Expression and phylogenetic analyses reveal paralogous lineages of putatively classical and non-classical MHC-I genes in three sparrow species (Passer)
© The Author(s). 2017
Received: 13 December 2016
Accepted: 17 May 2017
Published: 26 June 2017
The Major Histocompatibility Complex (MHC) plays a central role in immunity and has been given considerable attention by evolutionary ecologists due to its associations with fitness-related traits. Songbirds have unusually high numbers of MHC class I (MHC-I) genes, but it is not known whether all are expressed and equally important for immune function. Classical MHC-I genes are highly expressed, polymorphic and present peptides to T-cells whereas non-classical MHC-I genes have lower expression, are more monomorphic and do not present peptides to T-cells. To get a better understanding of the highly duplicated MHC genes in songbirds, we studied gene expression in a phylogenetic framework in three species of sparrows (house sparrow, tree sparrow and Spanish sparrow), using high-throughput sequencing. We hypothesize that sparrows could have classical and non-classical genes, as previously indicated though never tested using gene expression.
The phylogenetic analyses reveal two distinct types of MHC-I alleles among the three sparrow species, one with high and one with low level of polymorphism, thus resembling classical and non-classical genes, respectively. All individuals had both types of alleles, but there was copy number variation both within and among the sparrow species. However, the number of highly polymorphic alleles that were expressed did not vary between species, suggesting that the structural genomic variation is counterbalanced by conserved gene expression. Overall, 50% of the MHC-I alleles were expressed in sparrows. Expression of the highly polymorphic alleles was very variable, whereas the alleles with low polymorphism had uniformly low expression. Interestingly, within an individual only one or two alleles from the polymorphic genes were highly expressed, indicating that only a single copy of these is highly expressed.
Taken together, the phylogenetic reconstruction and the analyses of expression suggest that sparrows have both classical and non-classical MHC-I genes, and that the evolutionary origin of these genes predate the split of the three investigated sparrow species 7 million years ago. Because only the classical MHC-I genes are involved in antigen presentation, the function of different MHC-I genes should be considered in future ecological and evolutionary studies of MHC-I in sparrows and other songbirds.
The major histocompatibility complex (MHC) is a key component of adaptive immunity and holds the most polymorphic genes known in the vertebrate genome . MHC class I (MHC-I) proteins are expressed on all nucleated cells whereas MHC-II proteins are expressed only on antigen presenting cells . Typically, animals have a handful of functional MHC-I genes, as exemplified by humans (six genes), swine (Sus scrofa domesticus; six genes) and domestic chicken (Gallus gallus; four genes) [3–5]. On the contrary, songbirds of the order Passeriformes have a larger number of MHC-I genes than most other species investigated to date [6–8]. O’Connor et al. (2015) reported between four and 20 MHC-I genes per individual across Passerida (i.e. genomic MHC-I exon 3 sequences in open reading frame), and Biedrzycka et al. (2017) found 65 alleles per individual, i.e. at least 33 MHC-I genes, in the sedge warbler Acrocephalus schoenobaenus [8, 9]. The functional significance of all these MHC-I gene copies in songbirds is not known.
In most species studied to date — for example humans and other primates, swine, mice and chicken — MHC-I genes are categorized as classical or non-classical MHC-I genes [3, 5, 10, 11]. In mammals, classical MHC-I genes (MHC-Ia) are highly polymorphic and highly expressed, whereas non-classical (MHC-Ib) are less polymorphic and have low expression [12, 13]. The non-classical genes do not appear to have a common origin among distantly related mammals but seem to have arisen independently from recent duplications of classical MHC-I genes within species . MHC-Ia molecules play an important role in adaptive immunity by presenting peptides to T-cells , whereas MHC-Ib molecules have other immune functions [12, 13, 15, 16]. Humans have three MHC-Ia genes (HLA-A, -B and -C) and three MHC-Ib genes (HLA-E, -F and -G) . HLA-A, -B and -C have a much larger number of alleles (>9000 world-wide) than HLA-E,-F and -G (<90 world-wide) . HLA-A, -B and -C genes are expressed in most tissues, but there are gene-specific expression differences among the genes; HLA-C is expressed to a lower degree than HLA-A and -B resulting in high variation in expression levels among classical genes .
Classical and non-classical class I genes have also been reported in birds of the order Galliformes, e.g. in chicken, turkey (Meleagris gallopavo) and golden pheasant (Chrysolophus pictus) and here the MHC-Ia and MHC-Ib genes are referred to as MHC-B and MHC-Y, respectively [4, 19–21]. The chicken has two classical MHC-I genes at the MHC-B locus and two non-classical MHC-I genes at the MHC-Y locus . Both genes at the classical B-locus are expressed, but the ‘major’ gene is highly expressed compared to the ‘minor’ gene [22–24]. Only one of the non-classical Y-locus genes has been shown to be expressed and then specifically in spleen [25, 26]. The presence of classical and non-classical MHC-I genes is less established in non-galliform birds, but has been suggested in species of the orders Anseriformes, Charadriiformes and Pelecaniformes [27–30]. Moreover, in Anseriformes and Pelecaniformes there seem to be one putatively classical gene that is highly expressed [30, 31].
MHC genes evolve by frequent gene duplications, but there is also gene loss when gene copies become non-functional, and the evolution of MHC therefore fit a birth-and-death model of molecular evolution [32–34]. The large number of MHC-I genes in the genomes of songbirds indicates either a higher rate of gene duplications in songbirds compared to other animals, or that several copies of their MHC genes have been duplicated simultaneously [6–8]. Very little is known about neo-functionalization among the multiple gene copies in songbirds, though it seems likely that some gene copies have evolved different functions, like the classical and non-classical genes found in other species [27–30]. It is important to distinguish non-classical and classical MHC genes in evolutionary and ecological studies since only the latter are subject to balancing selection and expected to be associated with disease resistance and fitness.
Karlsson and Westerdahl  showed that house sparrows (Passer domesticus) have two types of MHC-I alleles that exhibit some of the hallmarks of classical and non-classical genes. The putatively non-classical house sparrow MHC-I alleles have low levels of polymorphism, few positively selected sites and form a distinct phylogenetic cluster, whereas the putatively classical MHC-I alleles have high polymorphism, many positively selected sites and do not form a supported phylogenetic cluster . The putatively non-classical alleles in house sparrows are easily identified by a six base pair deletion in exon 3 [35, 36]. However, the expression pattern of these putatively classical and non-classical genes in house sparrow has not been investigated. Hence, a way to further establish the occurrence of classical and non-classical MHC genes in house sparrows would be to measure their relative expression. Classical genes are often highly expressed compared to non-classical genes, and certain genes within each category might be more highly expressed, e.g. if ‘major’ and ‘minor’ loci are present as indicated in species of the bird orders Galliformes, Anseriformes and Pelecaniformes [25–30].
Previous studies on the evolution of MHC genes have shown that orthologous classical MHC genes often survive longer in the genome—over speciation events—than non-classical genes . A next step to continue studying putatively non-classical genes in Passerines is therefore to investigate their occurrence in species that are closely related to house sparrows. Finding putatively classical and non-classical genes in several species would not only make the finding more solid but also give an indication of their evolutionary age.
We set out to investigate the structure, number and expression patterns of MHC-I genes among three sparrow species, the house sparrow, the Spanish sparrow (Passer hispaniolensis) and the tree sparrow (Passer montanus), in order to identify neo-functionalization of MHC-I genes, in particular whether there are both putatively classical and non-classical genes. We firstly reconstruct allelic phylogenies where we classify the sparrow MHC-I alleles as putatively classical or non-classical genes and then test if these putatively classical and non-classical alleles differ in i) number of genomic alleles between species, ii) number of expressed alleles between species and iii) relative gene expression within species.
We study three species of sparrows in the Passer clade; house sparrow (Passer domesticus) (n = 5), Spanish sparrow (P. hispaniolensis) (n = 3) and tree sparrow (P. montanus) (n = 5). The native range of the house sparrow and tree sparrow covers most of Eurasia whereas the Spanish sparrow has a more restricted distribution around the Mediterranean Sea and in south-west Asia . All three species live in both urban and rural environments . In order to determine when house sparrow, Spanish sparrow and tree sparrow separated phylogenetically a maximum clade credibility tree was constructed based on data from 23 Passer species and an outgroup (Cyanistes caeruleus) from the Bird Tree website [38, 39], for details see Additional file 1: Method S1. House sparrows and Spanish sparrows split 3 million years ago, while the tree sparrows are more distantly related and split from the other two species 7 million years ago.
Sample collection and extractions
House sparrows and tree sparrows were caught, with mist nets, in Löberöd, Skåne, Sweden. The Spanish sparrows were kept and caught in aviaries at University of Oslo, Norway. All samples were collected during the autumn of 2012. Blood samples (20-40 μl) were taken from the brachial vein and then stored either at −20 °C in SET buffer (150 mM NaCl, 50 mM TRIS, 1 mM EDTA, pH 8.0), for DNA extraction, or at 4 °C in 100 μl K2EDTA and 500 μl TRIzol LS (Life Technologies, Carlsbad, CA, USA), for RNA extraction. DNA was extracted with ammonium acetate extraction . RNA was extracted with a combination of the TRIzol LS protocol (Life Technologies, Carlsbad, CA, USA) and the RNeasy Mini kit (QIAGEN, Hilden, Germany). Briefly, the homogenization and phase separation was done according to the TRIzol LS protocol, resulting in an aqueous phase. One volume of 70% EtOH was added to the aqueous phase and from this step the RNeasy protocol was followed, including an on-column DNase treatment . The RNA (mRNA) was reverse transcribed to complementary DNA (cDNA) using the RETROscript kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol.
High-throughput amplicon sequencing
We sequenced partial MHC-I exon 3 amplicons (185–226 bp) obtained from genomic DNA (gDNA) and cDNA (to examine gene expression) from house sparrows, tree sparrows and Spanish sparrows using 454 amplicon sequencing. Four different primer combinations were used to amplify MHC-I exon 3 alleles in each species to minimize the effects of amplification bias and allelic dropouts which is often a problem when using only one primer pair (Additional file 1: Table S1, Figure S1). Primer combinations 1 and 2 amplify both putatively classical and non-classical alleles whereas primer combination 3 and 4 exclusively amplifies putatively classical and non-classical alleles, respectively [8, 35, 42]. Each individual was represented by one gDNA and one cDNA sample and samples were technically duplicated (two PCRs from 40% of the samples). We performed PCR with individually tagged 454 fusion primers (6-bp tag on forward and reverse primer) . Each 15 μl PCR reaction contained either 25 ng gDNA or 10 ng cDNA, 0.2 μM of each primer and 1× QIAGEN Multiplex PCR Master Mix (QIAGEN, Hilden, Germany). The cycling conditions for primer combination 1 and 2 were set to 35 cycles at 95 °C (30s), 60 °C (90s), 72 °C (60s) followed by 72 °C for 10 min. For primer combination 3 and 4 the cycling conditions were 30 cycles at 95 °C (30s), 65 °C (60s), 72 °C (60s) followed by 72 °C for 10 min. The PCR products were verified on a 2% agarose gel and products were pooled semi-equimolarly based on the strength of the bands with a maximum of eight products per pool. These pools were purified on MinElute PCR purification columns (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol and quantified on a NanoDrop 2000/2000c (Thermo Fisher Scientific, Wilmington, DE, USA). The purified pools were pooled in equimolar DNA amounts in one final pool per primer combination. Amplicons were sequenced in two separate 454 sequencing runs (one for primer combination 1 and 2 amplicons and another for primer combination 3 and 4 amplicons) at the Lund University DNA Sequencing facility, Faculty of Science, Sweden.
Filtering of high-throughput sequencing data
There are errors associated with high-throughput sequencing techniques that will result in artefactual alleles (AA). The AAs were distinguished from the true alleles (TA) and removed from the dataset by a number of filtering steps, (for details see Additional file 1: Methods S2). Briefly, the first filtering step handled AA originating from homopolymer errors, these were identified by eye and if a possible AA always occurred with its parental sequences the read depth from the AA was added to that of the parental sequence. As a second step all amplicons with insufficient coverage was removed (the threshold was set to 110 for gDNA (and to 140 for cDNA, values in brackets) for primer combination 1, 70 (100) for primer combination 2, 100 (180) for primer combination 3 and 100 (140) for primer combination 4, see Additional file 1: Methods S2 for more details). Next, all sequences that had too few reads within an amplicon were deleted, this read depth was measured as percentage of the total read depth (varying from 1.1% to 3.0% depending on species and primer combination, see Additional file 1: Methods S2 for more details). All sequences that varied by 1–2 bp were identified and the read depth of the possible AA was only added to the parental sequences if the AA occurred only once in the entire data set, together with the parental sequence and when the read depth of the possible AA was less than half of the parental sequence. Chimeras and non-functional sequences were identified by eye and deleted from the data set. Possible chimeras were deleted from the data set only when they occurred with both parental sequences and when the read depth of the putative chimera was less than half of both parental sequences. Sequences that only occurred in one amplicon in the entire data set or in a single amplicon within an individual were deleted. Lastly low frequency sequences that were only amplified in cDNA were deleted since all cDNA sequences should also be found in the corresponding gDNA sample.
All sequences that remained after the strict filtering were considered TA. BLAST was used to determine which alleles had previously been published. When an allele was 100% identical to a previously published sequence and of the same length the allele was named according to the published sequence. If an allele was 100% identical to a previously published sequence but of different length the allele was given the name of the published sequence followed by ‘a’. Alleles that had not previously been published were given species specific names according to the recommended guide lines for naming MHC alleles . All new sequences were deposited in GenBank (GenBank Acc nr KY303944-KY304003). TA in the cDNA samples, i.e. transcribed alleles, are hereafter called expressed alleles. Thirty-six samples were run in duplicates and the repeatability between duplicates was calculated as the percentage of total number of alleles amplified (i.e. the concatenated number of alleles using both duplicates as ‘total number’).
Number and expression of MHC-I alleles
The use of multiple primer combinations enabled a detailed characterization of the total number of classical and non-classical MHC-I alleles per individual since the possibility of amplifying all alleles in an individual increases when different primer combinations are used [8, 42]. The number of alleles per individual was determined by combining the result from all four primer combinations. The amplification range of the four different primer combinations was calculated as the proportion of the total number of alleles amplified with all primer combinations. This was done separately for each individual and an average was calculated for classical and non-classical alleles in each species (Additional file 1: Table S2). We used primer combination 1 in the expression analysis since this primer combination amplified the majority of all the classical and non-classical alleles simultaneously. With the concatenated result from all four primer combinations we identified which alleles were expressed in each individual and in the expression analysis we only included individuals where primer combination 1 amplified the majority of all expressed alleles. Primer combination 1 fulfilled these strict criteria in six individuals (house sparrow, n = 3, tree sparrow, n = 3). The relative expression of each allele was estimated as the proportion of the total number of reads per individual. These six individuals expressed up to four classical and up to four non-classical alleles per individual, the maximum total number of expressed alleles was eight. In order to get an estimate of how many alleles that were highly expressed we set a custom threshold to define and separate highly expressed alleles from the remaining alleles based on the following reasoning: Given that there was a maximum of eight alleles per individual, each allele would with an even distribution contribute with 12.5% of the reads. We set the threshold for ‘high allelic expression’ to twice as high; hence highly expressed alleles should contribute with more than 25% of the reads in an individual.
Statistical analysis and phylogenetic relationship
Statistical analyses were performed in SPSS (IBM SPSS Statistics 22). The differences between species regarding number of alleles and number of expressed alleles were determined with one-way ANOVAs. The differences in variation in expression between classical and non-classical genes were determined in two ways. First, when only expressed alleles were included in the model, Levine’s test of equal variance was used. Second, when also non-expressed alleles (i.e. alleles with zero read depth in cDNA sample) were included in the model the data was no longer normally distributed and hence the Brown-Forsythe variance test was used. In order to determine the phylogenetic relationship between MHC-I alleles both a maximum likelihood tree and a neighbor-net network were construed. The maximum likelihood tree was constructed with the RAxML software (version 7.0.4) using the GTRGAMMA model and 1000 bootstraps and illustrated with iTOL (version 3.4.3) . The network was constructed with SplitsTree v. 4.14.4  using a GTR model with the α parameter for gamma distribution set to 0.3450, which was recommended by jModelTest 2.1.10  and 1000 bootstraps. Figures were produced in R, (version 2.15.3), using the built in packages barplot and plot .
Phylogenetic relationship of putatively classical and non-classical MHC-I genes
MHC-I alleles were genotyped in 13 individuals, five house sparrows, three Spanish sparrows and five tree sparrows, using high-throughput sequencing of both gDNA (genomic DNA) and cDNA (complementary DNA, i.e. reverse transcribed RNA, as a measure of gene expression). The average read depth of true alleles (alleles that remained after filtering the HTS data) per individual varied between 413 and 990 reads for gDNA and between 528 and 1108 reads for cDNA (combining three or four primer combinations, for further details on read depth see Additional file 1: Table S3). The repeatability in genotyping between duplicates varied between 91% and 100% across primers (Additional file 1: Table S4). Putatively classical and non-classical MHC-I genes were found in all three Passer species, and the putatively non-classical alleles were identified by a 6 bp deletion in exon 3, as described previously for house sparrows. In total, in all three species, 129 alleles were identified (Additional file 1: Figure S2).
Number of putatively classical and non-classical MHC-I alleles among sparrow species
Variance in expression of classical and non-classical MHC-I genes in house sparrows and tree sparrows
The subdivision of MHC-I genes into classical, highly polymorphic genes that present peptides to T-cells, and non-classical genes, less polymorphic genes that do not present peptides to T-cells, have been reported in many mammal species and also in several bird species [3–5, 10, 11, 19–21, 27–30]. However, classical and non-classical genes have not yet been confirmed in songbirds in the largest bird order Passeriformes, though such subdivision is likely since non-classical genes have been identified in other bird species from several different orders [25–30]. In the present study we found high numbers of MHC-I gene copies in sparrows; the maximum number of MHC-I alleles per individual that we identified was 21 (i.e sparrows have eleven or more MHC-I gene copies), though only about 50% of these alleles were expressed. In the maximum likelihood tree we identified one distinct strongly supported cluster (bootstrap = 92 and for the neighbor-net network the corresponding the bootstrap = 93) containing alleles (from all three species) with low polymorphism and a 6 bp deletion (putatively non-classical genes). The remaining alleles were more polymorphic and found in non-significantly supported groups (putatively classical genes). Several previous studies have reported considerably lower diversity estimates and lower rates of non-synonymous substitutions in the peptide binding region of putatively non-classical alleles compared with putatively classical alleles in house sparrows [35, 36, 49], and we found similar results in our data from three different sparrow species considering nucleotide diversity. Moreover, the analyses of gene expression showed that the polymorphic group with the putatively classical genes had variable expression, that is, some alleles were highly expressed while others had low expression. Strikingly, at most two alleles among these putatively classical MHC-I alleles were ever highly expressed in each individual. In contrast, the group with putatively non-classical genes had more uniformly low expression. Taken together, these results strongly indicate that we have identified classical and non-classical MHC-I genes in sparrows.
Phylogenetic ages of classical and non-classical genes
The phylogenetic reconstruction of sparrow MHC-I alleles places the non-classical genes in a single well-supported cluster, confirming that the subdivision of these putatively classical and non-classical genes predates the separation of the investigated sparrow species. This shows that orthologous gene copies of classical and non-classical genes have persisted in sparrows over several speciation events in a time frame of at least 7 million years. Classical (MHC-B) and non-classical (MHC-Y) genes have also been reported among a wide range of birds in the order Galliformes, including chicken, turkey and golden pheasant, species that split 28–40 million years ago, and non-classical alleles in chicken and turkey form a gene specific cluster [4, 20, 21, 50–52]. It is possible that these different sets of paralogous genes are even older and evolved in an ancient common ancestor of galliforms more than 65 million years ago . However, non-classical genes in sparrows of the order Passeriformes and species within the order Galliformes are not likely to be orthologous, presently available data suggest that the non-classical genes originate from more recent duplications of classical genes, a pattern seen also among distantly related mammals [14, 30]. Though, orthologous clusters of classical and non-classical MHC-I genes among relatively closely related mammals has been seen in hominids and here the classical and non-classical genes even cluster by locus . Classical (HLA-A, -B and -C) and non-classical (HLA-E, -F and -G) MHC-I genes in human and chimpanzee (Pan troglodytes), species that diverged 6 to 7 million years ago, form gene specific clusters at each of these six loci (A-G).
Number of MHC alleles in gDNA and cDNA in three sparrow species
We found no difference in the copy number of classical and non-classical alleles in the genome between house sparrows and Spanish sparrows (species that split 3 million years ago) but there was a difference in allele copy number relative to the tree sparrows, which diverged earlier (7 million years ago). This difference in gene copy number could have originated in two different ways, first the divergence time of house sparrows and Spanish sparrows may be too short for gene copy number to evolve while tree sparrows that are more distantly related have evolved further. Tree sparrows have a higher number of classical gDNA alleles and a lower number of non-classical gDNA alleles than house sparrows and Spanish sparrows. Alternatively, house sparrow and Spanish sparrow could have experienced different selection and lost classical gene copies. Without knowing the proportion of classical and non-classical genes in ancestral sparrows it is impossible to determine how this gene copy variation occurred. Moreover, there are certain problems associated with co-amplifying multiple genes at the same time which makes it more difficult to determine the exact number of genes and possible copy number variation [54, 55]. Here we have tried to overcome this problem by using several primer combinations.
The number of expressed alleles (cDNA) varied less between species than the number of alleles in the genome and, interestingly, there was no significant difference in any species comparisons in number of expressed classical alleles. One explanation could be that the number of expressed genes are more conserved than the total number of genes in the genome and there may be selection for expressing a certain number of genes, e.g. expressing an optimal number of classical genes . There was a significant difference in the number of expressed non-classical alleles among species; tree sparrows expressed fewer non-classical alleles than both house sparrows and Spanish sparrows. It is interesting to note that tree sparrows, which express significantly lower numbers of non-classical alleles, also have a lower number of non-classical alleles in the genome.
Variation in expression of classical and non-classical MHC-I alleles
The variance in gene expression was larger in putatively classical than non-classical MHC-I genes in sparrows. This finding is consistent with the existence of ‘major’ (highly expressed) and ‘minor’ (low expressed) loci for classical but not for non-classical MHC-I genes in sparrows, as previously reported for species within the order Galliformes [22, 23]. The highest numbers of classical alleles per individual in sparrows were 14 in gDNA but out of these only two alleles were highly expressed. Two highly expressed classical MHC-I alleles have been reported for several bird species in the order Galliformes (e.g. chicken and Japanese quail (Coturnix japonica)) and also in mallards belonging to the order Anseriformes. In chicken, Japanese quail and mallard the MHC genomic regions have been characterized and each species has a single major classical MHC-I locus, that is, one classical gene that is highly expressed [27, 57–59]. In all these species, the highly expressed gene is located next to the TAP gene (Transporter associated with antigen processing) and co-evolutionary processes between TAP and MHC is thought to explain why only a single MHC-I gene is highly expressed [4, 31, 57, 60–62]. Our findings on classical MHC-I genes in house sparrows and tree sparrows agree well with these previous findings from other birds, though with our data we cannot determine with certainty if sparrows have one single major classical MHC-I gene. It is possible that the six sparrows are homozygous for two classical MHC-I genes that are highly expressed, even though this is unlikely since heterozygosity is much more common than homozygosity at classical MHC-I loci. Alternatively, different genes could be highly expressed in the six individuals or the two alleles of one gene could be differently expressed, meaning that two genes could be highly expressed. It would be interesting to study this further and to determine if the highly expressed alleles belong to the same gene and if this gene is located next to TAP. Since we set strict criteria for including individuals in the expression analysis we could only investigate six of the individuals. Future analysis of more individuals would help determining how general our results are.
In our study of expression of MHC-I genes we only estimated gene expression in blood. Blood to some extent represent gene expression in different tissues but we do not claim that our results should be extrapolated to be representative for expression in all tissues. Non-expressed genes in our study could for example have specific expression under other conditions, in other tissues or in birds of different age classes. Chen et al.  recently characterized the genomic MHC region in the crested ibis (Nipponia nippon) and reported considerable differences in gene expression of five different MHC-I genes between tissues. Interestingly one particular MHC-I gene was highly expressed in all tissues in the crested ibis and this gene was the only gene situated in the core MHC genomic region . Expression in blood was however not reported in the crested ibis.
We have studied the highly duplicated MHC-I gene family in three species of sparrows and based on phylogeny and gene expression patterns found strong indications for the existence of classical and non-classical MHC-I genes. This subdivision of genes has previously been reported in many groups of vertebrates, for example in galliform birds and hominids, but never in songbirds. A majority of the sparrow MHC-I genes are putatively non-classical; hence they are presumably not involved in T-cell mediated immunity. Such a distinctly separated phylogenetic cluster of putatively non-classical genes is rarely found among songbirds, and within songbirds non-classical genes could be a unique feature for sparrows. However, we find it more likely that there are groups of non-classical MHC-I genes in most songbirds but that they often are missed. Therefore, it would be valuable if future studies of MHC-I in songbirds investigated the existence of putatively non-classical MHC-I genes, preferably using gene expression. Future ecological and evolutionary studies of MHC-I in wild birds would gain from considering the existence of classical and non-classical genes, since these two types of MHC-I genes have different functions.
We are grateful to Fredrik Haas for assistance during field work and to Emily O’Connor for assistance during lab work and data analysis.
This work was supported by the Swedish Research Council (grant 621–2011-3674 and 2015–05149) and by the Crafoord Foundation to H.W.
Availability of data and materials
All new sequences were submitted to GenBank (accession numbers: KY303944-KY304003). The datasets supporting the conclusions of this article are available in the Dryad Digital repository, [doi:10.5061/dryad.79t4b] .
HW and AD designed the study and conducted the field work. AD did the lab work. The data was analyzed by AD, HW and LR. All authors discussed the results and contributed to the writing of the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
All samples were collected with an ethical permission from the Malmö/Lund Committee for Animal Experiment Ethics (no. M45–14).
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Murphy K, Travers P, Walport M. Janeway’s immunobiology. 7th ed. New York: Garland Science; 2008.Google Scholar
- Neefjes J, Jongsma ML, Paul P, Bakke O. Towards a systems understanding of MHC class I and MHC class II antigen presentation. Nat Rev Immunol. 2011;11:823–36.PubMedGoogle Scholar
- Shiina T, Hosomichi K, Inoko H, Kulski JK. The HLA genomic loci map: expression, interaction, diversity and disease. J Hum Genet. 2009;54:15–39.View ArticlePubMedGoogle Scholar
- Kaufman J, Milne S, Göbel TW, Walker BA, Jacob JP, Auffray C, et al. The chicken B locus is a minimal essential major histocompatibility complex. Nature. 1999;401:923–5.View ArticlePubMedGoogle Scholar
- Lunney JK, Ho CS, Wysocki M, Smith DM. Molecular genetics of the swine major histocompatibility complex, the SLA complex. Dev Comp Immunol. 2009;33:362–74.View ArticlePubMedGoogle Scholar
- Westerdahl H. Passerine MHC: genetic variation and disease resistance in the wild. J Ornithol. 2007;148:469–77.View ArticleGoogle Scholar
- Sepil I, Moghadam HK, Huchard E, Sheldon BC, Kuduk K, Babik W, et al. Characterization and 454 pyrosequencing of major histocompatibility complex class I genes in the great tit reveal complexity in a passerine system. BMC Evol Biol. 2012;12:68.View ArticlePubMedPubMed CentralGoogle Scholar
- O’Connor EA, Strandh M, Hasselquist D, Nilsson J, Westerdahl H. The evolution of highly variable immunity genes across a passerine bird radiation. Mol Ecol. 2016;25:977–89.View ArticlePubMedGoogle Scholar
- Biedrzycka A, O’Connor E, Sebastian A, Migalska M, Radwan J, Zając T, et al. Extreme MHC class I diversity in the sedge warbler (Acrocephalus schoenobaenus); selection patterns and allelic distributions suggest that different genes have different functions. BMC Evol Biol. 2017. In press.Google Scholar
- Adams E, Parham P. Species-specific evolution of MHC class I genes in the higher primates. Immunol Rev. 2001;183:41–64.View ArticlePubMedGoogle Scholar
- Velten F, Rogel-Gaillard C, Renard C, Pontarotti P, Tazi-Ahnini R, Vaiman M, et al. A first map of the porcine major histocompatibility complex class I region. Tissue Antigens. 1998;51:183–94.View ArticlePubMedGoogle Scholar
- Shawar S, Vyas J. Antigen presentation by major histocompatibility complex class IB molecules. Annu Rev Immunol. 1994;12:839–80.View ArticlePubMedGoogle Scholar
- Rodgers JR, Cook RG. MHC class Ib molecules bridge innate and acquired immunity. Nat Rev Immunol. 2005;5:459–71.View ArticlePubMedGoogle Scholar
- Hughes ALL, Nei M. Evolution of the major histocompatibility complex: independent origin of nonclassical class I genes in different groups of mammals. Mol Biol Evol. 1989;6:559–79.PubMedGoogle Scholar
- Ishitani A, Sageshima N, Lee N, Dorofeeva N, Hatake K, Marquardt H, et al. Protein expression and peptide binding suggest unique and interacting functional roles for HLA-E, F, and G in maternal-placental immune recognition. J Immunol. 2003;171:1376–84.View ArticlePubMedGoogle Scholar
- Diefenbach A, Raulet DH. The innate immune response to tumors and its role in the induction of T-cell immunity. Immunol Rev. 2002;188:9–21.View ArticlePubMedGoogle Scholar
- Robinson J, Halliwell J, Hayhurst JD, Flicek P, Parham P, Marsh SGE. The IPD and IMGT/HLA database: Allele variant databases. Nucleic Acids Res. 2015;43:D423–31.View ArticlePubMedGoogle Scholar
- Apps R, Meng Z, Del Prete GQ, Lifson JD, Zhou M, Carrington M. Relative Expression Levels of the HLA Class-I Proteins in Normal and HIV-Infected Cells. J Immunol. 2015;194:3594–600.View ArticlePubMedPubMed CentralGoogle Scholar
- Briles WE, Goto RM, Auffray C, Miller MM. A polymorphic system related to but genetically independent of the chicken major histocompatibility complex. Immunogenetics. 1993;37:408–14.View ArticlePubMedGoogle Scholar
- Chaves LD, Krueth SB, Reed KM. Characterization of the turkey MHC chromosome through genetic and physical mapping. Cytogenet Genome Res. 2007;117:213–20.View ArticlePubMedGoogle Scholar
- Zeng Q, Zhong G, He K, Sun D, Wan Q. Molecular characterization of classical and nonclassical MHC class I genes from the golden pheasant (Chrysolophus pictus ). Immunogenetics. 2016;43:8–17.View ArticleGoogle Scholar
- Kaufman J, Jacob J, Shaw I, Walker B, Milne S, Beck S, et al. Gene organisation determines evolution of function in the chicken MHC. Immunol Rev. 1999;167:101–17.View ArticlePubMedGoogle Scholar
- Kaufman J. Co-evolving genes in MHC haplotypes: the “rule” for nonmammalian vertebrates? Immunogenetics. 1999;50:228–36.View ArticlePubMedGoogle Scholar
- Wallny H-J, Avila D, Hunt LG, Powell TJ, Riegert P, Salomonsen J, et al. Peptide motifs of the single dominantly expressed class I molecule explain the striking MHC-determined response to Rous sarcoma virus in chickens. Proc Natl Acad Sci U S A. 2006;103:1434–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Afanassieff M, Goto RM, Ha J, Sherman MA, Zhong L, Auffray C, et al. At least one class I gene in restriction fragment pattern-Y (Rfp-Y), the second MHC gene cluster in the chicken, is transcribed, polymorphic, and shows divergent specialization in antigen binding region. J Immunol. 2001;166:3324–33.View ArticlePubMedGoogle Scholar
- Hunt HD, Goto RM, Foster DN, Bacon LD, Miller MM. At least one YMHCI molecule in the chicken is alloimmunogenic and dynamically expressed on spleen cells during development. Immunogenetics. 2006;58:297–307.View ArticlePubMedGoogle Scholar
- Moon DA, Veniamin SM, Parks-Dely JA, Magor KE. The MHC of the duck (Anas platyrhynchos) contains five differentially expressed class I genes. J Immunol. 2005;175:6702–12.View ArticlePubMedGoogle Scholar
- Cloutier A, Mills JA, Baker AJ. Characterization and locus-specific typing of MHC class I genes in the red-billed gull (Larus scopulinus) provides evidence for major, minor, and nonclassical loci. Immunogenetics. 2011;63:377–94.View ArticlePubMedGoogle Scholar
- Buehler DMDM, Verkuil YIYI, Tavares ESES, Baker AJAJ. Characterization of MHC class i in a long-distance migrant shorebird suggests multiple transcribed genes and intergenic recombination. Immunogenetics. 2013;65:211–25.View ArticlePubMedGoogle Scholar
- Chen L-C, Lan H, Sun L, Deng Y-L, Tang K-Y, Wan Q-H. Genomic organization of the crested ibis MHC provides new insight into ancestral avian MHC structure. Sci Rep. 2015;5:7963.View ArticlePubMedPubMed CentralGoogle Scholar
- Mesa CM, Thulien KJ. Moon D a, Veniamin SM, Magor KE. The dominant MHC class I gene is adjacent to the polymorphic TAP2 gene in the duck, Anas platyrhynchos. Immunogenetics. 2004;56:192–203.View ArticlePubMedGoogle Scholar
- Ohno S. Evolution by Gene Duplication. Berlin, Heidelberg: Springer Berlin Heidelberg; 1970.View ArticleGoogle Scholar
- Eirin-Lopez JM, Rebordions L, Rooney AP, Rozas J. The Birth-and-Death Evolution of Multigene Families Revisited. Genome Dyn. 2012;7:170–96.View ArticlePubMedGoogle Scholar
- Nei M, Gu X, Sitnikova T. Evolution by the birth-and-death process in multigene families of the vertebrate immune system. Proc Natl Acad Sci National Acad Sciences. 1997;94:7799–806.View ArticleGoogle Scholar
- Karlsson M, Westerdahl H. Characteristics of MHC Class I Genes in House Sparrows Passer domesticus as Revealed by Long cDNA Transcripts and Amplicon Sequencing. J Mol Evol. 2013;77:8–21.View ArticlePubMedGoogle Scholar
- Bonneaud C, Sorci G, Morin V, Westerdahl H, Zoorob R, Wittzell H. Diversity of Mhc class I and IIB genes in house sparrows (Passer domesticus). Immunogenetics. 2004;55:855–65.View ArticlePubMedGoogle Scholar
- Mullarney K, Svensson L, Zetterström D, Grant PJ. Bird guide. The most complete field guide to the birds of britain and europe. London: HarperCollins Publishers Ltd; 2006.Google Scholar
- Jetz W, Thomas GH, Joy JB, Hartmann K, Mooers AO. The global diversity of birds in space and time. Nature. 2012;491:444–8.View ArticlePubMedGoogle Scholar
- A Global Phylogeny of Birds. http://birdtree.org. Accessed 25 Nov 2015.
- Sambrook J, Fritsch EFMT. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbour: Cold Spring Habour Laboratory Press; 1989.Google Scholar
- Chiari Y, Galtier N. RNA extraction from sauropsids blood: evaluation and improvement of methods. Amphibia-Reptilia. 2011;32:136–9.View ArticleGoogle Scholar
- Westerdahl H, Wittzell H, von Schantz T, Bensch S. MHC class I typing in a songbird with numerous loci and high polymorphism using motif-specific PCR and DGGE. Heredity (Edinb). 2004;92:534–42.View ArticleGoogle Scholar
- Kloch A, Babik W, Bajer A, Siński E, Radwan J. Effects of an MHC-DRB genotype and allele number on the load of gut parasites in the bank vole Myodes glareolus. Mol Ecol. 2010;19 Suppl 1:255–65.View ArticlePubMedGoogle Scholar
- Klein J, Bontrop RE, Dawkins RL, Erlich HA, Gyllensten UB, Heise ER, et al. Nomenclature for the major histocompatibility complexes of different species: a proposal. Immunogenetics. 1990;31:217–9.PubMedGoogle Scholar
- Letunic I, Bork P. Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res. 2016;44:W242–W245.View ArticlePubMedPubMed CentralGoogle Scholar
- Huson DH, Bryant D. Application of phylogenetic networks in evolutionary studies. Mol Biol Evol. 2006;23:254–67.View ArticlePubMedGoogle Scholar
- Posada D. jModelTest: Phylogenetic model averaging. Mol Biol Evol. 2008;25:1253–6.View ArticlePubMedGoogle Scholar
- R Core Team. In: RDC T, editor. R: A Language and Environment for Statistical Computing. Vienna: R Foundation for Statistical Computing; 2014.Google Scholar
- Borg AA, Pedersen SA, Jensen H, Westerdahl H. Variation in MHC genotypes in two populations of house sparrow (Passer domesticus) with different population histories. Ecol Evol. 2011;1:145–59.View ArticlePubMedPubMed CentralGoogle Scholar
- Reed KM, Bauer MM, Monson MS, Benoit B, Chaves LD, O’Hare TH, et al. Defining the turkey MHC: identification of expressed class I- and class IIB-like genes independent of the MHC-B. Immunogenetics. 2011;63:753–71.View ArticlePubMedGoogle Scholar
- Dimcheff DE, Drovetski SV, Mindell DP. Phylogeny of Tetraoninae and other galliform birds using mitochondrial 12S and ND2 genes. Mol Phylogenet Evol. 2002;24:203–15.View ArticlePubMedGoogle Scholar
- Van Tuinen M, Dyke GJ. Calibration of galliform molecular clocks using multiple fossils and genetic partitions. Mol Phylogenet Evol. 2004;30:74–86.View ArticlePubMedGoogle Scholar
- Jarvis E, Mirarab S, Aberer A, Li B, Houde P, Li C, et al. Whole-genome analyses resolve early branches in the tree of life of modern birds. Science. 2014;346:1320–31.View ArticlePubMedPubMed CentralGoogle Scholar
- Burri R, Promerová M, Goebel J, Fumagalli L. PCR-based isolation of multigene families: lessons from the avian MHC class IIB. Mol Ecol Resour. 2014;14:778–88.View ArticlePubMedGoogle Scholar
- Gaigher A, Burri R. Family-assisted inference of the genetic architecture of major histocompatibility complex variation; 2016. p. 1353–64.Google Scholar
- Milinski M. The Major Histocompatibility Complex, Sexual Selection, and Mate Choice. Annu Rev Ecol Evol Syst. 2006;37:159–86.View ArticleGoogle Scholar
- Kaufman J, Völk H, Wallny HJ. A “minimal essential Mhc” and an “unrecognized Mhc”: two extremes in selection for polymorphism. Immunol Rev. 1995;143:63–88.View ArticlePubMedGoogle Scholar
- Shiina T, Hosomichi K, Hanzawa K. Comparative genomics of the poultry major histocompatibility complex. Anim Sci J. 2006;77:151–62.View ArticleGoogle Scholar
- Fleming-canepa X, Jensen SM, Christine M, Diaz-satizabal L, Roth AJ, Parks-dely JA, et al. Extensive Allelic Diversity of MHC Class I in Wild Mallard Ducks. J Immunol. 2016;197:783–94.View ArticlePubMedGoogle Scholar
- Shiina T, Oka A, Imanishi T, Hanzawa K, Gojobori T, Watanabe S, et al. Multiple class I loci expressed by the quail Mhc. Immunogenetics. 1999;49:456–60.View ArticlePubMedGoogle Scholar
- Shiina T, Shimizu S, Hosomichi K, Kohara S, Watanabe S, Hanzawa K, et al. Comparative genomic analysis of two avian (quail and chicken) MHC regions. J Immunol. 2004;172:6751–63.View ArticlePubMedGoogle Scholar
- Walker BA, Hunt LG, Sowa AK, Skjødt K, Göbel TW, Lehner PJ, et al. The dominantly expressed class I molecule of the chicken MHC is explained by coevolution with the polymorphic peptide transporter (TAP) genes. Proc Natl Acad Sci U S A. 2011;108:8396–401.View ArticlePubMedPubMed CentralGoogle Scholar
- Drews A, Strandh M, Råberg L, Westerdahl H. Data from: Expression and phylogenetic analyses reveal paralogous lineages of putatively classical and non-classical MHC-I genes in three sparrow species (Passer). Dryad Digital Repository. http://0-dx.doi.org.brum.beds.ac.uk/10.5061/dryad.79t4b.