Abstract
The major histocompatibility complex (MHC) is central to the vertebrate immune system and its highly polymorphic genes are considered to influence several life-history traits of individuals. To characterize the MHC in a natural population of blue tits (Cyanistes caeruleus) we investigated the class I exon 3 diversity of more than 900 individuals. We designed two pairs of motif-specific primers that reliably amplify independent subsets of MHC alleles. Applying denaturing gradient gel electrophoresis (DGGE) we obtained 48 independently inherited units of unique band patterns (DGGE-haplogroups), which were validated in a segregation analysis within 105 families. In a second approach, we extensively sequenced 6 unrelated individuals to confirm that DGGE-haplogroup composition reflects individual allelic variation. The highest number of different DGGE-haplogroups in a single individual corresponded in 19 MHC exon 3 sequences, suggesting a minimum of 10 amplified MHC class I loci in the blue tit. In total, we identified 50 unique functional and 3 non-functional sequences. Functional sequences showed high levels of recombination and strong positive selection in the antigen binding region, whereas nucleotide diversity was comparatively low in the range of all passerine species. Finally, in a phylogenetic comparison of passerine MHC class I exon 3 sequences we discuss conflicting evolutionary signals possibly due to recent gene duplication, recombination events and concerted evolution. Our results indicate that the described method is suitable to effectively explore the MHC diversity and its ecological impacts in blue tits in future studies.
Similar content being viewed by others
References
Alcaide M, Edwards SV, Cadahía L, Negro JJ (2009) MHC class I genes of birds of prey: isolation, polymorphism and diversifying selection. Conserv Genet 10:1349–1355
Andersson S, Örnborg J, Andersson M (1998) Ultraviolet sexual dimorphism and assortative mating in blue tits. Proc R Soc Lond B 265:445–450
Anmarkrud JA, Johnsen A, Bachmann L, Lifjeld JT (2010) Ancestral polymorphism in exon 2 of bluethroat (Luscinia svecica) MHC class II B genes. J Evol Biol 23:1206–1217
Apanius V, Penn D, Slev PR, Ruff LR, Potts WK (1997) The nature of selection on the major histocompatibility complex. Crit Rev Immunol 17:179–224
Arriero E (2009) Rearing environment effects on immune defence in blue tit Cyanistes caeruleus nestlings. Oecologia 159:697–704
Ashelford AE, Chuzhanova NA, Fry JC, Jones AJ, Weightman AJ (2005) At least one in twenty 16S rRNA sequence records currently held in public repositories estimated to contain substantial anomalies. Appl Environ Microbiol 12:7724–7736
Ashelford KE, Chuzhanova NA, Fry JC, Jones AJ, Weightman AJ (2006) New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras. Appl Environ Microbiol 72:5734–5741
Balakrishnan CN, Ekblom R, Völker M, Westerdahl H, Godinez R, Kotkiewicz H, Burt DW, Graves T, Griffin DK, Warren WC (2010) Gene duplication and fragmentation in the zebra finch major histocompatibility complex. BMC Biol 8:29
Bensch S, Stjernman M, Hasselquist D, Ostmann O, Hansson B, Westerdahl H, Pinheiro RT (2000) Host specificity in avian blood parasites: a study of plasmodium and haemoproteus mitochondrial DNA amplified from birds. Proc R Soc Lond B 267:1583–1589
Bernatchez L, Landry C (2003) MHC studies in nonmodel vertebrates: what have we learned about natural selection in 15 years? J Evol Biol 16:363–377
Bodmer WF (1972) Evolutionary significance of the HL-A system. Nature 237:139–183
Bollmer JL, Vargas FH, Parker PG (2007) Low MHC variation in the endangered Galapagos penguin (Spheniscus mendiculus). Immunogenetics 59:593–602
Bollmer JL, Dunn PO, Wittingham LA, Wimpee C (2010) Extensive MHC class II B gene duplication in a passerine, the common yellowthroat (Geothlypis trichas). J Hered 101:448–460
Bonneaud C, Mazuc J, Chastel O, Westerdahl H, Sorci G (2004a) Terminal investment induced by immune challenge and fitness traits associated with major histocompatibility complex in the house sparrow. Evolution 58:2823–2830
Bonneaud C, Sorci G, Morin V, Westerdahl H, Zoorob R, Wittzell H (2004b) Diversity of Mhc class I and IIB genes in house sparrows (Passer domesticus). Immunogenetics 55:855–865
Bonneaud C, Chastel O, Federici P, Westerdahl H, Sorci G (2006a) Complex Mhc-based mate choice in a wild passerine. Proc R Soc B 273:1111–1116
Bonneaud C, Perez-Tris J, Federici P, Chastel O, Sorci G (2006b) Major histocompatibility alleles associated with local resistance to malaria in a passerine. Evolution 60:383–389
Bryant D, Moulton V (2004) Neighbor-net: an agglomerative method for the construction of phylogenetic networks. Mol Biol Evol 21(2):255–265
Burri R, Niculita-Hirzel H, Roulin A, Fumagalli L (2008a) Isolation and characterization of major histocompatibility complex (MHC) class II B genes in the Barn owl (Aves: Tyto alba). Immunogenetics 60:543–550
Burri R, Niculita-Hirzel H, Salamin N, Roulin A, Fumagalli L (2008b) Evolutionary patterns of MHC class II B in owls and their implications for the understanding of avian MHC evolution. Mol Biol Evol 25:1180–1191
Chaves LD, Krueth SB, Reed KM (2009) Defining the turkey MHC: sequence and genes of the B locus. Am Assoc Immnol 183:6530–6537
Delany ME, Robinson CM, Goto RM, Miller MM (2009) Architecture and organization of chicken microchromosome 16: order of the NOR, MHC-Y, and MHC-B subregions. J Hered 100(5):507–514
Doherty PC, Zinkernagel RM (1975) Enhanced immunological surveillance in mice heterozygous at the H-2 gene complex. Nature 256:50–52
Doutrelant C, Blondel J, Perret P, Lambrechts MM (2000) Blue tit song repertoire size, male quality and interspecific competition. J Avian Biol 31:360–366
Edwards SV, Dillon M (2004) Hitchhiking and recombination in birds: evidence from Mhc-linked and unlinked loci in Red-winged Blackbirds (Agelaius phoeniceus). Genet Res 84:175–192
Edwards SV, Wakeland EK, Potts WK (1995) Contrasting histories of avian and mammalian Mhc genes revealed by class II B sequences from songbirds. Proc Natl Acad Sci USA 92:12200–12204
Eimes JA, Bollmer JL, Dunn PO, Whittingham LA, Wimpee C (2010) Mhc class II diversity and balancing selection in greater prairie-chickens. Genetica 138:265–271
Ekblom R, Grahn M, Hoglund J (2003) Patterns of polymorphism in the MHC class II of a non-passerine bird, the great snipe (Gallinago media). Immunogenetics 54:734–741
Ekblom R, Stapley J, Ball AD, Birkhead T, Burke T, Slate J (2011) Genetic mapping of the major histocompatibility complex in the zebra finch (Taeniopygia guttata). Immunogenetics 63:523–530
Foerster K, Delhey K, Johnsen A, Lifjeld JT, Kempenaers B (2003) Females increase offspring heterozygosity and fitness through extra-pair matings. Nature 425:714–717
Foerster K, Valcu M, Johnsen A, Kempenaers B (2006) A spatial genetic structure and effects of relatedness on mate choice in a wild bird population. Mol Ecol 15:4555–4567
Freeman-Gallant CR, Johnson EM, Saponara F, Stanger M (2002) Variation at the major histocompatibility complex in Savannah sparrows. Mol Ecol 11(6):1125–1130
Garrigan D, Hedrick PW (2003) Perspective: detecting adaptive molecular polymorphism: lessons from the MHC. Evolution 57:1707–1722
Gasper JS, Shiina T, Inoko H, Edwards SV (2001) Songbird genomics: analysis of 45 kb upstream of a polymorphic Mhc class II gene in red-winged blackbirds (Agelaius phoeniceus). Genomics 75:26–34
Gill FB, Slikas B, Sheldon FH (2005) Phylogeny of titmice (Paridae): II. Species relationships based on sequences of the mitochondrial cytochrome-B gene. Auk 122:121–143
Gillingham MAF, Richardson DS, Løvlie H, Moynihan A, Worley K, Pizzari T (2009) Cryptic preference for MHC-dissimilar females in male red junglefowl, Gallus gallus. Proc R Soc B 276:1083–1092
Guillemot F, Billault A, Pourquie O, Behar G, Chausse AM, Zoorob R, Kreibich G, Auffray C (1988) A molecular map of the chicken major histocompatibility complex: the class II beta genes are closely linked to the class I genes and the nucleolar organizer. EMBO J 7:2775–2785
Guindon S, Gascuel O (2008) A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52(5):696–704
Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98
Hedrick PW (1999) Balancing selection and MHC. Genetica 104:207–214
Hess CM, Edwards SV (2002) The evolution of the major histocompatibility complex in birds. Bioscience 52:423–431
Hudson R, Kaplan N (1985) Statistical properties of the number of recombination events in the history of a sample of DNA sequences. Genetics 111:147–164
Hughes CR, Miles S, Walbroehl JM (2008) Support for the minimal essential MHC hypothesis: a parrot with a single, highly polymorphic MHC class II B gene. Immunogenetics 60:219–231
Huson DH, Bryant D (2006) Application of phylogenetic networks in evolutionary studies. Mol Biol Evol 23(2):254–267
Jarvi SI, Tarr CL, McIntosh CE, Atkinson CT, Fleischer RC (2004) Natural selection of the major histocompatibility complex (Mhc) in Hawaiian honeycreepers (Drepanidinae). Mol Ecol 13:2157–2168
Johansson US, Fjeldså J, Bowie RCK (2008) Phylogenetic relationships within Passerida (Aves: Passeriformes): a review and a new molecular phylogeny based on three nuclear intron markers. Mol Phylogenet Evol 48:858–876
Johnsen A, Delhey K, Andersson S, Kempenaers B (2003) Plumage colour in nestling blue tits: sexual dichromatism, condition dependence and genetic effects. Proc R Soc Lond B 270:1263–1270
Kaufman J, Salomonsen J, Flajnik M (1994) Evolutionary conservation of MHC class I and class II molecules-different yet the same. Semin Immunol 6:411–424
Kaufman J, Milne S, Gobel TWF, Walker BA, Jacob JP, Auffray C, Zoorob R, Beck S (1999) The chicken B locus is a minimal essential major histocompatibility complex. Nature 401:923–925
Kempenaers B, Verheyen GR, Vandenbroeck M, Burke T, Vanbroeckhoven C, Dhondt AA (1992) Extra-pair paternity results from female preference for high-quality males in the blue tit. Nature 357:494–496
Kempenaers B, Verheyren GR, Dhondt AA (1997) Extrapair paternity in the blue tit (Parus caeruleus): female choice, male characteristics, and offspring quality. Behav Ecol 8:481–492
Klein J (1986) Natural history of the major histocompatibility complex. Wiley, New York
Klein J (1987) Origin of major histocompatibility complex polymorphism: the trans-species hypothesis. Hum Immunol 19:155–162
Koch M, Camp S, Collen T, Avila D, Salomonsen J, Wallny HJ, van Hateren A, Hunt L, Jacob JP, Johnston F, Marston DA, Shaw I, Dunbar PR, Cerundolo V, Jones EY, Kaufman J (2007) Structures of an MHC class I molecule from B21 chickens illustrate promiscuous peptide binding. Immunity 27:885–899
Langefors Å, Lohm J, Von Schantz T, Grahn M (2000) Screening of Mhc variation in Atlantic salmon (Salmo salar): a comparison of restriction fragment length polymorphism (RFLP), denaturing gradient gel electrophoresis (DGGE) and sequencing. Mol Ecol 9:215–219
Loiseau C, Zoorob R, Garnier S, Birard J, Federici P, Julliard R, Sorci G (2008) Antagonistic effects of a Mhc class I allele on malaria-infected house sparrows. Ecology 11:258–265
Merino S, Moreno J, José Sanz J, Arriero E (2000) Are avian blood parasites pathogenic in the wild? A medication experiment in blue tits (Parus caeruleus). Proc R Soc Lond B 267:2507–2510
Mesa CM, Thulien KJ, Moon DA, Veniamin SM, Magor KE (2004) The dominant MHC class I gene is adjacent to the polymorphic TAP2 gene in the duck, Anas platyrhynchos. Immunogenetics 56:192–203
Milinski M (2003) The function of mate choice in sticklebacks: optimizing Mhc genetics. J Fish Biol 63:1–16
Milinksi M (2006) The major histocompatibility complex, sexual selection, and mate choice. Annu Rev Ecol Evol Syst 37:159–186
Miller HC, Lambert DM (2004) Gene duplication and gene conversion in class II MHC genes of New Zealand robins (Petroicidae). Immunogenetics 56:178–191
Miller KM, Ming TB, Schulze AD, Withler RE (1999) Denaturing gradient gel electrophoresis (DGGE): a rapid and sensitive technique to screen nucleotide sequence variation in populations. Biotechniques 27:1016–1030
Miller MM, Bacon LD, Hala K, Hunt HD, Ewald SJ, Kaufman J, Zoorob R, Briles WE (2004) 2004 Nomenclature for the chicken major histocompatibility (B and Y) complex. Immunogenetics 56:261–279
Miller HC, Bowker-Wright G, Kharkrang M, Ramstad K (2011) Characterisation of class II B MHC genes from a ratite bird, the little spotted kiwi (Apteryx owenii). Immunogenetics 63:223–233
Møller AP (1999) Good-genes effects in sexual selection. Proc Biol Sci 226:85–91
Murphy KP, Travers P, Walport M (2008) Janeway’s immunobiology, 7th edn. Taylor & Francis, London
Myers RM, Maniatis T, Lerman LS (1987) Detection and localization of single base changes by denaturing gradient gel electrophoresis. Meth Enzymol 155:501–527
Neff BD, Pitcher TE (2005) Genetic quality and sexual selection: an integrated framework for good genes and compatible genes. Mol Ecol 14:19–38
Nei M, Rooney AP (2005) Concerted and birth-and-death evolution of multigene families. Annu Rev Genet 39:121–152
Ohta T (1999) Effect of gene conversion on polymorphic patterns at major histocompatibility complex loci. Immunol Rev 167:319–325
Parker TH, Barr IR, Griffith SC (2006) The blue tit’s song is an inconsistent signal of male condition. Behav Ecol 17:1029–1040
Piertney SB, Oliver MK (2006) The evolutionary ecology of the major histocompatibility complex. Heredity 96:7–21
Poesel A, Foerster K, Kempenaers B (2001) The dawn song of the blue tit Parus caeruleus and its role in sexual selection. Ethology 107:521–531
Pond SLK, Frost SDW (2005a) Datamonkey: rapid detection of selective pressure on individual sites of codon alignments. Bioinformatics 21:2531–2533
Pond SLK, Frost SDW (2005b) Not so different after all: a comparison of methods for detecting amino acid sites under selection. SMBE 22:1208–1222
Pond SLK, Frost SDW, Muse SV (2005) HyPhy: hypothesis testing using phylogenies. Bioinformatics 21(5):676–679
Posada (2009) Selection of models of DNA evolution with jModelTest. Methods Mol Biol 537:93–112
Potts WK, Wakeland EK (1990) Evolution of diversity at the major histocompatibility complex. Trends Ecol Evol 5:181–187
Potts WK, Wakeland EK (1993) Evolution of Mhc genetic diversity—a tale of incest, pestilence and sexual preference. Trends Genet 9:408–412
Promerová M, Albrecht T, Bryja J (2009) Extremely high MHC class I variation in a population of a long-distance migrant, the Scarlet Rosefinch (Carpodacus erythrinus). Immunogenetics 61:451–461
Reusch TBH, Haberli MA, Aeschlimann PB, Milinski M (2001) Female sticklebacks count alleles in a strategy of sexual selection explaining MHC polymorphism. Nature 414:300–302
Richardson DS, Westerdahl H (2003) MHC diversity in two Acrocephalus species: the outbred Great reed warbler and the inbred Seychelles warbler. Mol Ecol 12:3523–3529
Richardson DS, Komdeur J, Burke T, von Schantz T (2005) MHC-based patterns of social and extra-pair mate choice in the Seychelles warbler. Proc R Soc B 272:759–767
Rozas J (1999) DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis. Oxford University Press, Oxford, pp 174–175
Sato A, Mayer WE, Tichy H, Grant PR, Grant BR, Klein J (2001) Evolution of Mhc class II B genes in Darwin’s finches and their closest relatives: birth of a new gene. Immunogenetics 53:792–801
Sato A, Tichy H, Grant PR, Grant BR, Sato T, O’hUigin C (2011) Spectrum of MHC Class II variability in Darwin’s finches and their close relatives. Mol Biol Evol 28(6):1943–1956
Sawyer SA (1989) Statistical tests for detecting gene conversion. Mol Biol Evol 6:526–538
Schut E, Aguilar JR, Merino S, Magrath MJL, Komdeur J, Westerdahl H (2011) Characterization of MHC-I in the blue tit (Cyanistes caeruleus) reveals low levels of genetic diversity and trans-population evolution across European populations. Immunogenetics 63:531–542
Seutin G, White BN, Boag P (1991) Preservation of avian blood and tissue for DNA analyses. Can J Zool 69:82–90
Sheldon FH, Gill FB (1996) A reconsideration of songbird phylogeny, with emphasis on the evolution of titmice and their sylvioid relatives. Syst Boil 45:473–495
Shiina T, Shimizu S, Hosomichi K, Kohara S, Watanabe S, Hanzawa K, Beck S, Kulski JK, Inoko H (2004) Comparative genomic analysis of two avian (quail and chicken) MHC regions. J Immunol 172:6751–6763
Slikas B, Sheldon FH, Gill FB (1996) Phylogeny of titmice (Paridae): I. Estimate of relationships among subgenera based on DNA–DNA hybridization. J Avian Biol 27:70–82
Sommer S (2005) The importance of immune gene variability (MHC) in evolutionary ecology and conservation. Front Zool 2:16–34
Stjernman M, Raberg L, Nilsson JA (2004) Survival costs of reproduction in the blue tit (Parus caeruleus): a role for blood parasites? Proc R Soc Lond 271:2387–2394
Strand T, Westerdahl H, Hoeglund J, Alatalo RV, Siitari H (2007) The Mhc class II of the Black grouse (Tetrao tetrix) consists of low numbers of B and Y genes with variable diversity and expression. Immunogenetics 59:725–734
Strandh M, Lannefors M, Bonadonna F, Westerdahl H (2011) Characterization of MHC class I and II genes in a subantarctic seabird, the blue petrel, Halobaena caerulea (Procellariiformes). Immunogenetics 63:653–666
Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. SMBE 24:1596–1599
Tomás G, Merino S, Moreno J, Sanz JJ, Morales J, García-Fraile S (2006) Nest weight and female health in the blue tit (Cyanistes caeruleus). Auk 123:1013–1021
Tomás G, Merino S, Moreno J, Morales J (2007) Consequences of nest reuse for parasite burden and female health and condition in blue tits, Cyanistes caeruleus. Anim Behav 73:805–814
Wallny H, Avila D, Hunt L, Powell T, Riegert P, Salomonsen J, Skjodt K, Vainio O, Vilbois F, Wiles M, Kaufman J (2006) Peptide motifs of the single dominantly expressed class I molecule explain the striking MHC-determined response to Rous sarcoma virus in chicken. Proc Natl Acad Sci USA 103:1434–1439
Wegner K, Kalbe M, Schaschl H, Reusch T (2004) Parasites and individual major histocompatibility complex diversity-an optimal choice? Microbes Infect 6:1110–1116
Westerdahl H (2004) No evidence of an MHC-based female mating preference in great reed warblers. Mol Ecol 13:2465–2470
Westerdahl H (2007) Passerine MHC: genetic variation and disease resistance in the wild. J Ornithol 148:469–477
Westerdahl H, Wittzell H, von Schantz T (1999) Polymorphism and transcription of Mhc class I genes in a passerine bird, the great reed warbler. Immunogenetics 49:158–170
Westerdahl H, Wittzell H, von Schantz T, Bensch S (2004) MHC class I typing in a songbird with numerous loci and high polymorphism using motif-specific PCR and DGGE. Heredity 92:534–542
Westerdahl H, Waldenström J, Hansson B, Hasselquist D, von Schantz T, Bensch S (2005) Associations between malaria and MHC genes in a migratory songbird. Proc R Soc B 272:1511–1518
Wittzell H, Madsen T, Westerdahl H, Shine R, von Schantz T (1998) MHC variation in birds and reptiles. Genetica 104:301–309
Wittzell H, Bernot A, Auffray C, Zoorob R (1999) Concerted evolution of two Mhc class II B loci in pheasants and domestic chickens. Mol Biol Evol 16:479–490
Yang Z (1997) PAML: a program package for phylogenetic analysis by maximum likelihood. Comput Appl Biosci 13(5):555–556
Yang Z (2007) PAML 4: a program package for phylogenetic analysis by maximum likelihood. Mol Biol Evol 24:1586–1591
Yang Z, Wong WSW, Nielsen R (2005) Bayes empirical bayes inference of amino acid sites under positive selection. Mol Biol Evol 22(4):1107–1118
Ziegler A, Kentenich H, Uchanska-Ziegier B (2005) Female choice and the MHC. Trends Immunol 26:496–502
Acknowledgments
We thank K. Persson, K. Teltscher, U. Holter and S. Kuhn for their excellent laboratory work and to all the field assistants who were involved in the Kolbeterberg project. We are grateful to H. Westerdahl for her help in designing the optimal primers and her support throughout the study. Furthermore we are indebted to C. Oppelt, for his inspiring comments on the manuscript. The present study was funded by the German Research foundation (DFG KE 867/1) and the Max Planck Society.
Conflict of interest
The authors declare that they have no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Wutzler, R., Foerster, K. & Kempenaers, B. MHC class I variation in a natural blue tit population (Cyanistes caeruleus). Genetica 140, 349–364 (2012). https://doi.org/10.1007/s10709-012-9679-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10709-012-9679-0