Bone-associated gene evolution and the origin of flight in birds

Bones have been subjected to considerable selective pressure throughout vertebrate evolution, such as occurred during the adaptations associated with the development of powered flight. Powered flight evolved independently in two extant clades of vertebrates, birds and bats. While this trait provided advantages such as in aerial foraging habits, escape from predators or long-distance travels, it also imposed great challenges, namely in the bone structure. We performed comparative genomic analyses of 89 bone-associated genes from 47 avian genomes (including 45 new), 39 mammalian, and 20 reptilian genomes, and demonstrate that birds, after correcting for multiple testing, have an almost two-fold increase in the number of bone-associated genes with evidence of positive selection (~52.8 %) compared with mammals (~30.3 %). Most of the positive-selected genes in birds are linked with bone regulation and remodeling and thirteen have been linked with functional pathways relevant to powered flight, including bone metabolism, bone fusion, muscle development and hyperglycemia levels. Genes encoding proteins involved in bone resorption, such as TPP1, had a high number of sites under Darwinian selection in birds. Patterns of positive selection observed in bird ossification genes suggest that there was a period of intense selective pressure to improve flight efficiency that was closely linked with constraints on body size.


Background
Powered flight evolved independently in birds and bats, but required similar trade-offs and limitations, including strong constraints on traits such body size [1,2] and skeletal structure to minimize energy requirements [3]. While body sizes have tended to increase through evolutionary time in many lineages [4], the size of flying vertebrates has been more constrained [5]. However, postcranial skeleton pneumatization (hollow air-filled bones) and bone modifications (such as bone fusion) may have provided increased evolutionary flexibility among birds [6] (Fig. 1a). In birds, hollow bones are formed with pneumatic foramina or openings in the wall of the bone that permit air sacs to perforate internal bone cavities [7,8]. The development of pneumatic bones in birds led to reductions in overall body mass and has also been associated with bone resorption [6,9].
These pneumatic bones have often been assumed to have lightened the entire avian skeleton relative to mammals [10] and to have reduced the metabolic cost of flight [3,[11][12][13][14]. However, some skeletal structures, such as the humerus, ulna-radius, tibio-tarsus and fibula, have more body mass in birds than mammals [15], suggesting that modern bird skeletons have experienced diverse bone-specific selection patterns.
Bats are the only mammals capable of sustained flight, but have distinct traits than birds that likely reflect key differences in ecological adaptations and distinct evolutionary histories [16]. Bats have elongated fingers instead of elongated forearms as seen in birds and have bones with high levels of mineral density that increases the stiffness of the skeleton [3]. On the other hand, as with birds, bats have relatively small bodies [17], fused bones and lightweight skeletons [3] (Additional file 1: Figure  S1). Many of the other shared traits among birds and bats are probably also associated with the challenges imposed by the evolution of powered flight (Additional file 1: Figure S1). These include improved respiratory systems [18], high metabolic output [19], hyperglycemia tolerance [20,21], diminished production of reactive oxidative species [22,23] and smaller intestines [24].
Here, we tested the evolutionary rate of change in 89 bone-associated genes in 47 avian and 39 mammalian genomes and evaluated genetic distinctions among flying versus non-flying species to assess patterns of selection in genes involved in bone development. Birds displayed a higher number of the bone-associated genes under positive selection, the majority of which were associated with regulatory process of bone remodeling. Of the 89 analyzed genes, 13 positively-selected genes in birds also had different evolutionary rates in bats relative to other mammals. These were mainly genes involved in bone fusion and bone-remodeling, which affirms the role of adaptive selection as a key process driving the evolution of flight.

Bone-associated gene locations and related phylogenetic analyses
The 89 bone-related genes (Additional file 2: Table S1) represent a subset of the genes associated with bone development [25]. These bone-associated genes were distributed widely across the genomes of mammals and birds (Additional file 3: Figure S2).
The inferred topology for bone-associated genes was significantly different from the avian species tree using the whole genome data [26,27], ΔlnL = 1891.34, but more similar to the tree topology obtained from protein coding only genes [27] ΔlnL = 537.06 (Fig. 2a). Both the avian species-tree and protein coding-genes tree showed significant differences under the tests 1sKH (one sided KH test based on pairwise SH tests), SH (Shimodaira-Hasegawa), and ELW (Expected Likelihood Weight) at a critical 5 % significance level relative to those obtained with the bone-associated gene-tree-based phylogeny. With the mammalian bone-associated genes the tree topology was slightly different from the mammalian species tree [28,29], since significant differences were obtained under the tests 1sKH, SH, and ELW at 5 % significance level, ΔlnL = 271.70 (comparison accepted species tree vs. obtained tree) (Fig. 2b). We note that the mammalian species tree was also generated mostly with protein coding sequences.
Site-models show a higher evolutionary rate in bird boneassociated genes In site models, of the 89 mammalian genes, 27 (~30.3 %) favored the alternate model (evolved under positive selection) ( Fig. 3; Additional file 4: Table S2), whereas in birds, 47 (52.8 %) were positively selected ( Fig. 3; Additional file 5: Table S3). This difference in the number of selected genes in birds compared to mammals was significant (Fisher's Exact Test, two-tailed, p-value = 0.003722). Additionally, we tested for signals of positive selection in reptiles. The observed positive selection in birds is a unique signature and not a ubiquitous tendency in sauropsida, since only 20 (~22 %) of 88 genes showed significant evidence of positive selection in reptiles (Additional file 6: Table S4). Furthermore, the presence of positive selection in bone-associated genes revealed different targets in the three different clades (Additional file 7: Figure S3). Of the 89 genes,~18 % (16) were positively selected in both birds and mammals, 34.8 % (31) were only positively selected in birds and only 12.4 % (11) were identified in only mammals (Fig. 4a). showing the key bone modifications observed in birds, and bones containing red-blood-cell-producing marrow (apneumatic bones). Most bones (except very small ones) are pneumatized. The structure of a pneumatic bone is highlighted in the light blue box (licensed by Rice University under a Creative Commons Attribution License (CC-BY 3.0)). b Positively selected genes in birds and those genes showing a dissimilar evolutionary rate in bats when compared to other mammals (lower evolutionary rate-colored in grey; and higher evolutionary rate-colored in white). Representation of the link between gene and physiological/development systems (colored accordingly: skeleton system (1), muscular system (2) and glucose (3) that are plausibly related with flight adaptation In birds the highest global omega values (0.53 and 0.71) were observed for AHSG (Alpha-2-HS-glycoprotein) and P2RX7 (P2X purinoceptor 7), respectively (Additional file 5: Table S3). Both genes are associated with bone mineral density and bone remodeling [30,31]. However, considering only the number of sites with omega > 1.0 and a Posterior Probability (pp) ≥ 0.95, two genes involved in bone resorption, TPP1 (Tripeptidyl peptidase I) and TFRC (Transferrin Receptor), had the highest number of positively selected sites, 95 and 33, respectively, corresponding to 19.8 % and 4.2 % of the alignment length (Additional file 5: Table S3). Since tpp1 protein is secreted by osteoclasts and Peptidase S53 is involved in bone collagen proteolysis [32], the positive selection may be related with the optimization of this proteolytic process during bone resorption.

Branch and branch-site models show increased selection in bone genes of flying species
For the branch-model analyses, the datasets were labeled according to their life-habits (flying vs. non-flying). Flightless birds [33] included those unable to sustain flight for long distances (such as turkey or chicken), aquatic-birds and running birds (e.g. ratites). This approach permitted the identification of genes evolving under different evolutionary rates in the different lineages of flightless and flying species. The correlation between mammals and birds had the lowest rho (ρ) value for flightless birds and flying mammals (Spearman's ρ = 0.579; p-value < 0.01) ( Table 1). The highest similarities in d N /d S values were obtained within each taxonomic clade; for bats and other mammals ρ = 0.833 (p-value <0.01) and for flightless and flying birds ρ = 0.883 (p-value <0.01). These patterns suggest that although a relatively small number of sites were affected, they were sufficient to be identified as evolving under positive selection, yet were insufficient to result in a significant different evolutionary rates between flying and flightless species. This is particularly evident in the branch-site models, since 10 of 86 genes (three genes were unreported in chiropterans species) were best fit the alternate model in branch-site analyses in flying birds and bats (Additional file 8: Table S5 and Additional file 9: Table S6). While 52 out of 86 genes best fit the null model in both flying birds and bats, in bats 59 out 86 genes and 63 out of 86 genes in flying birds had at least one site with an pp > =0.5 (Additional file 8: Table  S5 and Additional file 9: Table S6). This suggests that positive selection only affected a few sites while the majority of the proteins evolved under neutral and/or negative selection. Only 879 sites in flying birds (Additional file 8: Table S5) and 475 sites from a total of 53,526 analyzed positions were positively selected in flying  Table S6). The branch-site analyses also revealed four genes with the same positively-selected sites in both flying birds and bats, AHSG (two sites), ANKH, ANKH Inorganic Pyrophosphate Transport Regulator (one site), HOXA11, Homeobox protein Hox-A11, (three sites), MC4R, Melanocortin receptor 4 (one site).

Flying species have a high prevalence of positive selection in bone regulatory genes
In birds, the functional category analysis showed that genes under positive selection are mainly involved in processes regulating ossification (13 out of 19,~68 %), bone mineralization (10 out of 14,~71 %) and biomineral formation (10 out of 14,~71 %) (Fig. 5). These processes are significantly less represented in the list of positively-selected genes in mammals (Fisher's Exact Test p-value < 0.01). Notably, 13 genes that were positively selected in birds also had different evolutionary rates between bats and non-flying mammals ( Fig. 4b; Additional file 10: Table S7 and Additional file 11: Table  S8). Additionally, we identified five genes that had different evolutionary rate in flightless birds and were positively selected in terrestrial mammals and negatively selected in flying birds ( Fig. 4c; Additional file 12: Table  S9 and Additional file 13: Table S10).

Correlation between substitution rates and body mass
To determine if there is a possible correlation between evolution rates in flying species and body mass, we used the Bayesian method CoEvol that provides comparisons between rates of change in phenotypic traits and rates of molecular evolution [34]. In CoEvol, a high posteriorprobability of covariance between the rate of change in d S , d N /d S , GC nucleotide content and the change of a phenotypic trait would suggest that there is evidence of a link between molecular and phenotypic processes. The separate estimation of covariance for d S and d N /d S distinguishes mutational effects of d S from selective effects of d N /d S. In birds, high GC content has been associated with large population sizes and short generation times [35]. Therefore, GC content analysis can act as a control measure for the effects of small-bodied animals with putatively large populations that typically have lower the  [36]. Comparison between all birds vs. only flying birds was used to help understand the effect in the model estimation when flightless birds were included. A similar approach was employed for mammals, using a dataset including all mammals compared with other sets using only terrestrial mammals.
When only bird species that could fly were tested, a negative covariance was found between average body mass and d S (R = −0.507, posterior probability (pp) =0.023**), GC content and d N /d S (R = −0.9605, pp = 0**). When flightless species were included, in addition to the d S correlation with body mass (average) (R = −0.398, pp = 0.039*), there was also a negative covariance between GC content and body mass (R = −0.542, pp = 0.0405*), and a positive correlation between d N /d S and the body mass (R = 0.507, pp = 0.955*) ( Table 2; Additional file 14: Figure S4).
Mammals exhibited a different trend, since when bats were included, there was a negative correlation between body mass and d S (R = −0.534, pp = 0.0093**), and between body mass and GC content (R = −0.5035, pp = 0.01615**) and a positive correlation with body mass and d N /d S (R = 0.496, pp = 0.985**) ( Table 3). In contrast, when bats were excluded, d N /d S (R = 0.572, Fig. 4 Venn diagrams of positively-selected bone-associated genes. a Intersection between positively-selected genes shared in different combinations among mammals and birds, with the datasets including only terrestrial mammals and flying birds. b Intersection between positively-selected genes in terrestrial mammals, flying birds and those genes showing a different evolutionary rate in bats. c Intersection between positively-selected genes in terrestrial mammals, branch of flightless birds and flying birds. Asterisks (*) represent genes where the foreground branch was slower than background  For mammals and birds the results were also consistent under a different phylogenetic assumption, i.e., using the gene-based tree instead of the species tree (Additional file 15: Table S11 and Additional file 16: Table S12). These findings suggest that including or excluding bats has little effect on the results which can be partially explained by the relatively small number of bats in the dataset (~5 % of the total amount of sequences) compared with the larger percentage of flightless species (~87 %) in the avian comparison.
Additionally, the large flying fox is often reported as the largest bat, and therefore potentially introduces a slight bias in the analyses given its large body mass.

Discussion
We assessed the evolutionary patterns of 89 bonerelated genes in 47 avian and 39 mammalian genomes and demonstrate that there has been significantly higher positive selective pressure on several of the boneassociated genes of birds, particularly in those involved in bone-regulatory processes. Moreover, just as in birds, flying mammals (bats) had several genes with evolutionary rates that contrasted with the patterns observed in other mammals. These results highlight convergent changes in bone genes in the evolution of flight and the extensive selective pressure that flight triggered in bone-associated genes.  5 Functional annotation of positively-selected genes in birds and mammals. The heat map on the left represents the percentage of positivelyselected genes in birds and mammals for each GO category. Terms directly associated with bones are highlighted in bold, and those where there is a significant statistical difference between birds and mammals, upon Fisher's Exact Test, are marked with two asterisks (**). The heat map on the right presents the ratio obtained in heat map on the left for each GO term, divided by the ratio of positively-selected genes in birds and mammals respectively. A value great than one is indicative that there is evidence that the GO category has experienced positive selection

Body mass and bone-associated genes
The different evolutionary trajectories for developing the capacity to fly in birds and bats led to distinct mechanical and biochemical solutions to the adaptive challenges. Nevertheless, both birds and bats have bones with high mineral content [3] and both have body sizes that approach the predicted theoretical limit, i.e, the tradeoff between the mechanical power and the capacity for metabolic output essential for flight [37]. Among different avian orders, skeletal measurements and body mass are correlated, as they are limited by ecological and biomechanical constraints on bone dimensions [38]. The different life habits among birds partially explains the higher correlation between body mass and d N /d S that was observed when assessing the dataset including all the bird species. Since this covariance suggests a relaxation on the selective pressure on bone-associated genes in non-flying species, the findings are consistent with the hypothesis that the skeleton of flightless birds can be larger than in flying birds. The absence of this correlation among flying species may reflect their lower variation in the body mass and differences in the foraging habits irrespective of their body size, since bone structure is often associated with the life history of the species [39]. In contrast, extant mammals display a wider range of body mass than extant birds [40], supporting the observed correlation between d N /d S and average body mass.    Furthermore, the opposite trend in birds and mammals might partially be explained by the contrasting lifehistories of the species in the two clades. Bird evolution seems to have favored size reduction in Neoaves, while in mammals, trends in body mass vary among subclades [36]. This can also explain the higher correlation between d N /d S and body mass when bats are included. However, in both scenarios, either including or excluding bats, there was a positive and statistically significant correlation between body mass and d N /d S .

Evolutionary rate in flying versus non-flying species
Although vertebrate powered flight is not restricted to birds, flight is more ubiquitous in birds. Powered flight has been linked with low body mass [41], high metabolic rate [42], metabolic efficiency [43], and specialized mechanical systems supported by skeletal adaptations. Yet, many aspects of flight remain unclear, including how bone-related genes evolved in birds and other taxonomic groups such as bats. The high rates of selection that we found for several bone-related genes suggest that the observed variation among avian species is higher than would be expected under models of neutrality. Therefore, the presence of adaptive and positive selection in these genes is likely indicative of a fundamental feature of trait modeling in the evolution of the skeleton. The phylogeny also supports this observation since the incongruence between the species-tree and gene-tree reinforces the hypothesis that flight was a key event that had a noticeable impact on the evolution of boneassociated genes in birds and mammals.

Extended impact of flight on bone-associated genes
Our results suggest that a relatively small number of genes involved in bone structures may have independently evolved in birds and bats in similar ways that permitted the transition from terrestrial to aerial life styles. Of the 89 bone-associated genes, only 13 showed signatures of selection in both birds (site model) and bats (branch model exhibiting acceleration/deceleration relatively to terrestrial mammals with significant statistical support). The function of these 13 genes, summarized below, probably reflect key genetic pathways and adaptations that enable flight. However, since several of these bone-associated genes are also involved in other processes, the comparison between flying and non-flying species suggests that some of the genes involved in the evolution of flight may also have had other evolutionary constraints (Fig. 1b).
BMP2 (Bone morphogenetic protein 2) has been implicated in the stimulation of cartilage proliferation and differentiation and in the increase in digit length in bat embryonic forelimbs [44]. Similarly, PKDCC (protein kinase domain containing cytoplasmic) is implicated in the control of limbs length, since the target disruption of this gene leads to short limbs [45]. The lengthening of forelimbs was an essential step in the evolution of flight in vertebrates [46,47]. Birds also share several other features, including a fused cranial bone, which might be linked with BMP2 [48]. Importantly, several other examples of bone fusion (e.g. vertebrae fusion) have been cited as being crucial for the evolution of flight [49].
OSR2 (odd-skipped related 2) has been associated with forelimb, hindlimb and craniofacial development [50] and is a likely candidate gene for many of the fundamental changes in the limbs of birds and bats. At the beginning of avian evolution, the allometric coupling of forelimb and hindlimb with body size was disrupted, and as wings began to significantly elongate, they maintained a positive allometric relationship with body size, but their legs significantly shortened [47]. This would have facilitated the diversification of forelimb and hindlimb shapes and sizes that are currently observed in extant birds [47] and which are closely linked with foraging habits in birds and bats [47].
HOXA11 (homeobox A11) may also be related with bone fusion, as this gene has been reported to influence radio-ulnar fusion [51] and bats may also display partial fusion of those bones (see Additional file 1: Figure S1). Although birds presented no evidence of fusion of the radio and ulna, these bones are typically apneumatic in birds and therefore contain bone marrow; and HOXA11 has been associated with bone marrow failure syndrome [51]. Interestingly in this gene are detected three homologous sites under positive selection in bats and flying birds, suggestive of functional convergence, likely due to flight evolution (Additional file 8: Table S5 and Additional file 9: Table S6).
BMPR1A (bone morphogenetic protein type IA gene) is involved in bone remodeling, and the ablation of this receptor in osteoblasts increases bone mass [57]. This makes BMPR1A a prime candidate for the maintenance of bone strength, which is essential for a stiff, but lightweight skeleton system in flying species [3]. Similarly, ACVR2B (activin receptor type-2B) is involved in the control of bone mass, but interestingly is mediated by GDF-8 (myostatin) which is also involved in improving muscle strength [58].
PTK2B is involved in bone resorption [59], a process involved in bone remodeling, during which osteoclasts digest old bone [60]. Bone remodeling is essential to making the necessary adjustments of bone architecture for the mechanical needs of flight [60]. It may well be responsible for alterations that support the increased BMD levels [61] that are observed in both bats and birds.
CITED2 (Cbp/P300-interacting transactivator, with Glu/Asp-rich carboxyl-terminal) is involved in bone formation [62], but also plays a pivotal role in muscle mass regulation since it also counteracts glucocorticoidinduced muscle atrophy [63]. Flight in vertebrates requires powerful muscles, particularly those connected to sternum bones [64]. CITED2 has also been linked with some heart diseases [65], which may be of note since birds [66] and small bats [67] possess larger hearts relative to vertebrates of similar size.
TCF7L2 (Transcription factor 7-like 2) is associated with bone mineralization [68]. However, it is also considered to be the most significant genetic marker that has been linked with Diabetes mellitus Type 2 risk and it is a key regulator of glucose metabolism [69]. The signatures of selection observed in birds and bats in TCF7L are remarkable given the high blood glucose levels observed in birds [70] and fruit and nectar-feeding bats [21,71]. The tolerance of birds and bats to blood-hyperglycemia may therefore be related with the evidence for positive selection observed in our analyses, as flight requires efficient glucose metabolism and efficient transportation to the energy-demanding organs (e.g. flight muscles) that are involved in powered flight [71,72].
Despite the similarities between bats and birds, extensive positive selection is observed in some genes in birds but is absent in bats, including P2RX7 and TPP1, which are mainly involved in bone resorption [32,73]. In birds, the pneumatic epithelium that forms the diverticula is capable of extensive resorption of bone material given its close association with osteoclasts [74]. Bone remodeling through resorption may be crucial to the formation of the bone trabeculae and by extension, the formation of the pneumatic bones. Recently, polymorphisms described in P2RX7 have been associated with osteoporosis in humans [75], which is typically linked with increased bone resorption and a decrease in bone mineral density (BMD) [76]. Here we demonstrated that genes involved in bone remodeling (particularly evident in the subprocess bone resorption) had multiple signals of positive selection in birds, but contrary to osteoporosis, bird bones attain a high value of BMD [3].

Gene's functional categories, bone remodelling and their implication in life-habits
Although bone pneumaticity may have facilitated the transition to flight in birds, it may not have been a necessary step, since bats evolved the ability to fly without postcranial skeletal pneumaticity. Pneumatization preceded the origin of avian flight and evolved independently in several groups of bird-line archosaurs (ornithodirans) [77], and therefore cannot be exclusively the result of adaptation for flight [77]. It has been suggested that skeletal pneumaticity, in early evolutionary stages, provided no selective advantage [78] and also did not significantly affect the skeleton through the lightening or remodeling of individual bones [78]. Although skeletal density modulation would have resulted in energetic savings as part of a multi-system response to increased metabolic demands and the acquisition of an extensive postcranial skeleton, pneumaticity may have favored high-performance endothermy [77].
Nevertheless, the finding that genes involved in bone remolding have been subjected to a higher prevalence of positive selection is interesting because: 1) development of postcranial skeletal pneumaticity occurs after hatching [79]; 2) the skeleton is a metabolically active tissue that undergoes continuous remodeling throughout life [60]; and 3) bone remodeling may lead to a more porous bone structure [60]. Bone remodeling involves the removal of mineralized bone by osteoclasts followed by the formation of a bone matrix through the osteoblasts that is subsequently mineralized [60]. It is generally assumed that bone remodeling is essential for maintaining skeletal mechanical properties and mineral homeostasis [80]. Therefore the higher prevalence of positive selection in bone-remodeling genes suggests that bones with higher mineral density were attained as a response to the selective contingencies imposed by flying, including bone remodeling and bone resorption. The similarities among bats and flying birds, bones with high mineral content, suggests that genes involved in bone remodeling probably play a pivotal role in avian diversification and adaptation in a wide range of ecological and behavioral niches.

Conclusions
The evolution of flight in birds was a pivotal event in their successful adaptation to new ecological niches. However, the transition to flight imposed new challenges on their bone structure. The high rate of positive selection in bone-associated genes in birds suggests that there was a strong link among changes in these genes and the adaptations necessary for flight. Limitations imposed on body size were probably also a key factor in bird evolution, as we have shown here that body mass covaried significantly with the d N /d S value only when flightless birds were included. Evidence of adaptive selection in birds and bats also were apparent in genes plausibly linked with bone-remodeling, bone fusion, lengthening of forelimbs, as well as with functions outside the skeleton system, including glucose tolerance that also would have had a major influence on the capacity for powered flight. However, the examples of positive selection that were only observed in birds, such as the evolution of a more-diversified and richer-variety of protein-encoding genes involved in bone resorption (e.g. TPP1 and P2RX7) and the formation of bone trabeculae that are likely critical to the evolution of hollow or pneumatic bones, suggest that these might be crucial steps in the evolution of avian flight that are unique to birds.

Sequences and alignment
A list of bone-associated genes was retrieved from the GO database by querying the term "bone" in QuickGO [81]. The resulting list was filtered using unique terms and the correct gene name was mapped using the REST API available in bioDBnet [82]. The gene list was then used in Ensembl Biomart to retrieve the Ensembl Gene ID using Gallus gallus as reference. The gene name and/ or gene ID was used to search in each genome file that contained the annotated gene sequences from each bird species. The avian dataset derived from 47 bird genomes provided by the Avian Genome Consortium [26] encompasses 89 bone-associated genes (Additional file 2: Table  S1), resulting in a total of 3,388 sequences and~38 species sequences on average per multiple sequence alignment (MSA). Sequences for each gene were translated into amino acids, aligned using MUSCLE [83] and backtranslated to nucleotides. Aberrant sequences, containing frame-shifts (e.g. stop codons) and duplicated sequences, were removed from the MSA. The dataset from reptiles was retrieved from the NCBI nucleotide database [84], which encompassed 20 different species (Additional file 17: Figure S6). For MEPE only one sequence was retrieved and therefore 88 genes were successfully used (672 sequences,~7.6 sequences per gene). The sequences were aligned using MUSCLE [83] and back-translated to nucleotides. The mammalian dataset was derived from 39 genomes (2,903 sequences,~32 per gene) that were manually retrieved from ENSEMBL [28,29]. The MSA of each gene was built using the same strategy as with the avian genes. The 89 genes were concatenated using SequenceMatrix v 1.7.8 [85] to one MSA containing all the avian data, and a second MSA containing the 89 mammalian genes. A phylogenetic tree was built separately for birds and mammals using the 89 concatenated genes with PhyML v3.0 [86] under the Generalized Time-Reversible (GTR + Г + I) model and the branch-support was provided by aLRT [87]. The obtained phylogenetic trees were compared using TREE-PUZZLE [88].

Site models
CODEML, as implemented in PAML v4.7 [30,89], was used to test for selection signatures in the avian, mammalian and reptilian bone genes using three models (Models 0, 1 and 2). Model 0 was used to calculate the global d N /d S and Model 1 vs Model 2 to identify the sites under positive selection. Sites with significant signatures of selection were retrieved after a post-hoc analysis using Bayesian Empirical Bayes [90]. The tree topology used as the input for the CODEML models for mammals was the tree retrieved from ENSEMBL, for birds was the full-genome derived tree "species tree" [27] (Additional file 18: Figure S5) and for reptiles was adapted from recent publications [91,92] (Additional file 17: Figure S6). Estimations for d N and d S under Model 0 for each branch showed low levels of saturation (Additional file 19: Table S14).

Branch models
We tested for selection using branch models with a tworatio model that allow variation in the d N /d S ratio between the background and foreground branches. The two-ratio model was compared against a one-ratio model. In the bird and mammal datasets the "exceptions" (flightless birds and flying mammals) were compared against the flying birds and flightless mammals, providing an understanding of which genes were under differential selection patterns in the two clades. Spearman's correlations were performed in SPSS v20 [93].

Branch-site models
The branch-site model detects positive selection when it occurs in sites along particular lineages or labeled branches (foreground branches). This model allows the d N /d S ratio either to vary along the sites or the branches on the tree (foreground vs background branches). To compare the effect of flight in bone-associated genes, the terminal branches of flying species in birds were considered to be the foreground branches and the nonflying species the background branches. The sequences were aligned using all sequences and later separated into two different alignments. For each MSA was performed a branch-site model A with ω 2 = 1 fixed in the null model.

Correction for multiple testing
All the results from site, branch-site and branches models were corrected for possible multiple testing bias using the procedure of Benjamini and Hochberg [94] as implemented in the program Q-Value [95]. For each pvalue, we also estimated the corresponding q-value; where the q-value represents the false discovery rate using the critical value 0.05. When the q-value was below the critical p-value estimated for the Likelihood-Ratio Test value, the gene was considered to be under positive selection (1), and when above, the gene was considered negatively selected (0).

Functional classification of bone-associated genes
Functional annotation enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID v6.7) [96,97]. Each derived gene list was processed in DAVID for functional terms using Homo sapiens as background. Venn diagrams were generated using VENNY [98].
Correlation model between body mass and boneassociated genes CoEvol 1.3c [34] implements a phylogenetic model that correlates the evolution of substitution rates (e.g. d s , ω (d N /d S )) with continuous phenotypic characters (e.g. body mass, longevity). The MSA of the 89 bone-associated genes was divided into two different datasets, one including all birds and the other restricted to only the flying bird species. CoEvol was ran under two different phylogenetic assumptions: 1) using the species-tree used in the evolutionary analysis; 2) using the gene-based tree estimated for birds and mammals with the 89 concatenated genes in PhyML v3.0 and the Generalized Time-Reversible (GTR + Г + I) evolutionary model. To ensure convergence, we ran two different chains to at least an effective number of 50. Calibration of the tree was done using the divergencetime-based option in TimeTree [99] (Additional file 20: Table S15) and body mass estimates are provided in a supplemental table (Additional file 21: Table S16).
CoEvol models evolutionary rates of substitution and phenotypic characters as a multivariate Brownian diffusion process along the branches, correcting for the uncertainty about branch lengths and substitution history in the phylogenetic tree. Correlations among rates of substitution and phenotypic characters were calculated with posterior probabilities varying from 0 to 1 using a Bayesian Markov chain Monte Carlo and correcting for phylogenetic inertia using the independent contrast method. Posterior probabilities close to 0 indicate a negative correlation while values close to 1 indicate a positive correlation. Cut-offs of pp < 0.05 and pp > 0.95 suggest negative or positive covariance, respectively, between the substitution rates and the phenotypic trait. The CoEvol analyses were run for at least 2000 points for both phylogenetic trees (species tree and gene tree), for all genes and only positively selected genes in each clade (mammals and birds).