BACKGROUND
island colonizing species have long been an important target for evolutionary studies.
the transition into novel ecological environments sets the stage for ecological adaptation, and sometimes, speciation  <cit> .
on the other hand, the demographics of island colonization also pose challenges.
for example, island colonization may often involve only a small number of founders, in turn resulting in a loss of genetic variation and possibly inbreeding.
therefore, island populations may show reduced heterozygosity and divergent allele frequencies relative to source populations  <cit> .
over time, mating among this small pool of individuals may result in inbreeding depression, or reduced fitness from loss of genetic variation  <cit> .
the loss in genetic variation may have especially profound effects on the immune system where genetic diversity is thought to be particularly important for certain loci  <cit> .
reduced immunogenetic variation has been shown in island populations of mammals  <cit> , isolated populations of fish  <cit>  and bottlenecked and island populations of birds .
comparative studies of immune loci in island and mainland populations of birds, however, suggest that loss of immune function is not always the rule for island populations  <cit> .
if population bottlenecks are relatively mild, short in duration and if selection favors polymorphism, diversity at immune loci may be maintained following colonization  <cit> .
furthermore, in many cases an increased investment in the innate response is apparent, perhaps mitigating the consequences of reduced diversity in adaptive immune loci  <cit> .
a classic target of immune evolution studies has been the genes of the major histocompatibility complex   <cit> .
the mhc contains the most variable genes known in vertebrates, with hundreds of alleles in some species  <cit> .
mhc molecules play a very important role in the adaptive immune response.
these molecules bind and present peptides of degraded proteins on the surface of the cell to surveying t cells.
if these presented peptides are non-self, as in the case of pathogen derived peptides, the t cell will elicit an immune response based on the type of mhc molecule it recognizes  <cit> .
the high variability found in mhc molecules comes from the genes encoding the peptide-binding region , the site where peptides are loaded on to the molecule and presented on the cell surface.
this variability is believed to be driven by strong positive selection, which is thought to broaden the spectrum of pathogens a population can protect themselves against  <cit> .
thus, the “evolutionary arms race” between mhc and pathogens likely drives the high variation seen in mhc loci  <cit> .
much of the early work characterizing the mhc  <cit>  and assessing its immunological role  <cit>  in birds examined the domestic chicken .
the chicken was described to have a “minimal essential” mhc containing a single dominantly expressed mhc class i locus  <cit> .
recently, there have been a number of studies regarding mhc variation in non-model bird species, particularly in order passeriformes .
relative to chicken and other galliforms, passerines typically have more mhc genes overall, as well as longer introns and pseudogenes , which galliforms generally lack in the mhc.
this greater complexity found in passerines could be driven by strong pathogen-mediated balancing selection  <cit> .
among passerines, the zebra finch  has become an important model system for research in a diversity of fields, from ecology to neuroscience  <cit>  and was the second bird to have its genome sequenced  <cit> .
despite this, relatively little is known about patterns of genetic variation in zebra finch populations  <cit> .
two subspecies of zebra finch are found throughout australia  and the lesser sunda islands of southeast asia .
mayr  <cit>  originally hypothesized that the zebra finch colonized the lesser sunda islands from australia.
more recently, with the support of  <dig> genetic markers, balakrishnan & edwards  <cit>  showed that this colonization was associated with a severe genetic bottleneck that took place around one million years ago.
the two subspecies differ phenotypically, with timor finches being smaller and displaying reduced ornamentation  <cit> .
furthermore, these populations also differ at putatively neutral loci and differ significantly in the frequency of a large chromosomal inversion polymorphism  <cit> .
in addition to natural demographic changes in the wild, the zebra finch has also undergone multiple domestication events.
australian finches began the domestication process in the late 1800s  <cit>  and have since become a popular species of household pet, as well as a model species for neurobiology and song learning research  <cit> .
more recently, the timor subspecies has been brought into captivity.
the zebra finch is also the only passerine in which efforts have been made to reconstruct the structure and organization of the entire mhc region  <cit> .
despite this, little is known about mhc polymorphism in the zebra finch.
previous work on the zebra finch mhc has demonstrated that there are numerous mhc class ii loci, but only a single expressed mhc class i locus  <cit> .
in this study, we assess the role of historical bottlenecks on the variability of the functionally important mhc class i pbr in australian, timor and north american domesticated populations of zebra finches.
we also provide the first analysis of variation in the mitochondrial nadh dehydrogenase  <dig>  in the zebra finch as a comparative neutral marker.
in doing so, we not only continue the characterization of neutral genetic differences between the two wild zebra finch populations  <cit> , but also characterize potential differences in functional loci between the wild and domesticated populations that play such an important role in biological research.
methods
dna samples
twenty-nine dna samples from wild australian  birds were used in this study.
sixteen of these were previously analyzed by balakrishnan & edwards  <cit>  with field collection and permits described therein.
as in the previous study  <cit> , a subset of the samples from australia  were provided as purified dna by dave runciman .
tissue samples from balakrishnan and edwards  <cit>  are accessioned at the harvard university museum of comparative zoology and the academy of natural sciences at drexel university.
these wild australian birds were sampled at nine locations throughout the continent: queensland, selwyn range , longreach , brixton ; northern territory, alice springs ; western australia, fitzroy crossing , hamersley range , kununurra, ; victoria, wunghnu, .
we also used  <dig> dna samples from wild timor  birds sampled by runciman on the islands of west timor  and lombok .
we used three additional domesticated timor samples from the captive colony at the university of illinois at urbana-champaign .
due to the challenging logistics of fieldwork in the lesser sundas, and hence, small sample size, we lump wild and domesticated birds together for this subspecies.
we also sampled domesticated australian zebra finches from four captive colonies in the united states.
we used four samples from the east carolina university  colony, two samples from the university of illinois at urbana-champaign ,  <dig> samples from the university of chicago  and  <dig> samples from the usgs national wildlife health center in madison, wi .
the samples  collected at ecu were approved by the east carolina university institutional animal care and use committee  under protocol d <dig>  these four birds were sampled as part of ongoing neurogenomic research under protocol d <dig> and were euthanized via decapitation.
tissues were snap frozen on dry ice and dna was extracted using qiagen dneasy kits following manufacturer’s protocols.
additional file  <dig> provides information regarding sample location and locus genotyped for each sample.
seventy-one samples were sequenced for mhc class i and  <dig> of  <dig> samples used in the mhc analysis were also sequenced for nd <dig> along with five additional samples.
not all samples were genotyped at both loci, in part to due to limited dna availability for some samples.
mhc class i genotyping
previous studies utilizing bac libraries, southern blot  <cit>  and rnaseq  <cit>  revealed that only a single mhc class i locus is expressed in zebra finches.
thus, we amplified a  <dig> bp section of exon  <dig> of the mhc class i α chain using polymerase chain reaction .
for each pcr reaction, we combined  <dig>  μl of genomic dna with  <dig>  μl molecular grade ddh2o,  <dig>  μl each of 10x mgcl <dig> buffer and dntps,  <dig>  μl each of  <dig> μm mhc  <dig>  forward  and reverse  primers, and  <dig>  μl taq dna polymerase.
primers were designed to flank exon  <dig> based on the zebra finch genome assembly   <cit>  and the target regions are highly conserved in passerines  <cit> .
each reaction took place under the following pcr conditions:  <dig> min at  <dig> °c for denaturation, then  <dig> cycles of  <dig> s at  <dig> °c,  <dig> s at  <dig> °c and  <dig> s at  <dig> °c.
following pcr, we separated products on a  <dig> % agarose gel to ensure proper amplification of the dna during pcr.
we then purified pcr products using an epoch gencatch pcr extraction kit  and prepared for big dye terminator sequencing reaction.
each big dye reaction contained  <dig>  μl of  <dig> μm mhc  <dig>  forward or reverse primer,  <dig>  μl 5x extender buffer,  <dig>  μl big dye,  <dig> μl h <dig> and  <dig>  μl purified dna.
this  <dig> μl reaction underwent the following protocol:  <dig> min at  <dig> °c, then  <dig> cycles of  <dig> s at  <dig> °c,  <dig> s at  <dig> °c and  <dig> min at  <dig> °c.
we then purified big dye reaction products using sephadex columns and sequenced using an abi  <dig> at the ecu genomics core facility.
nd <dig> genotyping
mitochondrial genes are useful markers in reconstructing population histories and comparing patterns of divergence in markers experiencing high levels of selection, such as the mhc  <cit> .
thus, we sequenced a portion of the mitochondrial nd <dig> gene to provide a complementary perspective to contrast with our mhc class i analysis as well as to compare with mitochondrial studies in other taxa.
following the same protocol listed above, altering only the pcr annealing temperature to  <dig> °c, we genotyped a  <dig> bp region of the mitochondrial nd <dig> gene using the forward l <dig>  and reverse h <dig>  primers  <cit> .
dna sequence analysis
we used geneious v <dig> .
<dig>  to view sequence data, assemble and align sequences, remove primers, call polymorphic sites and perform consensus sequence alignment for downstream analyses.
after trimming, we obtained a  <dig> bp mhc class i and  <dig> bp nd <dig> consensus sequence for each individual.
we determined both mhc haplotypes using the phase software  <cit>  implemented in dnasp v <dig>  <cit> , which reconstructs haplotypes based on population genetic data.
measures of nucleotide diversity , synonymous  and nonsynonymous  nucleotide diversity, haplotype diversity   <cit> ,  <dig> % hd confidence interval by coalescent simulation  <cit>  and population differentiation   <cit>  were also calculated in dnasp v <dig>  we chose to estimate kst  <cit>  rather than fst, because kst is particularly suitable for smaller sample sizes, and for loci with high polymorphism, such as the mhc  <cit> .
we used our haplotypes to construct a network in the program popart  <cit>  to show relationships among the individuals sampled from different populations.
the tcs algorithm was selected to make this network based on its implementation of statistical parsimony  <cit> .
we identified signatures of positive selection in our mhc class i sequences with the program omegamap  <cit>  by calculating ω, the ratio of non-synonymous substitutions  to synonymous substitutions , where ω  >  <dig> indicates positive selection.
omegamap requires prior distributions for each parameter and we selected, as suggested by the manual, the following for ω: omega prior = inverse  and omega model = independent, which allows a unique ω value for each codon.
we ran the simulation twice for  <dig>  generations and combined each run with the suggested burn in of  <dig>   iterations using the summarize module provided by the program.
we visualized results using r v <dig> .
<dig>  using the homology based approach implemented by promerová et al.
<cit>  based on wallny et al.
<cit>  and koch et al.
<cit> , putative pbr sites were determined by aligning genbank sequences from the duck  mhc   <cit>  and scarlet rosefinch  mhc class i exon  <dig>   <cit>  with these zebra finch sequences.
we were then able to determine whether sites showing signs of positive selection correspond to putative peptide binding residues.
bottleneck simulations
to test whether patterns of variation could be explained by genetic drift acting on our mhc class i sequences during colonization of the lesser sunda islands, we ran bottleneck simulations in bottlesim v <dig>   <cit> .
we used the observed mhc allele frequencies in the australian subspecies to examine reductions of allelic diversity under neutrality in bottlenecks of varying severity.
we performed simulations using the following parameters: average lifespan =  <dig> years, age to sexual maturity =  <dig> year, generation overlap =  <dig> % and the observed australian mhc class i allele frequencies.
we varied rates of population growth post-bottleneck to see how the bottleneck affected allelic diversity during island colonization.
each simulation ran for  <dig>  iterations with a bottleneck that reduced populations to either  <dig> or  <dig> individuals, from an initial population size of  <dig> .
a bottleneck event with 10– <dig> founders is consistent with previous estimates of colonization of the lesser sunda islands  <cit> .
post bottleneck populations were allowed to grow exponentially at  <dig>   <dig> and  <dig> % for 34– <dig> years.
simulations of short duration  were run in some cases because population sizes became too large, making the simulation too computationally intensive.
RESULTS
mhc polymorphism & divergence
we successfully sequenced a total of  <dig> australian,  <dig> timor and  <dig> domestic zebra finches for exon  <dig> of the mhc class i gene .
using the phase software  <cit>  implemented in dnasp v <dig>  <cit> ,  <dig> unique nucleotide and amino acid haplotypes were reconstructed among the three populations .
within each of these populations, nucleotide diversity  is relatively high: timor =  <dig> , wild australia =  <dig> , domesticated =  <dig>  .
we also observe a similar pattern of synonymous  and nonsynonymous  site nucleotide diversity .
within the timor population, πa is still high , indicating maintenance of nonsynonymous diversity .
this is also reflected in the amino acid sequence alignment .
similarly, haplotype diversity  is high in each of the three sampled populations, with the timor population exhibiting the lowest diversity .
despite this, timor mhc haplotype diversity is higher than  <dig> out of  <dig>  of previously reported neutral loci .
additionally, the lower limit of the  <dig> % confidence interval, as estimated by coalescent simulation in dnasp, exceeds hd in 28/ <dig>  of the neutral loci sampled in timor birds .table  <dig> summary statistics for the three zebra finch populations
number of unique haplotypes  for the total number of individuals sampled , haplotype diversity , overall nucleotide diversity , synonymous  and nonsynonymous  nucleotide diversity, π for neutral loci reported by balakrishnan & edwards  <cit>  and average mhc class i ω reported here
fig.
<dig> parsimony network created in popart showing relationships between the different mhc alleles and populations.
the size of the nodes is proportional to the number of individuals with that particular allele.
coloration of the nodes represents the frequency of each allele among the study populations.
black circles represent missing alleles
fig.
<dig> island and mainland nucleotide diversity  plotted next to each other for all reported loci.
anonymous loci , introns  and z linked loci  reported by balakrishnan & edwards  <cit>  as well as mhc class i and nd <dig> from the present study
although each population exhibits high overall levels of nucleotide and haplotype diversity, there is a clear pattern of population divergence .
as expected, pairwise kst values reveal that the highest degree of divergence is between wild australian and timor zebra finch subspecies, which diverged 1– <dig> million years ago .
domesticated and wild australian populations showed the lowest levels of divergence, but the two populations were nevertheless significantly differentiated .
these patterns can also be observed in the tcs network , as australian zebra finches share fewer alleles with the timor subspecies relative to domesticated birds.table  <dig> pairwise kst values showing genetic divergence between populations with values previously reported
p-value of kst
p-value of kst
a <cit>
b <cit>
summary statistics describing polymorphism and diversity are also reflected in the tcs  <cit>  based parsimony network .
the high haplotype diversity is emphasized by the fact that most haplotypes are only carried by a small number of individuals .
the significant genetic divergence between timor and australian finches is reflected in the limited haplotype sharing among subspecies .
only two haplotypes were shared among subspecies, and the subspecies are therefore not reciprocally monophyletic for mhc class i alleles.
also of note is that timor alleles are dispersed across multiple parts of the network, rather than clustering together in one area.
we found broad overlap between alleles carried by domestic and wild australian zebra finches .
there are however, numerous  alleles found in wild population but not represented in our captive sample.
likewise,  <dig> alleles found in captive birds were not detected in wild-derived samples.
this is further reflected by the large number of unique amino acid sequences found in each population as well as the significant genetic differentiation among captive and wild birds .
nd <dig> polymorphism & divergence
we successfully sequenced  <dig> australian,  <dig> domestic and  <dig> timor individuals for a  <dig> bp region of the nd <dig> mitochondrial gene .
compared with the mhc, as expected, nd <dig> nucleotide diversity  is relatively low: australia =  <dig> , domestic =  <dig>  and timor =  <dig>  .
although the timor population exhibits the greatest nd <dig> nucleotide diversity, timor nd <dig> hd is the lowest .
pairwise kst values show significant divergence between wild australia and timor allele frequencies  and slight, but statistically significant, divergence between wild and domesticated populations .
the relatively low levels of diversity and significant population divergence are reflected in the large number of shared alleles between australian and domestic samples and separation of australian and timor samples in our nd <dig> haplotype network .
furthermore, the timor samples further segregate into two distinct haplotype groups based on sampling locality .
seven mutational steps separate these two clusters.fig.
<dig> parsimony network created in popart showing relationships between the different nd <dig> alleles and populations.
the size of the nodes is proportional to the number of individuals with that particular allele.
coloration of the nodes represents the frequency of each allele among the study populations.
black circles represent missing alleles.
timor individuals are represented by sampling location
tests of selection of mhc nucleotides
selection analyses using omegamap  <cit>  revealed strong evidence for positive selection on exon  <dig> of the mhc class i locus.
the average value of ω averaged across all codon sites is much higher than one; the expectation for neutrality , and 17– <dig> sites show signatures of positive selection with significant posterior probabilities in each population .
alignment of our sequences with those of previously reported mhc class i of the duck  and scarlet rosefinch  allowed us to determine which of our sites were putative residues of the pbr and whether these sites exhibited signatures of positive selection.
our sequences contained  <dig> putative pbr residues, seven of which exhibited strong signatures of positive selection in each population .fig.
<dig> output from omegamap plotting ω  against codon position for the wild australian population.
the black lines represent omega and the grey shading represents  <dig> % highest posterior probability densities .
stars represent sites encoding the pbr, with yellow stars indicating ω >  <dig> and green stars indicating ω < 1
neutral expectations following population bottlenecks
our bottleneck simulations revealed that based on drift alone, the timor zebra finch should carry between four and  <dig> mhc alleles given a founding population of  <dig> individuals, and over  <dig> alleles given a founding population of  <dig> individuals .
under a predicted scenario based on previous coalescent-based estimates  <cit> , in which a dramatic bottleneck down to  <dig> individuals was followed by exponential growth rate of  <dig> %, we observed a reduction from  <dig> initial alleles on the mainland to  <dig>   alleles after island colonization.
increasing the growth rate to  <dig> % post-bottleneck reduced the number of alleles from  <dig> to  <dig>  .
lastly, a  <dig> % exponential growth rate following the founder event reduced the number of alleles to  <dig>  , very similar to our observed value of  <dig> alleles.
under a less severe bottleneck scenario of  <dig> individuals, simulated allelic diversity estimates far exceeded our observed values .fig.
<dig>
bottlesim output with  <dig>   <dig> and  <dig> % growth rate post-bottleneck of  <dig> individuals
discussion
polymorphism maintenance in the mhc
population bottlenecks are expected to result in reduced neutral genetic variation  <cit> .
this pattern has previously been shown in both insular and domesticated zebra finch populations  <cit> .
of  <dig> nuclear loci, including intronic regions, sex-linked and anonymous loci, balakrishnan & edwards  <cit>  found over half  to be invariant in timor zebra finches.
on average, timor zebra finches exhibit a  <dig> % drop in nucleotide diversity of neutral markers.
we have shown here that even in the presence of this genome wide reduction in variation, timor zebra finches have maintained a high level of diversity in the functionally important mhc .
the mhc class i locus sequenced here thus stands in marked contrast to previously sampled putatively neutral loci in zebra finches .
one possible explanation for the elevated mhc diversity relative to neutral markers is if selection has acted to maintain polymorphism.
a few of lines of evidence support this conclusion.
first, we find strong evidence of selection acting on this mhc locus, as evidenced by elevated dn/ds values   <cit> .
second, the observed nucleotide diversity of the mhc locus in timor finches is outside the range of nucleotide diversity observed in previously sequenced neutral loci  <cit> .
even though loci that are highly polymorphic in the australian subspecies tend to be more polymorphic in the timor subspecies, the mhc locus has maintained more nucleotide diversity than all putatively neutral, autosomal loci .
further, our bottleneck simulations revealed that under many scenarios, including those that align with previous estimates of population demography  <cit> , neutral divergence would result in a more severe reduction in mhc diversity than we observed .
given the unique biology of the mhc and the uncertainty of zebra finch historical demography, completely ruling out neutral explanations for the observed high level of mhc diversity, however, is challenging.
under some demographic scenarios, neutral expectations based on simulations closely approximate observed patterns of mhc diversity.
for example, if the bottleneck associated with island colonization was less severe than previously estimated  <cit> , or if population growth were relatively fast following island colonization, higher levels of mhc diversity would be expected  <cit> .
indeed, we obtain higher than observed levels of post-bottleneck mhc allelic diversity under a less severe bottleneck scenario suggesting that drift alone could maintain allelic diversity .
previous, estimates suggest that a small number of individuals  likely colonized the islands.
confidence intervals around this estimate, however, are broad , making colonization of the islands by over  <dig> founders possible.
the process of domestication is also expected to reduce genetic diversity, particularly if populations are founded using relatively small numbers of individuals from the wild, or if selective breeding has been strong.
forstmeier et al.
<cit>  found domesticated zebra finches to have slightly reduced genetic variation at  <dig> microsatellite loci and significant population differentiation when compared with wild zebra finches.
however, forstmeier et al.
also found that a relatively high level of neutral genetic polymorphism has been maintained through domestication  <cit> .
we find the same pattern in the mhc class i , albeit with a lower degree of differentiation .
the domestic birds utilized in this study were domesticated relatively recently, breed randomly  and encounter pathogens found in captivity.
therefore, it is not surprising to see high levels of mhc class i sequence and haplotype diversity as seen in other domesticated vertebrates  <cit> .
we found  <dig> alleles in domesticated birds not found in the wild.
these novel alleles may indicate adaptation to the pathogen community found in captivity , although further sampling along with functional testing is needed for verification.
high diversity in the mhc suggests positive selection and indeed, there are abundant nonsynonymous polymorphisms throughout these class i sequences .
our sequences contained  <dig> putative pbr sites, seven of which exhibited strong evidence of positive selection in each population .
the sites we report to be under positive selection have also been previously reported in another passerine, the scarlet rosefinch , that displays high levels of mhc class i variation  <cit> .
we also observe several sites experiencing positive selection outside of the putative pbr, potentially revealing additional sites important in peptide binding.
as most reported estimates of mhc nucleotide diversity are from species with multiple expressed mhc loci  <cit> , direct comparisons of diversity among birds, in particular among passerines, are complicated by inter-species variation in mhc copy number.
the mhc plays a vital role in antigen presentation to the immune system, so selection is expected to maintain diversity to broaden pathogen recognition capability  <cit> .
although there have been a number of studies correlating bottlenecked populations with reduction in mhc variation  <cit> , there have also been several studies showing the opposite, as balancing selection appears to counteract drift  <cit> .
the specific details of population demography play a critical role in determining the outcome following colonization  <cit> .
recent work has revealed that balancing selection mediated either by negative frequency dependent selection or heterozygote advantage can actually decrease rather than maintain mhc polymorphism after a bottleneck  <cit> .
given sufficient time and recovery after a bottleneck, however, balancing selection will again act to maintain polymorphism  <cit> .
likewise, gene conversion has also been shown to maintain mhc variation  <cit> .
lastly, differential pathogen pressures faced in each of these populations may contribute to the high diversity and divergence among populations  <cit> .
divergence between australian, island and domesticated populations
the two zebra finch subspecies appear to have been isolated since the early pleistocene or late pliocene  <cit> .
thus, our estimates of genetic differentiation in the mhc class i and nd <dig> are consistent with the current understanding of zebra finch population history  <cit> .
this prolonged history of isolation of subspecies is also reflected in mhc class i haplotype diversity, as only two of  <dig> mhc class i haplotypes are shared between subspecies .
network analysis of mhc class i alleles shows that the timor alleles are distributed across multiple parts of the network, indicating that multiple colonizers carrying divergent alleles founded the island population.
kst estimates between subspecies for mhc class i are lower than from neutral markers.
this pattern, however, is expected, as measures of population divergence are necessarily lower for highly polymorphic markers  <cit> .
balancing selection could also contribute to this reduced kst  <cit> .
to provide a more complete picture of population genetic variation in this model system, we sequenced a portion of the mitochondrial nd <dig> gene.
mitochondrial genes have long been used as markers of population history  <cit> , and thus provide a point of contrast for loci under selection like the mhc  <cit> .
despite the utility of mitochondrial markers as a benchmark for taxonomic delineation and history, there has not previously been a mitochondrial dna  survey in zebra finches.
we found that zebra finch subspecies were reciprocally monophyletic for mtdna alleles , making this the first genetic marker to show monophyly of zebra finch subspecies  <cit> .
accordingly, there is highly significant population divergence between the subspecies .
nd <dig> polymorphism in zebra finches is comparable to previous reports of nd <dig> nucleotide diversity in other passerines  .
interestingly, nd <dig> nucleotide variation is twice as high in the timor subspecies  as in the australian subspecies  .
this high nucleotide diversity on the islands is a consequence of the existence of two divergent mtdna haplotype groups  in our sample of  <dig> birds.
each node in our timor network represents a distinct sampling locality  with the captive samples separated by one mutation from the wild timor samples.
the high divergence between samples from west timor and lombok islands suggests long-term isolation of those populations, but more detailed sampling throughout the timor subspecies range is still needed to formally test this.
zebra finch populations were domesticated in the 1800s.
as a result, domestic and wild populations have relatively low, but statistically significant, divergence in mhc class i allele frequencies .
these two populations also show statistically significant differences in nd <dig> allele frequencies .
together these finding provide evidence of genetic distinctiveness of wild populations in australia and domesticated populations in the usa.
CONCLUSIONS
domestication and artificial selection have important and predictable consequences .
however, we find high polymorphism despite a bottleneck in the colonization of the lesser sunda islands, and like forstmeier et al.
<cit> , we find high levels of nucleotide diversity in captive zebra finches as well.
we also found significant differences in mhc allele frequencies between zebra finch subspecies and domesticated populations, indicating potential immune adaptation to the pathogen pressures faced by each population  <cit>  or the impact of genetic drift.
although the discordance between neutral and mhc polymorphism is striking, improved resolution of historical demography is required in order to determine whether this disparity is driven by selection.
zebra finches are a model system in a diversity of fields.
genetic divergence in functional loci, such as that shown here for the mhc, may have important implications for research in the fields for which zebra finch is a model system.
for example there is already evidence that domesticated and wild zebra finches differ in aspects of social behavior  <cit> .
comparisons of these populations in terms immunological or gene expression responses to immune challenges will provide insight into the functional consequences of the mhc class i variability we report here.
availability of supporting data
all sequences used in this study have been deposited in genbank under accession numbers kt595736-kt <dig> for mhc class i and kt595878-kt <dig> for nd <dig>
additional files
additional file 1:
sample name, origin and locus genotyped for each sample used in this study.
additional file 2:
amino acid alignment of unique mhc class i sequences from each population.
for each unique allele, the first number represents allele id and the second number represents the total number of individuals in the population carrying the allele.
additional file 3:
island and mainland haplotype diversity  plotted next to each other for all reported loci.
anonymous loci , introns  and z linked loci  reported by balakrishnan & edwards  <cit>  as well as mhc class i and nd <dig> from the present study.
additional file 4:
output from
omegamap
plotting posterior probability of positive selection against codon position for the wild australian population.
stars represent sites encoding the pbr, with yellow stars indicating ω >  <dig> and green stars indicating ω <  <dig>
additional file 5:
output from
omegamap
plotting ω against codon position for the domesticated population.
the black lines represent omega and the grey shading represents  <dig> % highest posterior probability densities .
stars represent sites encoding the pbr, with yellow stars indicating ω >  <dig> and green stars indicating ω <  <dig>
additional file 6:
output from
omegamap
plotting posterior probability of positive selection against codon position for the domesticated population.
stars represent sites encoding the pbr, with yellow stars indicating ω >  <dig> and green stars indicating ω <  <dig>
additional file 7:
output from
omegamap
plotting ω against codon position for the timor population.
the black lines represent omega and the grey shading represents  <dig> % highest posterior probability densities .
stars represent sites encoding the pbr, with yellow stars indicating ω >  <dig> and green stars indicating ω <  <dig>
additional file 8:
output from
omegamap
plotting posterior probability of positive selection against codon position for the timor population.
stars represent sites encoding the pbr, with yellow stars indicating ω >  <dig> and green stars indicating ω <  <dig>
additional file 9:
bottlesim
output showing the final number of alleles  at  <dig>   <dig> and  <dig> % post-bottleneck growth rate for a bottleneck size of  <dig> and  <dig> individuals.
abbreviations
mhcmajor histocompatability complex
nd2nadh dehydrogenase 2
pbrpeptide-binding region
hdhaplotype diversity
mtdnamitochondrial dna
competing interests
the authors declare no competing interests.
authors’ contributions
cnb designed the study and provided samples.
djn performed laboratory methods and analyzed results.
djn and cnb drafted the manuscript.
both authors read and approved the final manuscript.

 island colonizing species have long been an important target for evolutionary studies. the transition into novel ecological environments sets the stage for ecological adaptation, and sometimes, speciation. on the other hand, the demographics of island colonization also pose challenges. for example, island colonization may often involve only a small number of founders, in turn resulting in a loss of genetic variation and possibly inbreeding. therefore, island populations may show reduced heterozygosity and divergent allele frequencies relative to source populations. over time, mating among this small pool of individuals may result in inbreeding depression, or reduced fitness from loss of genetic variation. the loss in genetic variation may have especially profound effects on the immune system where genetic diversity is thought to be particularly important for certain loci. reduced immunogenetic variation has been shown in island populations of mammals, isolated populations of fish and bottlenecked and island populations of birds. comparative studies of immune loci in island and mainland populations of birds, however, suggest that loss of immune function is not always the rule for island populations. if population bottlenecks are relatively mild, short in duration and if selection favors polymorphism, diversity at immune loci may be maintained following colonization. furthermore, in many cases an increased investment in the innate response is apparent, perhaps mitigating the consequences of reduced diversity in adaptive immune loci. a classic target of immune evolution studies has been the genes of the major histocompatibility complex. the mhc contains the most variable genes known in vertebrates, with hundreds of alleles in some species. mhc molecules play a very important role in the adaptive immune response. these molecules bind and present peptides of degraded proteins on the surface of the cell to surveying t cells. if these presented peptides are non-self, as in the case of pathogen derived peptides, the t cell will elicit an immune response based on the type of mhc molecule it recognizes. the high variability found in mhc molecules comes from the genes encoding the peptide-binding region, the site where peptides are loaded on to the molecule and presented on the cell surface. this variability is believed to be driven by strong positive selection, which is thought to broaden the spectrum of pathogens a population can protect themselves against. thus, the “evolutionary arms race” between mhc and pathogens likely drives the high variation seen in mhc loci. much of the early work characterizing the mhc and assessing its immunological role in birds examined the domestic chicken. the chicken was described to have a “minimal essential” mhc containing a single dominantly expressed mhc class i locus. recently, there have been a number of studies regarding mhc variation in non-model bird species, particularly in order passeriformes. relative to chicken and other galliforms, passerines typically have more mhc genes overall, as well as longer introns and pseudogenes, which galliforms generally lack in the mhc. this greater complexity found in passerines could be driven by strong pathogen-mediated balancing selection. among passerines, the zebra finch has become an important model system for research in a diversity of fields, from ecology to neuroscience and was the second bird to have its genome sequenced. despite this, relatively little is known about patterns of genetic variation in zebra finch populations. two subspecies of zebra finch are found throughout australia and the lesser sunda islands of southeast asia. mayr originally hypothesized that the zebra finch colonized the lesser sunda islands from australia. more recently, with the support of genetic markers, balakrishnan & edwards showed that this colonization was associated with a severe genetic bottleneck that took place around one million years ago. the two subspecies differ phenotypically, with timor finches being smaller and displaying reduced ornamentation. furthermore, these populations also differ at putatively neutral loci and differ significantly in the frequency of a large chromosomal inversion polymorphism. in addition to natural demographic changes in the wild, the zebra finch has also undergone multiple domestication events. australian finches began the domestication process in the late 1800s and have since become a popular species of household pet, as well as a model species for neurobiology and song learning research. more recently, the timor subspecies has been brought into captivity. the zebra finch is also the only passerine in which efforts have been made to reconstruct the structure and organization of the entire mhc region. despite this, little is known about mhc polymorphism in the zebra finch. previous work on the zebra finch mhc has demonstrated that there are numerous mhc class ii loci, but only a single expressed mhc class i locus. in this study, we assess the role of historical bottlenecks on the variability of the functionally important mhc class i pbr in australian, timor and north american domesticated populations of zebra finches. we also provide the first analysis of variation in the mitochondrial nadh dehydrogenase in the zebra finch as a comparative neutral marker. in doing so, we not only continue the characterization of neutral genetic differences between the two wild zebra finch populations, but also characterize potential differences in functional loci between the wild and domesticated populations that play such an important role in biological research. methods dna samples twenty-nine dna samples from wild australian birds were used in this study. sixteen of these were previously analyzed by balakrishnan & edwards with field collection and permits described therein. as in the previous study, a subset of the samples from australia were provided as purified dna by dave runciman. tissue samples from balakrishnan and edwards are accessioned at the harvard university museum of comparative zoology and the academy of natural sciences at drexel university. these wild australian birds were sampled at nine locations throughout the continent: queensland, selwyn range, longreach, brixton; northern territory, alice springs; western australia, fitzroy crossing, hamersley range, kununurra,; victoria, wunghnu,. we also used dna samples from wild timor birds sampled by runciman on the islands of west timor and lombok. we used three additional domesticated timor samples from the captive colony at the university of illinois at urbana-champaign. due to the challenging logistics of fieldwork in the lesser sundas, and hence, small sample size, we lump wild and domesticated birds together for this subspecies. we also sampled domesticated australian zebra finches from four captive colonies in the united states. we used four samples from the east carolina university colony, two samples from the university of illinois at urbana-champaign, samples from the university of chicago and samples from the usgs national wildlife health center in madison, wi. the samples collected at ecu were approved by the east carolina university institutional animal care and use committee under protocol d these four birds were sampled as part of ongoing neurogenomic research under protocol d and were euthanized via decapitation. tissues were snap frozen on dry ice and dna was extracted using qiagen dneasy kits following manufacturer’s protocols. additional file provides information regarding sample location and locus genotyped for each sample. seventy-one samples were sequenced for mhc class i and of samples used in the mhc analysis were also sequenced for nd along with five additional samples. not all samples were genotyped at both loci, in part to due to limited dna availability for some samples. mhc class i genotyping previous studies utilizing bac libraries, southern blot and rnaseq revealed that only a single mhc class i locus is expressed in zebra finches. thus, we amplified a bp section of exon of the mhc class i α chain using polymerase chain reaction. for each pcr reaction, we combined μl of genomic dna with μl molecular grade ddh2o, μl each of 10x mgcl buffer and dntps, μl each of μm mhc forward and reverse primers, and μl taq dna polymerase. primers were designed to flank exon based on the zebra finch genome assembly and the target regions are highly conserved in passerines. each reaction took place under the following pcr conditions: min at °c for denaturation, then cycles of s at °c, s at °c and s at °c. following pcr, we separated products on a % agarose gel to ensure proper amplification of the dna during pcr. we then purified pcr products using an epoch gencatch pcr extraction kit and prepared for big dye terminator sequencing reaction. each big dye reaction contained μl of μm mhc forward or reverse primer, μl 5x extender buffer, μl big dye, μl h and μl purified dna. this μl reaction underwent the following protocol: min at °c, then cycles of s at °c, s at °c and min at °c. we then purified big dye reaction products using sephadex columns and sequenced using an abi at the ecu genomics core facility. nd genotyping mitochondrial genes are useful markers in reconstructing population histories and comparing patterns of divergence in markers experiencing high levels of selection, such as the mhc. thus, we sequenced a portion of the mitochondrial nd gene to provide a complementary perspective to contrast with our mhc class i analysis as well as to compare with mitochondrial studies in other taxa. following the same protocol listed above, altering only the pcr annealing temperature to °c, we genotyped a bp region of the mitochondrial nd gene using the forward l and reverse h primers. dna sequence analysis we used geneious v. to view sequence data, assemble and align sequences, remove primers, call polymorphic sites and perform consensus sequence alignment for downstream analyses. after trimming, we obtained a bp mhc class i and bp nd consensus sequence for each individual. we determined both mhc haplotypes using the phase software implemented in dnasp v, which reconstructs haplotypes based on population genetic data. measures of nucleotide diversity, synonymous and nonsynonymous nucleotide diversity, haplotype diversity, % hd confidence interval by coalescent simulation and population differentiation were also calculated in dnasp v we chose to estimate kst rather than fst, because kst is particularly suitable for smaller sample sizes, and for loci with high polymorphism, such as the mhc. we used our haplotypes to construct a network in the program popart to show relationships among the individuals sampled from different populations. the tcs algorithm was selected to make this network based on its implementation of statistical parsimony. we identified signatures of positive selection in our mhc class i sequences with the program omegamap by calculating ω, the ratio of non-synonymous substitutions to synonymous substitutions, where ω > indicates positive selection. omegamap requires prior distributions for each parameter and we selected, as suggested by the manual, the following for ω: omega prior = inverse and omega model = independent, which allows a unique ω value for each codon. we ran the simulation twice for generations and combined each run with the suggested burn in of iterations using the summarize module provided by the program. we visualized results using r v. using the homology based approach implemented by promerová et al. based on wallny et al. and koch et al., putative pbr sites were determined by aligning genbank sequences from the duck mhc and scarlet rosefinch mhc class i exon with these zebra finch sequences. we were then able to determine whether sites showing signs of positive selection correspond to putative peptide binding residues. bottleneck simulations to test whether patterns of variation could be explained by genetic drift acting on our mhc class i sequences during colonization of the lesser sunda islands, we ran bottleneck simulations in bottlesim v. we used the observed mhc allele frequencies in the australian subspecies to examine reductions of allelic diversity under neutrality in bottlenecks of varying severity. we performed simulations using the following parameters: average lifespan = years, age to sexual maturity = year, generation overlap = % and the observed australian mhc class i allele frequencies. we varied rates of population growth post-bottleneck to see how the bottleneck affected allelic diversity during island colonization. each simulation ran for iterations with a bottleneck that reduced populations to either or individuals, from an initial population size of. a bottleneck event with 10– founders is consistent with previous estimates of colonization of the lesser sunda islands. post bottleneck populations were allowed to grow exponentially at and % for 34– years. simulations of short duration were run in some cases because population sizes became too large, making the simulation too computationally intensive.  mhc polymorphism & divergence we successfully sequenced a total of australian, timor and domestic zebra finches for exon of the mhc class i gene. using the phase software implemented in dnasp v, unique nucleotide and amino acid haplotypes were reconstructed among the three populations. within each of these populations, nucleotide diversity is relatively high: timor =, wild australia =, domesticated =. we also observe a similar pattern of synonymous and nonsynonymous site nucleotide diversity. within the timor population, πa is still high, indicating maintenance of nonsynonymous diversity. this is also reflected in the amino acid sequence alignment. similarly, haplotype diversity  is high in each of the three sampled populations, with the timor population exhibiting the lowest diversity. despite this, timor mhc haplotype diversity is higher than out of of previously reported neutral loci. additionally, the lower limit of the % confidence interval, as estimated by coalescent simulation in dnasp, exceeds hd in 28/ of the neutral loci sampled in timor birds.table summary statistics for the three zebra finch populations number of unique haplotypes for the total number of individuals sampled, haplotype diversity, overall nucleotide diversity, synonymous and nonsynonymous nucleotide diversity, π for neutral loci reported by balakrishnan & edwards and average mhc class i ω reported here fig. parsimony network created in popart showing relationships between the different mhc alleles and populations. the size of the nodes is proportional to the number of individuals with that particular allele. coloration of the nodes represents the frequency of each allele among the study populations. black circles represent missing alleles fig. island and mainland nucleotide diversity plotted next to each other for all reported loci. anonymous loci, introns and z linked loci reported by balakrishnan & edwards as well as mhc class i and nd from the present study although each population exhibits high overall levels of nucleotide and haplotype diversity, there is a clear pattern of population divergence. as expected, pairwise kst values reveal that the highest degree of divergence is between wild australian and timor zebra finch subspecies, which diverged 1– million years ago. domesticated and wild australian populations showed the lowest levels of divergence, but the two populations were nevertheless significantly differentiated. these patterns can also be observed in the tcs network, as australian zebra finches share fewer alleles with the timor subspecies relative to domesticated birds.table pairwise kst values showing genetic divergence between populations with values previously reported p-value of kst p-value of kst a b summary statistics describing polymorphism and diversity are also reflected in the tcs based parsimony network. the high haplotype diversity is emphasized by the fact that most haplotypes are only carried by a small number of individuals. the significant genetic divergence between timor and australian finches is reflected in the limited haplotype sharing among subspecies. only two haplotypes were shared among subspecies, and the subspecies are therefore not reciprocally monophyletic for mhc class i alleles. also of note is that timor alleles are dispersed across multiple parts of the network, rather than clustering together in one area. we found broad overlap between alleles carried by domestic and wild australian zebra finches. there are however, numerous alleles found in wild population but not represented in our captive sample. likewise, alleles found in captive birds were not detected in wild-derived samples. this is further reflected by the large number of unique amino acid sequences found in each population as well as the significant genetic differentiation among captive and wild birds. nd polymorphism & divergence we successfully sequenced australian, domestic and timor individuals for a bp region of the nd mitochondrial gene. compared with the mhc, as expected, nd nucleotide diversity is relatively low: australia =, domestic = and timor =. although the timor population exhibits the greatest nd nucleotide diversity, timor nd hd is the lowest. pairwise kst values show significant divergence between wild australia and timor allele frequencies and slight, but statistically significant, divergence between wild and domesticated populations. the relatively low levels of diversity and significant population divergence are reflected in the large number of shared alleles between australian and domestic samples and separation of australian and timor samples in our nd haplotype network. furthermore, the timor samples further segregate into two distinct haplotype groups based on sampling locality. seven mutational steps separate these two clusters.fig. parsimony network created in popart showing relationships between the different nd alleles and populations. the size of the nodes is proportional to the number of individuals with that particular allele. coloration of the nodes represents the frequency of each allele among the study populations. black circles represent missing alleles. timor individuals are represented by sampling location tests of selection of mhc nucleotides selection analyses using omegamap revealed strong evidence for positive selection on exon of the mhc class i locus. the average value of ω averaged across all codon sites is much higher than one; the expectation for neutrality, and 17– sites show signatures of positive selection with significant posterior probabilities in each population. alignment of our sequences with those of previously reported mhc class i of the duck and scarlet rosefinch allowed us to determine which of our sites were putative residues of the pbr and whether these sites exhibited signatures of positive selection. our sequences contained putative pbr residues, seven of which exhibited strong signatures of positive selection in each population.fig. output from omegamap plotting ω against codon position for the wild australian population. the black lines represent omega and the grey shading represents % highest posterior probability densities. stars represent sites encoding the pbr, with yellow stars indicating ω > and green stars indicating ω < 1 neutral expectations following population bottlenecks our bottleneck simulations revealed that based on drift alone, the timor zebra finch should carry between four and mhc alleles given a founding population of individuals, and over alleles given a founding population of individuals. under a predicted scenario based on previous coalescent-based estimates, in which a dramatic bottleneck down to individuals was followed by exponential growth rate of %, we observed a reduction from initial alleles on the mainland to alleles after island colonization. increasing the growth rate to % post-bottleneck reduced the number of alleles from to. lastly, a % exponential growth rate following the founder event reduced the number of alleles to, very similar to our observed value of alleles. under a less severe bottleneck scenario of individuals, simulated allelic diversity estimates far exceeded our observed values.fig. bottlesim output with and % growth rate post-bottleneck of individuals discussion polymorphism maintenance in the mhc population bottlenecks are expected to result in reduced neutral genetic variation. this pattern has previously been shown in both insular and domesticated zebra finch populations. of nuclear loci, including intronic regions, sex-linked and anonymous loci, balakrishnan & edwards found over half to be invariant in timor zebra finches. on average, timor zebra finches exhibit a % drop in nucleotide diversity of neutral markers. we have shown here that even in the presence of this genome wide reduction in variation, timor zebra finches have maintained a high level of diversity in the functionally important mhc. the mhc class i locus sequenced here thus stands in marked contrast to previously sampled putatively neutral loci in zebra finches. one possible explanation for the elevated mhc diversity relative to neutral markers is if selection has acted to maintain polymorphism. a few of lines of evidence support this conclusion. first, we find strong evidence of selection acting on this mhc locus, as evidenced by elevated dn/ds values. second, the observed nucleotide diversity of the mhc locus in timor finches is outside the range of nucleotide diversity observed in previously sequenced neutral loci. even though loci that are highly polymorphic in the australian subspecies tend to be more polymorphic in the timor subspecies, the mhc locus has maintained more nucleotide diversity than all putatively neutral, autosomal loci. further, our bottleneck simulations revealed that under many scenarios, including those that align with previous estimates of population demography, neutral divergence would result in a more severe reduction in mhc diversity than we observed. given the unique biology of the mhc and the uncertainty of zebra finch historical demography, completely ruling out neutral explanations for the observed high level of mhc diversity, however, is challenging. under some demographic scenarios, neutral expectations based on simulations closely approximate observed patterns of mhc diversity. for example, if the bottleneck associated with island colonization was less severe than previously estimated, or if population growth were relatively fast following island colonization, higher levels of mhc diversity would be expected. indeed, we obtain higher than observed levels of post-bottleneck mhc allelic diversity under a less severe bottleneck scenario suggesting that drift alone could maintain allelic diversity. previous, estimates suggest that a small number of individuals likely colonized the islands. confidence intervals around this estimate, however, are broad, making colonization of the islands by over founders possible. the process of domestication is also expected to reduce genetic diversity, particularly if populations are founded using relatively small numbers of individuals from the wild, or if selective breeding has been strong. forstmeier et al. found domesticated zebra finches to have slightly reduced genetic variation at microsatellite loci and significant population differentiation when compared with wild zebra finches. however, forstmeier et al. also found that a relatively high level of neutral genetic polymorphism has been maintained through domestication. we find the same pattern in the mhc class i, albeit with a lower degree of differentiation. the domestic birds utilized in this study were domesticated relatively recently, breed randomly and encounter pathogens found in captivity. therefore, it is not surprising to see high levels of mhc class i sequence and haplotype diversity as seen in other domesticated vertebrates. we found alleles in domesticated birds not found in the wild. these novel alleles may indicate adaptation to the pathogen community found in captivity, although further sampling along with functional testing is needed for verification. high diversity in the mhc suggests positive selection and indeed, there are abundant nonsynonymous polymorphisms throughout these class i sequences. our sequences contained putative pbr sites, seven of which exhibited strong evidence of positive selection in each population. the sites we report to be under positive selection have also been previously reported in another passerine, the scarlet rosefinch, that displays high levels of mhc class i variation. we also observe several sites experiencing positive selection outside of the putative pbr, potentially revealing additional sites important in peptide binding. as most reported estimates of mhc nucleotide diversity are from species with multiple expressed mhc loci, direct comparisons of diversity among birds, in particular among passerines, are complicated by inter-species variation in mhc copy number. the mhc plays a vital role in antigen presentation to the immune system, so selection is expected to maintain diversity to broaden pathogen recognition capability. although there have been a number of studies correlating bottlenecked populations with reduction in mhc variation, there have also been several studies showing the opposite, as balancing selection appears to counteract drift. the specific details of population demography play a critical role in determining the outcome following colonization. recent work has revealed that balancing selection mediated either by negative frequency dependent selection or heterozygote advantage can actually decrease rather than maintain mhc polymorphism after a bottleneck. given sufficient time and recovery after a bottleneck, however, balancing selection will again act to maintain polymorphism. likewise, gene conversion has also been shown to maintain mhc variation. lastly, differential pathogen pressures faced in each of these populations may contribute to the high diversity and divergence among populations. divergence between australian, island and domesticated populations the two zebra finch subspecies appear to have been isolated since the early pleistocene or late pliocene. thus, our estimates of genetic differentiation in the mhc class i and nd are consistent with the current understanding of zebra finch population history. this prolonged history of isolation of subspecies is also reflected in mhc class i haplotype diversity, as only two of mhc class i haplotypes are shared between subspecies. network analysis of mhc class i alleles shows that the timor alleles are distributed across multiple parts of the network, indicating that multiple colonizers carrying divergent alleles founded the island population. kst estimates between subspecies for mhc class i are lower than from neutral markers. this pattern, however, is expected, as measures of population divergence are necessarily lower for highly polymorphic markers. balancing selection could also contribute to this reduced kst. to provide a more complete picture of population genetic variation in this model system, we sequenced a portion of the mitochondrial nd gene. mitochondrial genes have long been used as markers of population history, and thus provide a point of contrast for loci under selection like the mhc. despite the utility of mitochondrial markers as a benchmark for taxonomic delineation and history, there has not previously been a mitochondrial dna survey in zebra finches. we found that zebra finch subspecies were reciprocally monophyletic for mtdna alleles, making this the first genetic marker to show monophyly of zebra finch subspecies. accordingly, there is highly significant population divergence between the subspecies. nd polymorphism in zebra finches is comparable to previous reports of nd nucleotide diversity in other passerines. interestingly, nd nucleotide variation is twice as high in the timor subspecies as in the australian subspecies. this high nucleotide diversity on the islands is a consequence of the existence of two divergent mtdna haplotype groups in our sample of birds. each node in our timor network represents a distinct sampling locality with the captive samples separated by one mutation from the wild timor samples. the high divergence between samples from west timor and lombok islands suggests long-term isolation of those populations, but more detailed sampling throughout the timor subspecies range is still needed to formally test this. zebra finch populations were domesticated in the 1800s. as a result, domestic and wild populations have relatively low, but statistically significant, divergence in mhc class i allele frequencies. these two populations also show statistically significant differences in nd allele frequencies. together these finding provide evidence of genetic distinctiveness of wild populations in australia and domesticated populations in the usa.  domestication and artificial selection have important and predictable consequences. however, we find high polymorphism despite a bottleneck in the colonization of the lesser sunda islands, and like forstmeier et al., we find high levels of nucleotide diversity in captive zebra finches as well. we also found significant differences in mhc allele frequencies between zebra finch subspecies and domesticated populations, indicating potential immune adaptation to the pathogen pressures faced by each population or the impact of genetic drift. although the discordance between neutral and mhc polymorphism is striking, improved resolution of historical demography is required in order to determine whether this disparity is driven by selection. zebra finches are a model system in a diversity of fields. genetic divergence in functional loci, such as that shown here for the mhc, may have important implications for research in the fields for which zebra finch is a model system. for example there is already evidence that domesticated and wild zebra finches differ in aspects of social behavior. comparisons of these populations in terms immunological or gene expression responses to immune challenges will provide insight into the functional consequences of the mhc class i variability we report here. availability of supporting data all sequences used in this study have been deposited in genbank under accession numbers kt595736-kt for mhc class i and kt595878-kt for nd additional files additional file 1: sample name, origin and locus genotyped for each sample used in this study. additional file 2: amino acid alignment of unique mhc class i sequences from each population. for each unique allele, the first number represents allele id and the second number represents the total number of individuals in the population carrying the allele. additional file 3: island and mainland haplotype diversity plotted next to each other for all reported loci. anonymous loci, introns and z linked loci reported by balakrishnan & edwards as well as mhc class i and nd from the present study. additional file 4: output from omegamap plotting posterior probability of positive selection against codon position for the wild australian population. stars represent sites encoding the pbr, with yellow stars indicating ω > and green stars indicating ω < additional file 5: output from omegamap plotting ω against codon position for the domesticated population. the black lines represent omega and the grey shading represents % highest posterior probability densities. stars represent sites encoding the pbr, with yellow stars indicating ω > and green stars indicating ω < additional file 6: output from omegamap plotting posterior probability of positive selection against codon position for the domesticated population. stars represent sites encoding the pbr, with yellow stars indicating ω > and green stars indicating ω < additional file 7: output from omegamap plotting ω against codon position for the timor population. the black lines represent omega and the grey shading represents % highest posterior probability densities. stars represent sites encoding the pbr, with yellow stars indicating ω > and green stars indicating ω < additional file 8: output from omegamap plotting posterior probability of positive selection against codon position for the timor population. stars represent sites encoding the pbr, with yellow stars indicating ω > and green stars indicating ω < additional file 9: bottlesim output showing the final number of alleles at and % post-bottleneck growth rate for a bottleneck size of and individuals. abbreviations mhcmajor histocompatability complex nd2nadh dehydrogenase 2 pbrpeptide-binding region hdhaplotype diversity mtdnamitochondrial dna competing interests the authors declare no competing interests. authors’ contributions cnb designed the study and provided samples. djn performed laboratory methods and analyzed results. djn and cnb drafted the manuscript. both authors read and approved the final manuscript. 
the australian subspecies has been domesticated and is now an important model system for research. as a benchmark for neutral divergence, we also report the first mitochondrial nadh dehydrogenase sequences in this important model system. two subspecies of zebra finch, taeniopygia guttata castanotis and t. g. guttata are native to australia and the lesser sunda islands, respectively. whereas the mhc class i locus shows broad haplotype sharing across populations, nd is the first locus surveyed to date to show reciprocal monophyly of the two subspecies. the diversity at the mhc class i locus in the lesser sundan subspecies contrasts sharply with the lack of diversity in previously examined neutral loci, and may thus be a result of selection acting to maintain polymorphism. as expected, we find higher levels of nucleotide diversity in the mhc locus relative to neutral loci, and strong evidence of positive selection acting on important residues forming the peptide-binding region. the surveyed populations also differ in mhc class i allele frequencies, and future studies are needed to assess whether these changes result in functional immune differences. both the lesser sundan subspecies and domesticated australian zebra finches have undergone population bottlenecks in their history, and previous analyses using neutral markers have reported reduced neutral genetic diversity in these populations. despite genetic bottlenecks and genetic drift, all surveyed zebra finch populations have maintained high mhc class i diversity. despite natural and human-mediated population bottlenecks, we find that high mhc class i polymorphism persists across all populations. clear population differentiation of mhc allele frequencies is also evident, and this may be due to adaptation to new habitats and associated pathogens and/or genetic drift. bottleneckimmunend2positive selectiongenetic driftevolutionissue-copyright-statement© the author 2015
 two subspecies of zebra finch, taeniopygia guttata castanotis and t. g. guttata are native to australia and the lesser sunda islands, respectively. the australian subspecies has been domesticated and is now an important model system for research. both the lesser sundan subspecies and domesticated australian zebra finches have undergone population bottlenecks in their history, and previous analyses using neutral markers have reported reduced neutral genetic diversity in these populations. here we characterize patterns of variation in the third exon of the highly variable major histocompatibility complex class i α chain. as a benchmark for neutral divergence, we also report the first mitochondrial nadh dehydrogenase sequences in this important model system.  despite natural and human-mediated population bottlenecks, we find that high mhc class i polymorphism persists across all populations. as expected, we find higher levels of nucleotide diversity in the mhc locus relative to neutral loci, and strong evidence of positive selection acting on important residues forming the peptide-binding region. clear population differentiation of mhc allele frequencies is also evident, and this may be due to adaptation to new habitats and associated pathogens and/or genetic drift. whereas the mhc class i locus shows broad haplotype sharing across populations, nd is the first locus surveyed to date to show reciprocal monophyly of the two subspecies.  despite genetic bottlenecks and genetic drift, all surveyed zebra finch populations have maintained high mhc class i diversity. the diversity at the mhc class i locus in the lesser sundan subspecies contrasts sharply with the lack of diversity in previously examined neutral loci, and may thus be a result of selection acting to maintain polymorphism. given uncertainty in historical population demography, however, it is difficult to rule out neutral processes in maintaining the observed diversity. the surveyed populations also differ in mhc class i allele frequencies, and future studies are needed to assess whether these changes result in functional immune differences. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords bottleneckimmunend2positive selectiongenetic driftevolutionissue-copyright-statement© the author 2015 