Gene duplication has been an important factor for shaping the immune defence against the high diversity of pathogens faced by vertebrates [1–4]. This can be seen in the gene clusters of the major histocompatibility complex (MHC) class I and II [2], but also in the innate immune system in, for example, genes coding for toll-[1] or anti microbial peptides, i.e. defensins [5].

Defensins are a group of small cationic peptides that play an important role in the innate immune system of vertebrates, as well as in invertebrates [6–8]. Defensins provide a broad pathogenic defence against bacteria, viruses, fungi as well as showing activity against protozoan parasites [9–11], primarily by binding to and disrupting the membrane of the invading organism. However, recent studies have shown that they also might play an important role in the link between other parts of the immune system by signalling to macrophages, lymphocytes and mast cells [12, 13]. Moreover, the peptides might play a more complex role in destroying pathogens by not only disrupting cell membranes, but also by being able to block viral infections by disrupting the replication cycle or altering host cell recognition sites [10, 14]. Depending on the distribution of sulphide bonds within the mature peptide, defensins can be divided into three subfamilies; θ-, α- and β-defensin. In the chicken genome, 14 genes coding for β-defensins have been found in a dense cluster located on the end of the 3^rd chromosome (3q3.5-q3.7, [15–17]). These genes consist of four exons (E1-E4), E1 coding for 5'UTR, E2 the signal peptide and a part of the propiece (i.e. the part of the prepeptide that is later cleaved off to get the mature peptide), E3 codes for the rest of the propiece and the mature peptide and the fourth exon codes for the 3'-UTR. β-defensins are expressed and excreted by neutrophils and epithelia cells lining various organs [18–20]. In chicken, the tissue-specific pattern seems to vary across the different defensin genes with some showing expression in a wide array of tissues (e.g. AvBD9 with found expression in 22 different tissues, ranging from brain to different intestines and testis [16]), whereas other seem to have more limited expression patterns (e.g. AvBD8 with expression only observed in the liver and gall-bladder [16]). For complete summary, see [16]. Tissue specific patterns might however be hard to fully understand, without experimental exposure to pathogens, as the genes as a group, seems to vary in terms of having constitutive or induced expression patterns [21–24]. Constitutively expressed AvBDs can potentially be observed in all tissues in which they are active in, whereas AvBDs with induced expression would only be observed when exposed by pathogens or other external triggers, thus possibly biasing comparisons of tissue specificity between different AvBDs.

Evolutionary studies have shown that the formation of defensin genes has been driven by ancient gene duplication events where, for example, all α-defensins are thought to have evolved by gene duplication after the bird-mammal split and the θ-defensins to have arisen in the primate lineage, originating from the α-defensins [17]. A comparison of β-defensin genes across distant species (i.e. birds and mammals) yields weak phylogenetic signals of species [17], suggesting that many of these genes have been duplicated and became defined in function long before the bird-mammal split. However, a comparison of more closely related mammal species has yielded evidence of more recent gene duplications [25].

By comparing the chicken genome with a more closely related species, the zebra finch, we investigated how β-defensin genes have been evolving since the G-P split in the late Cretaceous period, i.e. about 100 million years ago [26], in terms of duplication events, gene losses and rates of evolution. More specifically, we tested whether the gene cluster of β-defensins have been conserved across the ancestral lineages of the birds species, and the extent to which the avian β-defensin complex had acquired novel genes through duplications along the two ancestral lineages. We also tested whether duplicated zebra finch β-defensin genes where evolving according to different selection pressures compared to unduplicated genes and investigated tissue specific expression patterns of this gene cluster using a digital transcriptomic approach [27, 28]

Throughout the paper the nomenclature for avian β-defensins (AvBDs) proposed by Lynn et al. (2007) is used, where avian β-defensins (also known as gallinacins) found in the chicken formally named Gall1-14 have been renamed to AvBD1-14 [15]. Genes found in the zebra finch that have ortholog chicken defensins are numbered accordingly but with z as a suffix in order to tell them apart and newly found genes without an orthologous chicken gene named with a number greater than 100.

The zebra finch genome contains at least 22 different genes coding for β-defensins. All 22 genes cluster within 125 Kbp on the 3^rd chromosome (figure 1, table 1). Ten of these have an ortholog gene in the chicken genome. In these cases the zebra finch genes were grouped together, with a "sister gene" from the chicken genome, with a posterior probability > 95% (Baysian phylogeny) or a bootstrap value > 95 (neighbour-joining tree) (figure 2 and 3). Further support of the relationship between the genes can be found when investigating the gene location and order of the two sets of genes (figure 1) where the 10 orthologous genes have kept their orientation and relative location in comparison to each other on the chromosome. This suggests that these genes have been duplicated long before the G-P split and have thereafter been conserved along the ancestral lineages of the two different bird species. In the case of AvBD6 found in chicken, it seems to have originated by a duplication of AvBD7 after the G-P split (figure 2 and 3), whereas the AvBD14 seem to have been lost in the ancestral line of the zebra finch. The remaining 12 genes found in the zebra finch genome stem from two ancestral genes, likely AvBD1 and AvBD3 based on the phylogenetic relationship, which correspond to the physical location of the genes on the chromosome (figure 1, 2 and 3). The 12 unique zebra finch genes were all found within a cluster located between gene AvBD2 and AvBD5, the same location where AvBD1 and AvBD3 are located in the chicken genome. The gene cluster can be divided into two separate groups, one consisting of AvBD123-AvBD125 (referred to as cluster A) that clustered together with AvBD1, and where all three genes shared identical signal peptide (figure 2, 3 and 4). The second cluster (cluster B) consists of 9 genes located next to each other (AvBD115-122) on the chromosome (figure 1) and clustered together in the phylogeny (figure 2, 3 and 4); these probably share an ancestral origin with AvBD3. Five of the genes in cluster B share one type of signal peptide, whereas two others (AvBD121 and 122) share a signal peptide that differs by one amino acid substitution (figure 2, 3 and 4). All other β-defensin genes found in the zebra finch genome had unique signal peptides. Within cluster B two sets of genes are likely to be the product of very recent gene duplication events. The genes AvDB117α and β share identical amino acid composition for signal and mature peptide and differ only at the nucleotide level (including the signal and mature peptide sequences), with one synonymous substitution in the signal peptide and an additional 21 SNP when including the intron, resulting in a 3.6% difference along the full gene. The other case is AvBD121 and 122 that differs at three different amino acid positions in the mature peptide (figure 4) and has three synonymous substitutions, two of which are in the signal peptide. We can't exclude that the case of AvBD117α and β is a misassemble of two different alleles or a case of gene copy variation, as seen in human β-defensins [29]. However, that they do not map next to each other (figure 1) together with the fairly diverged intron reduces that chance. It would therefore be of importance for future study to investigate the occurrence of gene-copy variation, not only for AvBD117 but also for the gene group as a whole.

A-defensin	Length chicken	Length zebra finch	GeneBank nr chicken	GeneBank nr zebra finch	Net charge ph 7.0	Isoelectric point	Average hydrophility
AvBD1	700		NM_204993		7.7	9.9	0.1
AvBD 123		544		BK006977	5.8	9.2	0.1
AvBD124		543		BK006978	0.7	7.6	0
AvBD125		545		BK006979	5.7	9.2	0.1
AvBD2c	300	459	NM_204992		3.9	8.6	-0.3
AvBD2z		459		BK006967	4.9	8.9	-0.1
AvBD3	1200		NM_204650		5.8	9.4	-0.2
AvBD115		254		BK006966	5.9	9.2	0
AvBD116		449		BK006965	6.7	9.9	0.3
AvBD117α		611		BK006961	5.7	9.4	-0.1
AvBD117β		611		BK006962	5.7	9.4	-0.1
AvBD118		603		BK006963	7.7	10	0.1
AvBD119		641		BK006964	5.8	9.3	-0.2
AvBD120		689		BK006981	7.7	11	0.3
AvBD121		706		BK006975	5.8	9.4	-0.2
AvBD122		710		BK006976	7.7	10.6	0
AvBD4c	1350		NM_001001610		5.8	9.1	-0.3
AvBD4z		1944		BK006982	6.8	9.4	-0.1
AvBD5c	600		NM_001001608		1.8	8	0.3
AvBD5z		783		BK006969	0.8	7.6	0.3
AvBD6c	1650	-	NM_001001193		5.8	9.2	-0.6
AvBD7c	400		NM_001001194		4.7	8.9	-0.2
AvBD7z		268		BK006970	5.7	9.2	-0.3
AvBD8c	500		NM_001001781		1.1	7.7	0.1
AvBD8z		700		BK006968	2.8	8.3	0.2
AvBD9c	1800		NM_001001611		3.8	8.6	0
AvBD9z		2544		BK006972	3.7	8.6	0
AvBD10c	500		NM_001001609		1.8	8	-0.2
AvBD10z		440		BK006971	1.8	8	0
AvBD11c	1200		NM_001001779		4.8	8.9	0
AvBD11z		1095		BK006973	3.9	8.6	0
AvBD12c	700		NM_001001607		-0.2	6.8	0
AvBD12z		731		BK006980	-1.1	6	-0.3
AvBD13c	4700		NM_001001780		4	8.6	0
AvBD13z		1919		BK006974	4.9	8.9	0.5
AvBD14		-	AM 402954		6.7	9.6	0.1

Lengths correspond to the start of the signal peptide to the end of the mature peptide.

The d_N/d_S ratio (ω) calculated pairwise over all obtained genes together with chicken defensins showed evidence of purifying selection, i.e. values of ω < 1 (mean dS = 1.59 S.E. = 0.03, mean dN = 0.54, S.E. = 0.01, mean ω = 0.67 S.E. = 0.01, figure 5). However, looking at the distribution of the ω values in Figure 5 together with having a very deep phylogenetic tree (figure 2) indicate that the faster evolving non-synonymous sites have become saturated, thus making comparisons between paralog genes in the tree biased. For the following analysis we therefore only used pairwise comparisons between ortholog genes (i.e. for the same gene but between species), or comparisons between recently duplicated genes after the G-P split.

Pairwise comparisons between AvBD1/AvBD3 and its duplicated homolog genes in the zebra finch genome showed higher values of ω than comparisons between orthologous chicken - zebra finch genes that had not undergone duplication (mean ω = 0.53 (S.E. = 0.04) vs. mean ω = 0.3 (S.E. = 0.05), Mann-Whitney U-test, Z = -3.26, P < 0.001, Monte Carlo, test P < 0.001, Figure 6a). Testing the rate between more recent and phylogenetically more robust clusters of duplicated genes in the zebra finch genome in relation to the rate of evolution of the unduplicated orthologous genes revealed an even bigger relaxation of the selection pressure of the duplicated genes (mean ω = 1.0 (S.E. = 0.08) vs. mean ω = 0.3 (S.E. = 0.05), Mann-Whitney U-test, Z = -4.6, P < 0.0001, Monte Carlo, test P < 0.001, Figure 6b). The mean value of ω for the duplicated genes was close to unity, indicating neutrality, although several cases are observed with ω > 1 (Figure 6b). One reason for neutral evolution to occur may be if the genes have lost their function after the duplication events and thus any selective forces acting upon it. However, if only a few amino acid positions are responsible for the differences in antimicrobial properties of the peptide, positive selection acting upon these specific sites might be might be masked by negative selection acting on other sites that keep the structure of the peptide.

Several of the genes included in the β-defensin cluster of the chicken have been highly conserved both in the zebra finch and in the chicken since they split around 100 million years ago. However, new avian β-defensin genes have been acquired through duplications along the two ancestral lineages, especially in the zebra finch genome. After the duplications, the new genes have evolved under relaxed purifying selection (although some amino acids show evidence of positive selection) compared to the non-duplicated genes.

The zebra finch genome contains a cluster of 22 β-defensin genes, of which ten were found to have orthologous genes in the chicken genome, originating from ancient gene duplications before the G-P split. The remaining 12 genes have evolved by a series of more recent gene duplication events. The long branches and low basal resolution in the phylogenies (figure 2 and 3) suggests that the different orthologous genes have occurred by ancient gene duplication events and then been conserved along the evolutionary lineages of the chicken and the zebra finch, i.e., in the region of 100 million years. That β-defensin genes arose by ancient gene duplications has been suggested by earlier studies [5, 17, 25]. The functions of the avian β-defensins in chicken are increasingly well understood, both in terms of where in the body they are being expressed but also how effective they are against different pathogens (for summary see [16, 33]). The overall patterns of tissue specific expression of zebra finch AvBDs do coincide to large extent to that found in chicken (for detailed comparison see van Dijk 2008 [16] and table 2). Similar to chicken, AvBD2, 8, 9, and 10 was expressed in the liver, AvBD9 and 10 in testis and skin, AvBD2 and9 in spleen. The discrepancies are AvBD2 and 13 that in the zebra finch that also was found to be expressed in the skin, AvBD7 and 10 was additionally found in the spleen (table 2).

Within a small region of 56 Kbp in the zebra finch genome a series of more recent gene duplications have occurred. Within this short physical distance on chromosome 3, there are two AvDB genes in the chicken genome. These two genes have undergone several duplication events and can now comprise 12 different AvBD genes in the zebra finch genome. That these duplicated genes have not lost their function is indicated by several factors.

Firstly, our data on expressed genes together with a similar study on expressed genes in the brain of zebra finch [26] identify four or possible five of the duplicated genes (with two and three expressed genes from each of the duplication clusters). In this study, we identified the genes AvBD123 (expressed in spleen) and 125 (expressed in the testis) that are a result of a duplication event (Table 2). The third expressed gene belonging to duplication cluster identified in this study was AvBD115 (also with expression in the spleen). AvBD115 was together with AvBD117α/β also found to be expressed in the separate study of [26]. In their study the expressed sequence tag, FE7232851, was identical with AvBD115 and ESTs, DV954612.1 and FE728335.1, are likely alleles of AvBD 117α/β as it only differed by three SNPs, two of which have caused nonsynonomous substitutions at position 34 and 56 (figure 4) changing Threonine (T) to Serine (S, both amino acids with uncharged polar R groups) and FE728335.1 had an additional SNP causing a nonsynonomous substitution on position 41 changing an Phenylalanine (F) to Tyrosine (Y). It should be noted that a blast search of the EST against the zebra finch genome yielded AvDB 117α and β as the closest genes. A possible reason that some of the identified genes were not found to be expressed in the investigated zebra finches could be due to tissue specific expression in organs not investigated by us. In fact, in chickens several of the β-defensins are expressed in tissues not evaluated in our study. These are manly tissues associated with internal organs such as bursa, the intestines, lungs but also bone marrow and leucocytes [16]. Further, if no infection or immune response associated with the investigated genes have been initiated then we would not expect to see the gene being expressed.

Secondly, several of the genes share identical signal peptides, although the mature peptides have evolved new amino acid sequences (figure 2, 3 and 4). Thirdly, within cluster B the amino acids under positive selection (Table 3) are similar to the sites known to be under selection in the functional chicken AvBD genes (Table 3 and figure 2 and 3, [34]).

Several models have been put forward about how new genes and gene functions might arise through gene duplication. Initial models (different versions of the neo-functionalization model) [35, 36] suggested that after duplication a gene is free from selection pressure and can thereby accumulate mutations that could lead to new functions. However, the main problem with these models is that they do not explain why the original (neutral) duplication is not lost by drift in the population [37, 38]. If the original gene have evolved to conduct more than one function then the two functions can be divided between the two new genes thus escaping adaptive conflicts between the two functions (sub-functionalization models) [35, 39, 40]. Even here, the duplicated genes are released from selection until one of the functions has been lost, although this time might be shorter than in the neo-functionalization model, as in this case mutations have to cause losses of a function, instead of gaining new specialised ones [37]. However, a model (the innovation, amplification, duplication, IAD model), that maintains duplicated genes under continuous selection has recently been proposed by Bergthorsson and co-workers [37]. First, innovation, in which the original gene product has both a primary function and a secondary function which have neither deleterious nor beneficial properties. With a change in ecological niche, or for immune genes, the encounter of a new pathogen fauna, the secondary function becomes beneficial and an increase of the activity is selected for. In the second step, amplification, an increase of the secondary activity is achieved by gene duplication and thus selected for. Lastly, divergence, being freed from its original function the duplicated gene can now evolve improvements in what previously was a secondary function, thereby diverging from its original gene. In the present dataset there are several indications that the IAD model might be able to explain how the AvBD genes have duplicated and diverged in the zebra finch genome, as discussed below.

Individual AvBD genes have been shown to have activity against several different groups of pathogens [16, 41]. The effectiveness against the different groups of pathogens might, however, differ between the different AvBD genes [41–43] thus depending on the environment having primary and secondary function against different pathogens. In two cases in the zebra finch genome very recent events of gene duplication can be observed, in one case the mature peptides are identical (i.e. AvBD117 α and β) and in the second case (AvBD121 and 122) the mature peptide is highly similar. If having multiple copies of an AvBD gene increases the gene expression, as found when investigating gene-copy variation of human β-defensins [29]. Then the effectiveness against certain pathogens might increases simply due to an increase in dosage, as seen in vitro [41]. Of interest would be to investigate whether the expression of the AvBD117 α and β is associated with an increased expression, in relation to other single copy genes. Once duplicated, the genes should start to diverge in order to be optimized for the function that was formerly only a secondary function. In the cluster of duplicated genes in the zebra finch genome selection has been relaxed (Figure 6a and 6b), compared to the genes that have not been duplicated. It should also be mentioned that in the case of the more recent duplication event in the chicken genome (AvBD6 and 7, figure 2) strong positive selection has acted upon the genes since the duplication event (pairwise comparison, ML, ω = 1.8). As defensins have been found to have broader functions such as signalling to the innate immune system [12, 13, 44], it might also be that the diversification of the duplicated genes in the zebra finch genome is associated with selection of other functions than direct disruption of pathogen cell membranes. In the chicken genome the expression patterns of the duplicated genes AvBD6 and 7 show similar tissue expression pattern [16], suggesting that they have retained similar functions and both have kept strong antimicrobial activity in vitro [16, 43].

It can not be excluded that our finding that AvBD14 has been lost somewhere along the zebra finch lineage is an artefact from a misassemble of the zebra finch genome. It may also be that our method for finding β-defensin genes was not general enough to find this gene. Furthermore, AvBD14 is yet to be annotated in the chicken genome (although the coding sequence for this gene was deposited in GenBank in 2006 there is still no publication describing the finding of this gene). However, an ortholog gene to AvBD 14 has been identified in the Turkey genome indicating that the chicken AvBD14 is not an assembly artefact (David Burt, pers. comm.)

We have shown here, together with previous studies on mammals and frogs, that antimicrobial peptides such as β-defensins are evolving through gene duplication and positive selection in vertebrates [45, 46]. This stand in contrast the counterpart of AMPs in the immune system in insects (i.e. Drosophila spp.) where studies several times failed to detect positive selection, although duplications seems to have been common [47–49]. As insects lack the adaptive immune system, selection might work on other mechanisms that regulate the gene expression instead of making the peptides more specific [49]. To fully understand the different evolutionary pressures imposed on the immune system of different organisms, we call for studies that examine not only the selective forces acting upon the peptide itself but also studies examining the selection on regulatory elements associated with the peptide expression.

Studying the evolution of immune genes is associated with additional challenges compared to other genes as they might lose functionality by not evolving as pathogens constantly are evolving in order to evade the function of the immune genes. However, as shown in this study, the steady increase in the availability of genomic data makes comparative studies possible in order to understand the underlying mechanisms of evolution and diversification of genes in the immune system.

The genomic comparisons of the β-defensins gene clusters of the chicken and zebra finch illuminate the evolutionary history of this gene complex. Along their ancestral lines several gene duplication events have occurred in the passerine line after the galliformes-passeriformes split giving rise to 12 novel genes compared to a single duplication event in the galliformes line. After the duplication events the duplicated genes been subject to a relaxed selection pressure, compared to the non-duplicated genes.