subfamilies becomes more restricted to specific appendages [15,16].

Interestingly, the four subfamilies of β-keratin genes form a cluster on microchromosome 25 (GGA25), and form monophyletic groups [8]. In the case of the chicken, members of the feather subfamily are located on 6 different chromosomes in addition to GGA25 [8]. Although we have a genomic map of the β-keratins in the chicken, we are far from understanding how the individual genes in these specific β-keratin subfamilies are utilized to build all the epidermal appendages such as the beak, spur, egg tooth, lingual nail or the numerous types of feathers [17-19].

In addition to the four subfamilies of β-keratins, two novel β-keratins have also been identified in separate experimental approaches; one from serially cultured chicken keratinocytes [20] and another from jun-transformed quail fibroblasts [21].

Structure of the β-keratin Protein

Feather β-keratins are fibrous proteins that have four repeating units of two β-sheets that form a helical structure. This structure is surrounded by a matrix that makes up the filament-matrix texture that is seen in the structure of feathers. Fraser and Parry [22] found through X-ray diffraction studies that a 32 amino acid segment, of the total 97 amino acids that comprise the feather β-keratin coding region, makes up the 2-3 nm filament and that the remaining residues comprise the matrix (Figure 1). This is in contrast to the alpha (α)-keratins (intermediate filaments), which have a coiled coil α-helix structure and have associated amorphous proteins [23]. Based on sequence similarity, this 32 amino acid residue has also been identified in the β-keratins of scales and claws from reptiles and birds in addition to the chicken, suggesting that it is an important region and should be under intense purifying selection.

thumbnailFigure 1. Amino Acid Alignment of β-keratins showing the 32aa Filament Segment of Feathers: Alignment of the two most diverged feather β-keratins from each chromosome of G. gallus and T. guttata. Annotation of the sequences includes the three letter abbreviation of the species, the chromosome number, type of β-keratin, and the number indicating position in the 5' to 3' direction on each chromosome. The 31 amino acids in the box comprise those described by Fraser and Parry [22] as the 32-residue segment constituting the filament framework of feather β-keratins. Both the G. gallus and T. guttata β-keratins possess a deletion in position 3 of the 32-residue segment.

Gene duplication

Evolution of multigene families is believed to occur through gene duplication. Duplication is relatively common and occurs by several methods, including unequal crossing over, gene conversion, and transposition by genomic elements. [24,25]. Unequal crossing over and gene conversion are often linked to tandem duplication, which results in arrays of similar regions of DNA. Transpositions are the result of transposable elements and can result in tandem duplication or the duplication of genes to other loci in a genome or species [25-28]. Lynch et al. [24] points out three possible outcomes of gene duplication: non-functionalization, in which one gene is silenced; neo-functionalization, where one of the copies acquires a new function; and sub-functionalization, in which case both copies become partially compromised [29-31]. It has been proposed that the feather β-keratin subfamily evolved from the scale β-keratin subfamily through a deletion event followed by gene duplication [11,28], but other authors suggest that the feather genes are basal to the avian scale genes [4].

Genomic Organization of β-keratins in Gallus gallus domesticus

In the White Leghorn chicken (Gallus gallus domesticus), a genomic region spanning ~100 kb containing the claw, feather, and feather-like subfamilies of the β-keratin multigene family was described by Presland et al [12] (Figure 2). This region was reconstructed from a chicken cosmid library and a feather probe was used to identify β-keratins. The library revealed 4 claw β-keratins, 18 feather β-keratins, and 3 feather-like β-keratins in this 5' to 3' order, respectively. The scale β-keratins were not mapped in that study.

thumbnailFigure 2. The Genomic Organization of the β-keratin Subfamilies of Gallus gallus domesticus: Reproduction of the ~100 kb region containing the β-keratin gene cluster of Gallus gallus domesticus from Presland et al [12]. Arrows indicate the transcriptional orientation of the coding regions, where known. The boxes represent coding regions of unknown transcriptional orientation. The three β-keratin subfamilies (feather-like, feather, and claw) are labeled.

The genomes of Gallus gallus and Taeniopygia guttata have been sequenced to a 6.6 X coverage and a 5.5 X coverage respectfully [7,32]. The G. gallus genome was the first avian genome to be sequenced and recently the 2.1 build has been released. With information about β-keratin genes in the chicken [8,12] and the recent publication of the zebra finch genome, an in depth comparison of avian β-keratin genes and their molecular evolution is now possible in these species which differ phenotypically and are separated by ~87-111mya [33-36].

Results

We have identified a total of 111 complete β-keratin gene sequences (coding regions) in the G. gallus genome [7,8], which are distributed on three macrochromosomes (GGA1, 2 and 5), one intermediate chromosome (GGA6), two microchromosomes (GGA25, and 27), and "chromosome unknown" (GGA_Un) (see Table 2) [37,38]. In the genome of T. guttata a total of 108 β-keratin genes are located on one macrochromosome (TGU2), two microchromosomes (TGU25 and 27), and "chromosome unknown" (TGU_Un). We also identified the number of probable genes and pseudogenes in each subfamily of β-keratin found in both genomes. These probable genes contain reasonable stop and start codons, no in-frame stop codons, no frame shift mutations, or regions of unknown genomic sequence, and they meet stringent E-values (Table 2, 3).

Table 2. Comparison of the Type and Number of β-keratin Genes Found on Each Chromosome.

Table 3. Query Sequences used to Perform BLAST Searches of the G. gallus and T. guttata genomes.

Molecular Phylogeny

The molecular phylogeny of 219 probable avian β-keratin sequences, using crocodilian β-keratin genes as the outgroup demonstrates that the avian scale β-keratins are the basal group in birds (Figure 3). The β-keratin from cultured chicken keratinocytes, found only on GGA_25, is basal to the avian scale; however its expression in normal tissues in vivo has not been shown. The avian claw genes are basal to the feather genes, and the claw and scale genes are monophyletic, and form sister groups between chicken and zebra finch.

thumbnailFigure 3. Phylogenetic Tree Reconstruction of all β-keratin Sequences Located on the Genomes of both G. gallus and T. guttata: Three nile crocodile (Crocodylus niloticus) β-keratin genes are constrained as the out group [4] with all 219 β-keratin genes found in the two avian genomes. The BS (bootstrap values) from the Neighbor-Joining method are listed for each major branch when they are above 70 percent. The bootstrap values from the Maximum Likelihood method, are listed in parenthesis for each major branch when they are above 70 percent (see Methods) [56,60]. All individual taxa names have been removed and instead only the names of major groups are displayed. The subfamilies are colored with the following scheme: GGA25_Keratinocyte = red, scale β-keratin genes = blue, claw β-keratin genes = yellow, feather β-keratin genes = green and feather-like β-keratin genes = magenta. Please refer to Additional File 5, Figure S2 to view all bootstrap values and taxa labels for the Neighbor-Joining phylogeny and Additional File 6, Figure S3 to view all bootstrap values and taxa labels for the Maximum Likelihood phylogeny.

The feather genes on macrochromosome 2 form a monophyletic group and form sister groups between the two species. Twelve feather genes from TGU_Un sorted out with the monophyletic group of TGU2 and 5 feather genes from GGA_Un sorted out with the monophyletic group of GGA2. The β-keratin genes on GGA6, identified as being similar to those isolated from jun-transformed fibroblasts form a paraphyletic group with the monophyletic group of feather genes on macrochromosome 2, the monophyletic group of feather genes on GGA25 (which includes the feather gene on GGA1 and one feather gene from GGA_Un), the monophyletic group of feather genes on TGU25, and the feather-like genes on TGU25 and GGA25. Except for the feather-like genes, all sister groups are monophyletic. These sister groups and the feather-like genes are basal to all of the feather genes on microchromosome 27 including those from "chromosome unknown". TGU27_clade 1 is basal to 2 other clades of TGU27 and 3 clades of GGA27 feather β-keratins (Figure 3).

Characterization and Identification of β-keratins on Microchromosome 25

Microchromosome 25 of both species displays all four subfamilies. The gene order seen on microchromosome 25, in a 5' to 3' direction, is claw, feather, feather-like, and scale (Figure 4). The overall size of the β-keratin cluster on microchromosome 25 varied between the two species with a base pair range of ~178 kb and ~120 kb for G. gallus and T. guttata respectfully. The chromosomal position of the β-keratin cluster differs for each species with the region on GGA25 being ~307 kb upstream from the β-keratin genomic cluster on TGU25.

thumbnailFigure 4. Comparison of the β-keratin Genomic Regions in the G. gallus and T. guttata Genomes: This figure was constructed using the Artemis program [53] and shows both the positive and negative strands of all genomic loci containing more than three β-keratin genes. Each chromosomal region is annotated above by the three letter species name and the chromosome number. The β-keratin coding regions are colored with the following scheme: scale β-keratin genes = blue, claw β-keratin genes = yellow, feather β-keratin genes = green and feather-like β-keratin genes = magenta. The arrow and strand indicate the directional orientation of each β-keratin gene. Base ranges for each chromosome are listed between the two strands. All labels are based on NCBI BLAST results with cDNA from previous expression studies [8,52,53].

β-keratin from cultured keratinocytes

One β-keratin from cultured keratinocytes was identified in the genome of G. gallus. This one coding region was found ~10 kb downstream of the scale β-keratins on the negative strand of GGA25 (Figure 4). This β-keratin sequence has very low overall similarity to other avian β-keratins, but of particular interest is an approximate 49 amino acid region beginning at the twentieth amino acid of the keratinocyte β-keratin coding region that shows high similarity with other β-keratin family members. This region includes the 32 amino acids described by Fraser and Parry [22] that make up the filament framework of β-keratins. Several strong hits resulted from a BLAST search of the zebra finch genome, but these hits either lacked a reasonable stop codon or contained in frame stop codons.

Scale β-Keratins

The genomic location, orientation and number of the scale β-keratins on chromosome 25 of G. gallus and T. guttata share striking similarities. Both species contain four scale β-keratin coding regions, which are downstream of the feather-like β-keratins on chromosome 25 (Figure 4). Both have similar orientations of the scale β-keratins, with a negative and positive strand alternating pattern. The distance between the feather-like β-keratins and the scale β-keratins is 34,176 and 19,033 bps for G. gallus and T. guttata, respectively. The total distance covered by scale β-keratins on their respective chromosome 25 is shorter on G. gallus having an 11,260 bp range and T. guttata having a 13,231 bp range. A ~6.5 kb center to center distance is seen between both GGA25_scale1 and 3; and GGA25_scale2 and 4, which are the coding regions found on the same strands. T. guttata lacks the conserved distance between sequences on the same strand, with TGU25_scale1 and 3 having ~7 kb and TGU25_scale2 and 4 having ~8 kb center to center distance.

Claw β-keratins

While Presland et al. [12] found four claw genes in G. gallus domesticus; we have identified 8 complete genes in G. gallus. Both lineages display positive and negative strand orientation. Regular spacing of less than 1 kb is observed for the claw β-keratins in the Galliformes. However, in G. gallus we removed three claw genes from the final dataset because they contained regions of unknown sequence or frame shift mutations and might be pseudogenes. Two of these potential pseudogenes are located between GGA25_Claw1 and 2 with a negative and positive strand orientation, and a third is located on the positive strand between GGA25_Claw6 and 7. The presence of these pseudogenes supports the view that the claw β-keratin genes have undergone duplication events [39].

Significant differences are seen between the claw β-keratin genes of G. gallus and T. guttata. GGA25 contains eight claw genes while TGU25 only has one (Figure 4). The sequence similarity of TGU25_Claw1 to the claw genes found in the chicken is rather low with only 49-55% identity observed from an amino acid alignment (data not shown).

β-keratin in jun-transformed cells (BKJ)

The only β-keratins found on GGA6 were found to be highly similar to the β-keratin isolated from quail (Coturnix japonica) fibroblast cells that were jun-transformed [21]. No significant blastn results were obtained from a search of the T. guttata genome, although the highest similarity was to TGU25_FL2. The coding regions found in the G. gallus genome are tandemly arrayed on chromosome 6 with unequal spacing over a ~11.7 kb range and all having a length of 109 amino acids, which is consistent with the cDNA of C. japonica. These coding regions have the highest similarity to feather-like β-keratins at 63.3% in an alignment with all subfamilies of the multigene β-keratin family (data not shown). A nucleotide alignment (data not shown) shows that the three BKJ coding regions only differ by one or two nucleotide changes and therefore have a greater than 99% identity between the three sequences.

Feather β-keratins on Macrochromosome 2

Chicken macrochromosome 2 has five complete coding regions spanning ~2.6 kb and in contrast TGU2 has twenty-two feather β-keratins located in a region spanning ~101 kb (Figure 4). The molecular phylogeny demonstrates that additional feather genes similar to those on macrochromosome 2 may be found on chromosome unknown of both species (Figure 3). Five feather genes on GGA_Un sort with the feather genes found on GGA2 and have not yet been placed in the current build of the chicken. Additionally, 12 feather genes from TGU_Un sort with those on TGU2 making the total number of genes on TGU2 34.

Feather-like β-keratins

There are 4 feather-like β-keratin genes on microchromosome 25 of G. gallus (Figure 4), while G. gallus domesticus contains three feather-like coding regions [12], as seen in T. guttata. Feather-like pseudogenes were found on chromosomes 7 and 10 in G. gallus. No additional feather-like genes were found outside of TGU25 in the zebra finch.

Feather β-keratins on Microchromosome 25

We found 15 feather β-keratin genes on microchromosome 25 of G. gallus, which is in contrast to the 18 identified by Presland et al [12] for G. gallus domesticus. All 15 coding regions are found on the positive strand and have equal spacing of ~3 kb in agreement with Presland et al [12] (see Figure 2, 4). A large gap is seen in the feather β-keratin region on GGA25, which is located between GGA25_FK2 and 3. A pseudogene is present here, but was excluded from our data because it contains a frame shift mutation. This type of mutation is evidence of a duplication event and in an array of genes indicates that some of these genes are the product of tandem duplication [39].

Only two feather coding regions are located on microchromosome 25 of T. guttata and they have a negative and positive strand orientation with approximately 3.5 kb separating the two genes (Figure 4).

Feather β-keratins on GGA1 and 5

Both avian genomes contain coding regions at other loci with high similarity to the feather β-keratins found on chromosome 25. All loci outside of chromosome 25 have tandem arrays with the exception of GGA1 and GGA5. Both of these loci contain one feather β-keratin coding region.

The molecular phylogeny (Figure 3) shows that the sequence on macrochromosome 1 sorts with the feather β-keratins of GGA25. Similar to the relationship of GGA1 and GGA25 is that of GGA5_FK1 with its paralogues on GGA27_clade2 (Figure 3). In fact, the single feather β-keratin sequence on GGA5 has a very high similarity to GGA27_FK35 with only one synonymous substitution (data not shown).

Feather β-keratins on Microchromosome 27

Both species have the largest number of β-keratins with the greatest similarity to feather keratins on microchromosome 27. A tandem array of 61 coding sequences is located on GGA27, in which 11 are found on the negative strand toward the 3' end of the cluster (Figure 4). The remaining 50 are found on the positive strand. TGU27 has two tandem arrays totaling 41 feather β-keratins. The array farthest downstream in the 3' direction has a similar orientation to that of GGA27 (Figure 4), but has a reduced number of coding regions. Unique to TGU27 is a second array found ~2,309 kb upstream, containing eleven coding regions.

Analysis of Selection acting on the Feather β-keratins

Fraser and Parry, through X-ray diffraction studies, described the structure of the feather molecule. The central region, 32 amino acids of the total 97 amino acid protein, forms the filament with the remaining amino acids comprising the matrix (see Figure 1) [22]. We hypothesize that the filament region should be under purifying selection for the proper formation of feathers. In order to determine whether the large numbers of feather β-keratin genes found throughout the avian genome are actively transcribed for feather formation, the filament region was analyzed at the amino acid level by determining the individual dN/dS ratio.

The PAML program was used to analyze each chromosomal locus separately that consists of more than two feather genes (see Methods and Additional File 1, Table S1). The only significant results for high dN/dS ratios at individual amino acid sites in the filament region, using the PAML Program, were found at sites 37 and 38 of the feather genes on GGA2. However, the Likelihood Ratio Tests (LRTs) to determine the fit of the models, using four separate tests (M0 vs. M2a; M0 vs. M3; M1a vs. M2a; and M7 vs. M8), to the data did not significantly differ from the chi-squared value (see Methods and Additional File 2, Table S2), indicating a false positive result. This result may be due to the low number of feather genes analyzed on GGA2 (5 feather β-keratin genes).

Additional file 1. Annotation of β-keratins resulting from BLAST Searches of the Gallus gallus and Taeniopygia guttata Genomes. List of all sequences used in this study that resulted from a BLAST search of the G. gallus and T. guttata genomes [7,8]. Annotation of each sequence used in this study, its position on its respective chromosome, and strand orientation is indicated by a lowercase c (complement) if it was found on the complementary strand. Additionally, the Ensembl Gene identification numbers were listed for every β-keratin sequence that was annotated by Ensembl, and was not eliminated from this study by criteria described in the Methods section.

Format: XLS Size: 34KB Download file

This file can be viewed with: Microsoft Excel ViewerOpen Data

Additional file 2. PAML Analysis of all Feather β-keratin Loci that Resulted in Positively Selected Sites: The six models are listed in the first column with a brief description, and the values obtained from each analysis are listed in their respective rows. The dN/dS ratios are the average of the sum of all branches. All positively selected sites above 95% are listed, with those reaching 99% shown in bold. The Naïve Empirical Bayes (NEB) and Bayes Empirical Bayes (BEB) are shown when appropriate. The M3 model only uses the NEB analysis [61]. Additionally, the tables for the likelihood ratio test (LRT) results for each locus are included (see Methods).

Format: PDF Size: 111KB Download file

This file can be viewed with: Adobe Acrobat ReaderOpen Data

Expression of β-keratins

The Expressed sequence tag (EST) databases for the chicken and zebra finch were downloaded via NCBI and contained 599,999 and 91,801 sequences, respectably. Seventy chicken EST sequences were found to be highly similar to β-keratin sequences found in the genome of G. gallus (see Methods). These results include a total of forty-three separate chicken β-keratin genomic sequences, which consists of feather β-keratin sequences found on GGA1, 2, 25, and all clades of GGA27, and claw, scale, and keratinocyte β-keratin sequences of GGA25. Thus, genes of feather β-keratins are expressed from all major clades (Figure 3). However, the tissue sources of the chicken EST data are extremely diverse; ovaries, testes, eye, fat, spleen, breast muscle, and various other glands. The only significant result for the β-keratin sequences of T. guttata was to the TGU2_FK22 sequence, in which the resulting EST only covered the first 291 nucleotides of the 336 base pair sequence with 98% similarity. The majority of the T. guttata ESTs are from brain tissues (Additional File 3, Table S3).

Additional file 3. Expressed Sequence Tag BLAST results. List of all β-keratin sequences in the genomes of G. gallus and T. guttata that had at least an E-value score of 1e-160 and 1e-150, respectably, for an expressed sequence tag (EST). EST database was downloaded via NCBI for each species. The GI number for each EST, region of the EST matching the coding region of the β-keratin, the 5' region outside of the coding region for each matching EST, the 3' region outside of the coding region for each matching EST, the tissue source of the EST, the developmental stage of the collected EST source and the sex are listed.

Format: XLS Size: 43KB Download file

This file can be viewed with: Microsoft Excel ViewerOpen Data

Gene Conversion

In order to investigate occurrences of gene conversion and/or unequal crossing over, the program GENECONV was used to analyze all 219 β-keratin genes in this data set (see Methods for details). The main questions addressed here were: are the β-keratin subfamilies (scale, claw, feather, feather-like) formed through unequal crossing over and subsequent divergence, and did gene conversion play a significant role in the homogenization of the these subfamilies in the avian genome [31]? Seven fragments in our data set were found to be statistically significant and support the view that unequal crossing over has occurred on GGA25. Five of these fragments, which are 33 nucleotides long, were found between GGA25_Claw6 and GGA25_FK1, 7, 8, 10 and 12. Two were between GGA25_FL1 andGGA25_FK6 and 9 and were 72 nucleotides long. These seven fragments demonstrate that the feather and feather-like β-keratins on GGA25 were formed through unequal crossing over. In contrast, a fragment of 146 nucleotides was found between two TGU_Un feather genes, which demonstrates homogenization of the feather β-keratins in the zebra finch.

Discussion

Genome Assembly

The actual number of β-keratin genes at any locus may not be correct because diploid genomes may contain mis-assembled contigs that result from allelic variation [40]. This variation is often localized to "chromosome unknown", but may occur when a second allelic copy is inserted next to a sister copy on a chromosome, which results in an apparent duplication of the gene or sequence [40]. In fact, the loci containing β-keratins are made up of several separate contigs. For example the genomic contig that contains the feather β-keratin clades of GGA27 consists of 162 separate contigs (see Table 4). Therefore conclusions in this study rely more heavily on phylogenetic results (Figure 3) than on the actual number of β-keratin genes. The EST data in combination with molecular phylogenies demonstrate that the β-keratin multigene family does consist of multiple clades of feather β-keratins found on many loci throughout the genomes of the chicken and zebra finch.

Table 4. Genomic Contigs that contain Clusters of β-keratins and their Composition.

In the case of G. gallus microchromosome 25, a similar number of β-keratin genes from three subfamilies (claw, feather, and feather-like) with the same genomic organization was observed in G. gallus domesticus by Presland et al. [12]. They [12] reconstructed this region from a chicken cosmid library using restriction enzymes to map the ~100 kb genomic fragments (see Figure 2). Comparison of this region with the region we have identified on GGA25 (from the start of GGA25_claw6 to the end of GGA25_FL3 spanning ~99 kb distance, see Figure 4) indicates that this region of the chicken genome build 2.1 is of high quality.

Molecular phylogeny

A comparison of the trees generated by the Neighbor-Joining method (see Additional file 5) and the Maximum Likelihood method (see Additional file 6) shows that generally the Maximum Likelihood has more conservative bootstrap values. Although the Maximum Likelihood phylogeny shows that the claw and scale β-keratin genes are monophyletic it does not support the paraphyletic grouping by species. Furthermore, the bootstrap support for TGU27 clade 1 being monophyletic and the basal group for the other feather β-keratin genes on microchromosome 27 is very weak. GGA27 clade 2 and 3 also lack strong bootstrap support for their monophyly. Overall the trees are very similar, but the low bootstrap support from Maximum Likelihood analysis may reflect the high similarity (indicating fewer segregating sites) within the subfamilies of β-keratins and the robustness of this method.

Expression of β-keratins and Phenotypic Variation between the Chicken and Zebra Finch

Studies of protein expression using two-dimensional gel electrophoresis have demonstrated that overlap exists between the proteins expressed by the claw, egg tooth, beak and scale from 19-20 day chick embryos (Additional File 4, Figure S1). These tissues express different levels of β-keratins. For example, the scale and beak express at least 7 different β-keratins and several phosphorylated β-keratins, while the egg tooth and claw have significantly reduced levels of two of these β-keratins and two phosphorylated β-keratins [13]. The beak, scale and claw tissues display very similar expression patterns for their β-keratins on two-dimensional gels (Additional File 4, Figure S1). These data suggest that not only do the scale epidermal cells express scale β-keratin genes but they also express β-keratin genes from other subfamilies such as claw. Furthermore, some β-keratin genes are expressed at the same level in multiple epidermal appendages (claw, egg tooth, beak, and scale), while others are expressed at significantly reduced levels [13]. Interestingly, we have found that the gene isolated from embryonic chicken beak (Wu et al, unpublished results [41]) has 97% identity with GGA25_Claw6 (results not shown) further demonstrating that the beak epidermis expresses the claw subfamily of β-keratins.

Additional file 4. Two-dimensional Gels of β-keratin Expression in Chick Epidermal Appendages. Reprint of Figure 2 in Shames et al [19]. Two-dimensional gels of protein extracted from 19-20 day embryonic chick (A) scutate scale epidermis, (B) cornified beak, (C) egg tooth, (D) periderm, and (E) claw. The acidic (Ac) and basic (Ba) ends of the gel and the molecular weight markers are indicated for the second dimension. The protein spots labeled 1 and 2, 3 are scale β-keratins identified by hybrid-selection using a scale specific oligonucleotide probe [65].

Format: JPEG Size: 12KB Download fileOpen Data

No information is available on the expression of β-keratins in the claws or beaks of passeriformes. It may be that the single claw gene (TGU25_Claw1) in the zebra finch genome is expressed in both structures. However, a recent study of claw development in the zebra finch suggests that the zebra finch claw is a modified scale [42]. Perhaps the zebra finch claw epidermis uses β-keratins from the scale subfamily and/or other subfamilies for its cornification. Furthermore since the beaks of chickens express both scale and claw genes (Table 1), perhaps the beaks of passeriformes express mainly scale β-keratins [19].

Passeriformes are altricial birds [10], and are mostly naked when they hatch. In the zebra finch a few embryonic feather filaments are scattered over the body of the hatchling [43]. Assuming that the number of genes on microchromosome 25 is correct for the chicken and zebra finch, the differences in the number of feather β-keratin genes on microchromosome 25 for these two species may be related to the altricial nature of passeriformes. Studies do demonstrate that the feather β-keratin genes on GGA25 are expressed in developing feathers [11,12]. If the feather genes on microchromosome 25 are used mainly to produce the structural proteins for the embryonic down feathers, then the demand for multiple copies of feather genes [44-46] may be relaxed in altricial birds.

Recombination

The results of the gene conversion test (See Results) suggest that the feather genes on microchromosome 25 arose through unequal crossing over from either the claw or feather-like genes or both. An alternative hypothesis is that the claw genes, through unequal crossing over, gave rise to the feather genes which in turn gave rise to the feather-like genes found on GGA25. These results indicate that many episodes of recombination have occurred on GGA25 and that gene conversion may be very rare among β-keratin sequences. In fact, the only results that indicate gene conversion are between two feather β-keratin genes found on TGU_Un.

The high number of genes found for the clades on microchromosome 27 in both species may be due to the high rate of recombination seen on avian microchromosomes. The negative correlation between recombination and chromosome size, results in a higher gene density on microchromosomes [7,37]. The high number of feather β-keratins on TGU2 may relate to the high rate of recombination that occurs toward the ends of the macrochromosomes in the zebra finch genome [47].

Implications for Evolution of Avian Genomes

Recently, Hu et al. [48] described a small, crow-sized theropod, Anchiornis huxleyi, dated to ~155mya, with long pennaceous feathers on its forelimbs and hindlimbs. They point out that large pennaceous feathers located on the hind limb is a feature known for the basal members of the three major paravian groups [see also [49]]. Hu et al. [48] further proposes that feathering of the foot was a critical step in the evolution of birds [see [50]]. Since numerous studies demonstrate that adult feathers are made of feather β-keratins [9,11,12], it is reasonable to assume that the feather and/or feather-like β-keratins were present in the pennaceous feathers on Anchiornis and other Paraves. Considered in light of our phylogenetic analyses of the β-keratin subfamilies, the presence of pennaceous feathers on Anchiornis supports the view that a β-keratin multigene cluster similar to that seen on microchromosome 25 in today's birds may have existed in archosaurians as early as the Middle Jurassic [48] (Figure 5).

thumbnailFigure 5. Proposed Evolution of the β-keratin Genomic Region. This figure illustrates a proposed scenario for the evolution of the β-keratin subfamilies in extant birds from their archosaurian ancestor. The vertical arrows indicate evolutionary time and the horizontal arrows in the boxes indicate possible unequal crossing over events. The subfamilies are colored with the following scheme: scale β-keratin genes = blue, claw β-keratin genes = yellow, feather β-keratin genes = green and feather-like β-keratin genes = magenta.

Conclusion

Our results suggest the following scenario for the evolution of the β-keratin gene family (Figure 5). The genome of early archosaurians contained a cluster of β-keratin genes, closely related to the scale β-keratin genes seen in today's crocodilians and birds [4,6,8,51]. Duplication and diversification lead to the subfamily known as claw, which provided additional building blocks for the evolution of archosaurian appendages; i. e., claws, beaks, spurs, etc [17-19]. In fact, members of both the scale and claw subfamilies of β-keratin are present in developing claws, beaks, scales, and even feathers of birds (Table 1 and Additional File 4, Figure S1) [11,12,19]. As the development and morphogenesis of the epidermal appendages diversified further, recombination in the β-keratin gene cluster provided the raw material for the evolution of new β-keratin genes, such as feather and feather-like, which would eventually provide the structural proteins for appendages with novel functions, such as the feather. In fact, our molecular phylogenies demonstrate that the avian claw genes evolved from the scale genes, and form a basal group to the feather-like and feather genes (Figure 3 and 5).

Methods

Identification and localization of the β-keratin multigene family

The β-keratin nucleotide sequences, amino acid sequences, and unique features associated with the β-keratin genes were obtained from NCBI and published sources [8]. All GI numbers or references (without GI numbers) for β-keratins are listed in Table 3. The NCBI Basic Local Alignment Search Tool (BLAST) was used to search the 2.1 build of G. gallus and the 1.1 build of T. guttata [52]. The genomic data was downloaded via the NCBI ftp site. We used Artemis 8.1 [53] to view the β-keratin genes graphically.

To identify the genomic region in G. gallus that contained the claw, feather, and feather-like β-keratins described by Presland et al [12] we used very strict E-values and BLAST scores. This strict use of identity was also applied to all the unique features associated with the β-keratins, including the scale β-keratins and the β-keratin from cultured keratinocytes. Table 3 identifies the highest and lowest E-value and BLAST score used for each feature of the genomic region identified by Presland et al [12] for G. gallus [8] and for the β-keratin in jun-transformed cells. The sequences used to search T. guttata were identical to those used for G. gallus (Table 3).

In addition to the cluster of β-keratins identified by Presland et al [12], preliminary analysis of the genomes of both G. gallus and T. guttata revealed additional genomic loci (including chromosome unknown of both the chicken and zebra finch) containing feather or feather-like β-keratins. To identify and classify these genes fully, more lenient parameters were used: an E-value cut-off of 1e-10, reasonable stop codons and start codons, no in-frame stop codons, no frame shift mutations, and no regions of unknown genomic sequence (see Table 2).

All sequences in this study were obtained from the genomic sequences of G. gallus and T. guttata and use a simple annotation pattern. Since all data presented in this paper is from the genomic sequences of G. gallus and T. guttata, the numbering of the β-keratins will follow a 5' to 3' pattern. This annotation also includes the species (abbreviated as GGA or TGU), chromosome number and β-keratin subfamily (feather = FK, feather-like = FL, claw = Claw, or scale = Scale and β-keratin from cultured keratinocytes = keratinocyte). For example, the claw sequence which is found at the 5' end of the cluster on the positive strand, located on microchromosome 25 of G. gallus is annotated as GGA25_claw1 (See Additional File 1 Table S1).

Expressed Sequence Tag Analysis

Expressed sequence tag (EST) databases for both avian species were downloaded via NCBI and contained 599,999 and 91,801 sequences for chicken and zebra finch, respectably. The β-keratin databases for chicken and zebra finch were used as queries for blastn searches of the EST databases. An E-value cutoff of 1e-160 was used for G. gallus β-keratin sequences and an E-value cutoff of 1e-150 was used for the T. guttata β-keratin sequences. The GI number for the EST, the range of the nucleotides corresponding to the β-keratin sequence, the range of the 5' and 3' EST base pairs, and the EST tissue collection information is listed in Additional File 3, Table S3.

Phylogenetic Analysis

Alignments were accomplished using the program CLUSTAL W Multiple Sequence Alignment Program [54] and PAL2NAL [55]. All default parameters were used in PAL2NAL and CLUSTAL W with the option SLOW/ACCURATE selected for CLUSTAL W alignments. Visual inspection confirmed an adequate alignment. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 4 [56].

Tree reconstruction was done using a total of 222 taxa, which included all probable feather β-keratins (located on GGA1, GGA2, TGU2, GGA5, GGA27, TGU27, GGA_Un and TGU_Un) and all probable β-keratins on GGA6 and chromosome 25 of both avian species (see Table 2). As an outgroup for tree reconstruction, three β-keratin nucleotide coding sequences were used from Crocodylus niloticus (Nile crocodile) and were obtained via NCBI and have the following Genbank numbers: 215541571, 215541573 and 187942180[4]. The Modeltest program, using the Akaike Information Criterion (AIC), selected the generalized time reversible evolutionary model with gamma distributed rate heterogeneity and a proportion of invariant sites as the best fit model for these taxa [57]. Using this suggested evolutionary model, tree reconstruction was accomplished using both distance based (Neighbor-Joining) and character based (Maximum Likelihood) methods.

MEGA version 4 [56] was used for Neighbor-Joining tree reconstruction, which was accomplished with 1000 bootstrap replicates, pairwise deletion, all codon positions selected, the LogDet evolutionary model, substitutions including transitions and transversions, and a heterogeneous pattern among lineages [58,59]. The nile crocodile sequences was chosen as the outgroup. The resulting evolutionary tree was used for figure construction (Additional file 5).

Additional file 5. Tree Reconstruction of all β-keratin genes found in the Gallus gallus and Taeniopygia guttata Genomes. Neighbor-Joining tree reconstruction of the 219 β-keratin nucleotide sequences from both avian genomes and the three nile crocodile sequences as the outgroup. The subfamilies are colored with the following scheme: GGA25_Keratinocyte = red, scale β-keratin genes = blue, claw β-keratin genes = yellow, feather β-keratin genes = green and feather-like β-keratin genes = magenta. The taxa nomenclature and methodology is detailed in the Methods section. Only bootstrap values from the Neighbor-Joining method are listed.

Format: PDF Size: 14KB Download file

This file can be viewed with: Adobe Acrobat ReaderOpen Data

The RA×ML 7.0.3 edition was used for Maximum Likelihood analyses. 1000 bootstrap replicates was accomplished using the GTR+I+G model (GTRGAMMAI) with C. niloticus chosen as the outgroup. The first run was done using the -f i option, which performs a really thorough standard bootstrap procedure (1000 bootstrap replicates). A second run was done with -f d option, which performs a fast rapid hill-climbing algorithm. Bootstrap values from the first run were added to the resulting tree of the second run (Additional file 6) [60].

Additional file 6. Tree Reconstruction of all β-keratin genes found in the Gallus gallus and Taeniopygia guttata Genomes. Maximum Likelihood tree reconstruction of the 219 β-keratin nucleotide sequences from both avian genomes and the three nile crocodile sequences as the outgroup. The subfamilies are colored with the following scheme: GGA25_Keratinocyte = red, scale β-keratin genes = blue, claw β-keratin genes = yellow, feather β-keratin genes = green and feather-like β-keratin genes = magenta. The taxa nomenclature and methodology is detailed in the Methods section. Only bootstrap values from the Maximum Likelihood method are listed.

Format: PDF Size: 13KB Download file

This file can be viewed with: Adobe Acrobat ReaderOpen Data

The large scale duplication seen among the feather β-keratin sequences were analyzed to determine amino acid sites under varying selective pressures using the PAML 4.0 package, in which six models of evolution were used [61]. Models tested were: M0 (one ratio), M1a (nearly neutral), M2a (positive selection), M3 (discrete), M7 (beta) and M8 (beta + ω). Three models allow for the possibility of positive selection, M2a, M3, and M8, which is the dN/dS ratio of ω being greater than 1. To test the fit of these models to the data and therefore the occurrence of a false positive, likelihood ratio tests (LRTs) were used. LRTs were only used when a model (M2a, M3, or M8) suggested a high dN/dS ratio for amino acid sites. These three models were tested against a null model, using four separate tests (M0 vs. M2a; M0 vs. M3; M1a vs. M2a; and M7 vs. M8), by comparing (2ln Δl) against the χ2-distribution, with the degrees of freedom equal to the number of parameters between models. Each major locus that contained more than two feather β-keratins were analyzed separately (TGU2, GGA2, GGA25, GGA27 and TGU27). This allowed for the possibility that each chromosome/locus of each species (GGA and TGU) has evolved independent or temporally separate and would then be under different selective pressures. The PAL2NAL alignment tool was used to convert a nucleotide alignment into a codon alignment for input into the PAML program in association with the Neighbor-Joining (N-J) tree output from CLUSTAL W [54,56,62].

Gene Conversion

To search for duplicated segments shared between pairs of genes, by mechanisms such as gene conversion and/or unequal crossing over, the program GENCONV 1.81a was utilized and applied to all probable genes in the final data set (See Identification and localization of the β-keratin multigene family above). Global Bonferroni corrected P-values were calculated against a simulated distribution of 10,000 iterations to determine the statistical significance of a shared observed fragment from a pair of sequences. To be considered a significant result the p-value must be lower than 0.01 [31,63].

Authors' contributions

MJG generated the data as part of his Ph. D. research. RHS as the major professor assisted in preparation of the manuscript. Both authors read and approved the final manuscript.

Acknowledgements

The authors thank Robert Friedman, Austin Hughes and Joseph Quattro for their insightful discussions of vertebrate evolution and bioinformatics. We also thank the 3 anonymous reviewers for their critical reviews and helpful suggestions.

References

  1. Bereiter-Hahn J, Matoltsy AG, Richards KS: Biology of the integument. Volume 2. Vertebrates. Berlin: Springer-Verlag; 1986. OpenURL

  2. Valle LD, Nardi A, Belvedere P, Toni M, Alibardi LJ: β-Keratins of Differentiating Epidermis of Snake Comprise Glycine-Proline-Serine-Rich Proteins With an Avian-Like Gene Organization.

    Developmental Dynamics 2007, 236:1939-1953. PubMed Abstract | Publisher Full Text OpenURL

  3. Hallahan DL, Keiper-Hrynko NM, Shang TQ, Ganzke TS, Toni M, Valle LD, Alibardi L: Analysis of gene expression in gecko digital adhesive pads indicates significant production of cysteine- and glycine-rich beta-keratins.

    J Exp Zool (Mol Dev Evol) 2009, 312B:58-73. Publisher Full Text OpenURL

  4. Valle LD, Nardi A, Gelmi C, Toni M, Emera D, Alibardi L: β-Keratins of the Crocodilian epidermis: composition, structure, and phylogenetic relationships.

    J Exp Zool (Mol Dev Evol) 2009, 312B:42-57. Publisher Full Text OpenURL

  5. Valle LD, Nardi A, Toni M, Emera D, Alibardi L: Beta-keratins of turtle shell are glycine-proline-tyrosine rich proteins similar to those of crocodilians and birds.

    J Anat 2009, 214:284-300. PubMed Abstract | Publisher Full Text OpenURL

  6. Ye C, Wu X, Yan P, Amato G: β-Keratins in crocodiles reveal amino acid homology with avian Keratins.

    Mol Biol Rep 2009.

    DOI 10.1007/s1103-009-9480-z.

    PubMed Abstract | Publisher Full Text OpenURL

  7. International Chicken Genome Sequencing Consortium: Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution.

    Nature 2004, 432:695-716. PubMed Abstract | Publisher Full Text OpenURL

  8. Huth TJ: The Genomic Organization of the Four Subfamilies of β-keratin (Claw, Feather, Feather-like, and Scale) in the Chicken, Gallus gallus. In Masters Thesis. University of South Carolina, Department of Biology; 2008. OpenURL

  9. Glenn TC, French JO, Heincelman TJ, Jones KL, Sawyer RH: Evolutionary relationships among copies of feather beta (α) keratin genes from several avian orders.

    Integrative and Comparative Biology 2008, 48(4):463-475. Publisher Full Text OpenURL

  10. Gill FB: Ornithology. New York: W.H. Freeman and Company; 1995. OpenURL

  11. Presland RB, Gregg K, Molloy PL, Morris CP, Crocker LA, Rogers GE: Avian keratin genes, I. A molecular analysis of the structure and expression of a group of feather keratin genes.

    J Mol Biol 1989, 209:549-560. PubMed Abstract | Publisher Full Text OpenURL

  12. Presland RB, Whitbread LA, Rogers GE: Avian keratin genes, II. Chromosomal arrangement and close linkage of three gene families.

    J Mol Biol 1989, 209:561-576. PubMed Abstract | Publisher Full Text OpenURL

  13. Whitbread LA, Gregg K, Rogers GE: The structure and expression of a gene encoding chick claw keratin.

    Gene 1991, 101:223-229. PubMed Abstract | Publisher Full Text OpenURL

  14. Knapp LW, Shames RB, Barnes GL, Sawyer RH: Region-Specific Patterns of Beta Keratin Expression During Avian Skin Development.

    Developmental Dynamics 1993, 196:283-290. PubMed Abstract | Publisher Full Text OpenURL

  15. Byrne C, Hardman M, Nield K: Covering the limb - formation of the integument.

    J Anat 2003, 202:113-124. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  16. Dhouailly D, Sawyer RH: Avian Scale Development: XI. Initial Appearance of the Dermal Defect in Scaleless Skin.

    Developmental Biology 1984, 105:343-350. PubMed Abstract | Publisher Full Text OpenURL

  17. Smoak KD, Sawyer RH: Avian Spur Development: Abnormal Morphogenesis and Keratinization in the Scaleless (sc/sc) Mutant.

    Trans Am Microsc Soc 1983, 102(2):135-144. Publisher Full Text OpenURL

  18. Carver WE, Sawyer RH: Immunocytochemical Localization and Biochemical Analysis of α and β Keratins in the Avian Lingual Epithelium.

    The American Journal of Anatomy 1989, 184:66-75. PubMed Abstract | Publisher Full Text OpenURL

  19. Shames RB, Knapp LW, Carver WE, Sawyer RH: Region-Specific Expression of Scutate Scale Type Beta Keratins in the Developing Chick Beak.

    The Journal of Experimental Zoology 1991, 260:258-266. PubMed Abstract | Publisher Full Text OpenURL

  20. Vanhoutteghem A, Londero T, Ghinea N, Djian P: Serial cultivation of chicken keratinocytes, a composite cell type that accumulates lipids and synthesizes a novel β-keratin.

    Differentiation 2004, 72:123-137. PubMed Abstract | Publisher Full Text OpenURL

  21. Hartl M, Bister K: Specific activation in jun-transformed avian fibroblasts of a gene (bkj) related to the avian β-keratin gene family.

    Proc Natl Acad Sci USA 1995, 92:11731-11735. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  22. Fraser RD, Parry AD: Molecular packing in the feather keratin filament.

    Journal of Structural Biology 2008, 162:1-13. PubMed Abstract | Publisher Full Text OpenURL

  23. Dhouailly D: A new scenario for the evolutionary origin of hair, feather, and avian scales.

    J Anat 2009, 214:587-606. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  24. Lynch M, Conery JS: The Evolutionary Fate and Consequences of Duplicate Genes.

    Science 2000, 290:1151-1155. PubMed Abstract | Publisher Full Text OpenURL

  25. Friedman R, Hughes AL: Gene Duplication and the Structure of Eukaryotic Genomes.

    Genome Research 2001, 11:373-381. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  26. Zimmer EA, Martin SL, Beverley SM, Kan YW, Wilson AC: Rapid duplication and loss of genes coding for the α chains of hemoglobin.

    Proc Natl Acad Sci USA Genetics 1980, 77(4):2158-2162. Publisher Full Text OpenURL

  27. Hughes AL, Friedman R, Ekollu V, Rose JR: Non-random association of transposable elements with duplicated genomic blocks in Arabidopsis thaliana.

    Molecular Phylogenetics and Evolution 2003, 29:410-416. PubMed Abstract | Publisher Full Text OpenURL

  28. Koszul R, Fischer G: A prominent role for segmental duplications in modeling Eukaryotic genomes.

    C R Biologies 2009., 332 PubMed Abstract | Publisher Full Text OpenURL

  29. Cornman RS: Molecular Evolution of Drosophila Cuticular Protein Genes.

    PLoS ONE 2009, 4(12):e8345.

    doi:10.1371/journal.pone.0008345

    PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  30. Woelk CH, Frost SD, Richman DD, Higley PE, Pond SL: Evolution of the interferon alpha gene family in eutherian mammals.

    GENE 2007, 397:38-50. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  31. Wu D, Irwin DM, Zhang Y: Molecular evolution of the keratin associated protein gene family in mammals, role in the evolution of mammalian hair.

    BMC Evolutionary Biology 2008, 8:241.

    doi:10.1186/1471-2148-8-241.

    PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  32. Warren WC, Clayton DF, Ellegren H, Arnold AP, Hillier LW, Kunster A, Searle S, White S, Vilella AJ, Fairly S, Heger A, Kong L, Ponting CP, Jarvis ED, Mello CV, Minx P, Lovell P, Velho TAF, Ferris M, Balakrishnan CN, Sinha S, Blatti C, London SE, Li Y, Lin Y, George J, Sweedler J, Southey B, Gunaratne P, Watson M, Nam K, Backstrom N, Smeds L, Nabholz B, Itoh Y, Whitney O, Pfenning AR, Howard J, Volker M, Skinner BM, Griffin DK, Ye L, McLaren WM, Flicek P, Quesada V, Velasco G, Lopez-Otin C, Puente XS, Olender T, Lancet D, Smit AFA, Hubley R, Konkel MK, Walker JA, Batzer MA, Gu W, Pollock DD, Chen L, Cheng Z, Eichler EE, Stapley J, Slate J, Ekblom R, Birkhead T, Burke T, Burt D, Scharff C, Adam I, Hughes R, Sultan M, Soldatov A, Lehrach H, Edwards SV, Yang S, Li X, Graves T, Fulton L, Nelson J, Chinwalla A, Hou S, Mardis ER, Wilson RK: The genome of a songbird.

    Nature 2010., 464

    doi:10.1038/nature08819.

    PubMed Abstract | Publisher Full Text OpenURL

  33. Chojnowski JL, Kimball RT, Braun EL: Introns outperform exons in analyses of basal avian phylogeny using clathrin heavy chain genes.

    Gene 2008, 410:89-96. PubMed Abstract | Publisher Full Text OpenURL

  34. Ericson GP, Anderson CL, Britton T, Elzanowski A, Johansson US, Kallersjo M, Ohlson JI, Parsons TJ, Zuccon D, Mayr G: Diversification of Neoaves: integration of molecular sequence data and fossils.

    Biol Lett 2006, 2:543-547. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  35. Pereira SL, Baker AJ: A Mitogenomic Timescale for Birds Detects Variable Phylogenetic Rates of Molecular Evolution and Refutes the Standard Molecular Clock.

    Mol Biol Evol 2006, 23(9):1731-1740. PubMed Abstract | Publisher Full Text OpenURL

  36. Tuinen MV, Hedges SB: The Effect of External and Internal Fossil Calibrations on the Avian Evolutionary Timescale.

    J Paleont 2004, 78(1):45-50. Publisher Full Text OpenURL

  37. Burt DW: Origin and evolution of avian microchromosomes.

    Cytogenet Genome Res 2002, 96:97-112. PubMed Abstract | Publisher Full Text OpenURL

  38. Douaud M, Feve K, Gerus M, Fillon V, Bardes S, Gourichon D, Dawson DA, Hanotte O, Burke T, Vignoles F, Morisson M, Tixier-Boichard M, Vignal A, Pitel F: Addition of the microchromosome GGA25 to the chicken genome sequence assembly through radiation hybrid and genetic mapping.

    BMC Genmoics 2008, 9:129. BioMed Central Full Text OpenURL

  39. Graur D, Li W: Fundamentals of Molecular Evolution. 2nd edition. Sunderland, MA. Sinauer Associates, Inc; 2000. OpenURL

  40. Kelley DR, Salzberg SL: Detection and correction of false segmental duplications caused by genome mis-assembly.

    Genome Biology 2010, 11:R28. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  41. Wu P, Jiang T, Suksaweang S, Widelitz RB, Chuong C: Molecular Shaping of the Beak.

    Science 2004, 305:1465-1466. PubMed Abstract | Publisher Full Text OpenURL

  42. Alibardi L: Claw development and cornification in the passeraceous bird zebrafinch (Taeniatopygia guttata castanotis).

    Anat Sci Int 2009.

    DOI 10.1007/s12565-009-0015-4.

    OpenURL

  43. Alibardi L, Sawyer RH: Cell structure of developing downfeathers in the zebrafinch with emphasis on barb ridge morphogenesis.

    J Anat 2006, 208(5):621-642. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  44. Bell E, Thathachari YT: Development of feather keratin during embryogenesis of the chick.

    J Cell Biol 1963, 16:215-223. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  45. Bell E, Schuler M, Merrill C: Feather formation and synthesis of keratin by primary skin cells and by skin cells grown in vitro. In The Epidermis. Edited by Montagna W, Lobitz WC. New York: Academic Press; 1964:35-56. OpenURL

  46. Haake AR, König G, Sawyer RH: Avian feather development: Relationships between morphogenesis and keratinazation.

    Dev Biol 1984, 106:406-413. PubMed Abstract | Publisher Full Text OpenURL

  47. Backstrom N, Forstmeier W, Schielzeth H, Mellenius H, Nam K, Bolund E, Webster MT, Ost T, Schneider M, Kempenaers B, Ellegren H: The recombination landscape of the zebra finch Taeniopygia guttata genome.

    Genome Research 2010, 20:485-495. PubMed Abstract | Publisher Full Text OpenURL

  48. Hu D, Hou L, Zhang L, Xu X: A pre-Archaeopteryx troodontid theropod from China with long feathers on the metatarsus.

    Nature 2009, 461:640-643. PubMed Abstract | Publisher Full Text OpenURL

  49. Witmer LM: Feathered dinosaurs in a tangle.

    Nature 2009, 461:601-602. PubMed Abstract | Publisher Full Text OpenURL

  50. Sawyer RH, Knapp LW: Avian Skin Development and the Evolutionary Origin of Feathers.

    Journal of Experimental Zoology (MOL DEV EVOL) 2003, 298B:57-72. Publisher Full Text OpenURL

  51. Alibardi L, Toni M: Cytochemical and molecular characteristics of the process of cornification during feather morphogenesis.

    Progress in Histochemistry and Cytochemistry 2008, 43:1-69. PubMed Abstract | Publisher Full Text OpenURL

  52. Altschul SF, Gish W, Miller EW, Lipman DJ: Basic Local Alignment Search Tool (BLAST) Version 2.2.16. NCBI division of NIH; 2006.

  53. Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA, Barrell B: Artemis: sequence visualization and annotation.

    Bioinformatics 2000, 16:944-945. PubMed Abstract | Publisher Full Text OpenURL

  54. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice.

    Nucleic Acids Research 1996, 22:4673-4680. Publisher Full Text OpenURL

  55. Suyama M, Torrents D, Bork P: PAL2NAL: robust conversion of protein sequences alignments into the corresponding codon alignments.

    Nucleic Acids Research 2006, 34:W609-W612. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  56. Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0.

    Molecular Biology and Evolution 2007, 24:1596-1599. PubMed Abstract | Publisher Full Text OpenURL

  57. Posada D, Crandall KA: Modeltest: testing the model of DNA substitution.

    Bioinformatics 1998, 14(9):817-818. PubMed Abstract | Publisher Full Text OpenURL

  58. Lake JA: Reconstructing evolutionary trees from DNA and protein sequences: Paralinear distances.

    PNAS 1994, 91:1455-1459. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  59. Lockhart PJ, Steel MA, Hendy MD, Penny D: Recovering Evolutionary Trees under a More Realistic Model of Sequence Evolution.

    Mol Biol Evol 1994, 11(4):605-612. PubMed Abstract | Publisher Full Text OpenURL

  60. Stamatakis A: Maximum Likelihood-based Phylogenetic Analyses with Thousands of Taxa and Mixed Models.

    Bioinformatics 2006, 22(21):2688-2690. PubMed Abstract | Publisher Full Text OpenURL

  61. Yang Z: PAML 4: Phylogenetic Analysis by Maximun Likelihood.

    Mol Biol Evol 2007, 24(8):1586-1591. PubMed Abstract | Publisher Full Text OpenURL

  62. Yang Z, Nielsen R, Goldman N, Pedersen AK: Codon-Substitution Models for Heterogeneous Selection Pressure at Amino Acid Sites.

    Genetics 2000, 155:431-449. PubMed Abstract | PubMed Central Full Text OpenURL

  63. Sawyer SA: Statistical tests for detecting gene conversion.

    Molecular Biology and Evolution 1989, 6:526-538. PubMed Abstract | Publisher Full Text OpenURL

  64. Barnes GL: Differentiation of the Embryonic skin of the chicken. In PhD Thesis. University of South Carolina, Department of Biological Sciences; 1993. OpenURL

  65. Shames RB, Knapp LW, Carver WE, Sawyer RH: Identification, expression, and localization of β keratin gene products during development of avian scutate scales.

    Differentiation 1988, 38:115-123. PubMed Abstract | Publisher Full Text OpenURL