Blood was collected from 36 individuals of wild bird Carduelis species (Table 1). Distribution and cytochrome b GenBank sequence accession numbers are given in Table 1. Blood was obtained from wild birds in their natural thriving areas, and kept at 4°C with an ethylenediaminetetraacetic acid (EDTA) solution until use [26Arnaiz-Villena A, Ruiz-del-Valle V, Reche P, et al. Songbirds conserved sites and intron size of MHC Class I molecules reveal a unique evolution in vertebrates. Open Ornithol J 2010; 3: 156-65.
[http://dx.doi.org/10.2174/1874453201003010156] ].

Table 1
Origin and Cytochrome b GenBank accession numbers of the species analyzed in this study.

DNA was isolated from whole blood with an automatic DNA extracting device (Nucleic Acid Extraction System. QuickGene-810, FUJIFILM) after treating samples with a commercial kit (QuickGene Whole Blood Extraction Kit S, FUJIFILM). DNA concentration was measured with a spectrophotometer (ND-1000, NANODROP), and adjusted to about 100 ng/μl. Finally, samples were stored at -20°C.

Sequences of the MHC molecule most variable region (exon 2, intron 2, exon 3) were amplified by the polymerase chain reaction (PCR) method [27Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 1987; 155: 335-50.
[http://dx.doi.org/10.1016/0076-6879(87)55023-6] [PMID: 3431465] ] in 42 cycles (10 s at 95°C, 30 s at 65°C, 60 s at 72°C) using an EPPENDORF thermocycler and AmpliTaq DNA Polymerase (APPLIED BIOSYSTEMS) [28Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985; 230(4732): 1350-4.
[http://dx.doi.org/10.1126/science.2999980] [PMID: 2999980] ]. Primers were used as described in [26Arnaiz-Villena A, Ruiz-del-Valle V, Reche P, et al. Songbirds conserved sites and intron size of MHC Class I molecules reveal a unique evolution in vertebrates. Open Ornithol J 2010; 3: 156-65.
[http://dx.doi.org/10.2174/1874453201003010156] ]. Fragments of about 850 base pairs were obtained; some of them were purified by electrophoresis in a 2% agarose gel in order to verify the amplification process. Sequencing was performed using the Sanger method [29Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977; 74(12): 5463-7.
[http://dx.doi.org/10.1073/pnas.74.12.5463] [PMID: 271968] ], using the same primers as for amplification, plus an internal primer: 5'- GGATTGATGTGGCTCCAAGG-3'. Ambiguities due to heterocygosity were solved by cloning in competent Escherichia coli cells. An average of 12 different cloned sequences per individual were obtained. Amplification and sequencing of cyt b gene 924 base pairs (bp) was performed as previously described [30Zamora J, Lowy E, Ruiz-del-Valle V, et al. Rhodopechys obsoleta (desert finch): A pale ancestor of greenfinches (Carduelis spp.) according to molecular phylogeny. J Ornithol 2006; 147: 448-56.
[http://dx.doi.org/10.1007/s10336-005-0036-2] ].

Table 2
MHC class I DNA alleles found in wild Carduelis individuals. GenBank accession numbers are also shown.

MEGA 5.0 software [31Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011; 28(10): 2731-9.
[http://dx.doi.org/10.1093/molbev/msr121] [PMID: 21546353] ] was used to align MHC sequences and translate DNA sequences into protein, for each individual and clone. Different alleles were identified and characterized manually.

Regarding to cyt-b gene, sequences were further analyzed with MEGA 5.0 as described [31Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011; 28(10): 2731-9.
[http://dx.doi.org/10.1093/molbev/msr121] [PMID: 21546353] ]. Phylogenetic dendrograms were obtained using Maximum Likelihood (ML) methodology [32Felsenstein J. Evolutionary trees from DNA sequences: A maximum likelihood approach. J Mol Evol 1981; 17(6): 368-76.
[http://dx.doi.org/10.1007/BF01734359] [PMID: 7288891] ] with PAUP* v. 4.0b10 program [33Swofford DL. PAUP* Phylogenetic Analysis Using Parsimony (* and other methods) version 4 2002.] and Bayesian Inference (BI) methodology using MrBayes program [34Huelsenbeck JP, Ronquist F. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 2001; 17(8): 754-5.
[http://dx.doi.org/10.1093/bioinformatics/17.8.754] [PMID: 11524383] , 35Ronquist F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 2003; 19(12): 1572-4.
[http://dx.doi.org/10.1093/bioinformatics/btg180] [PMID: 12912839] ]. Model test v. 3.7 [36Posada D, Crandall KA. MODELTEST: Testing the model of DNA substitution. Bioinformatics 1998; 14(9): 817-8.
[http://dx.doi.org/10.1093/bioinformatics/14.9.817] [PMID: 9918953] ] was used to find out a DNA substitution model that fits the data best. Also, best model was used prior to both ML and BI analyses. Linearized ML dendrograms were obtained with PAUP* v. 4.0b10 [33Swofford DL. PAUP* Phylogenetic Analysis Using Parsimony (* and other methods) version 4 2002.] with the estimated branch length [37Thorne JL, Kishino H, Painter IS. Estimating the rate of evolution of the rate of molecular evolution. Mol Biol Evol 1998; 15(12): 1647-57.
[http://dx.doi.org/10.1093/oxfordjournals.molbev.a025892] [PMID: 9866200] ] which assumes that the rates among the evolutionary lineages may not be constant. Tree calculation strategy consisted of a heuristic search with NNI (Nearing Neighbour Interchange) swapping algorithm. Robustness of nodes was assessed by 1000 bootstrap replicates in the ML analyses. The parameters rates defining the model of evolution were allowed to change in the BI analysis after each generation in order to increase the likelihood of resulting trees. Therefore, none of the parameters were a priori fixed. In BI analyses, two independent runs (with one cold and three heated chains each) were performed along with 5 million generations. Trees were sampled every 100 generations and the first 12,500 samples were discarded as ‘burn-in’. Split frequencies average standard deviation approached to zero being around 0.01 at the end of the analysis. Posterior probability values (ppv) indicate the robustness of the nodes in the BI. In Phylogenetic analyses chaffinch (Fringilla coelebs) (family Fringillidae, subfamily Fringillinae), was used as outgroup.

The peopling of America continent by genus Carduelis species could be carried out by three rapid radiations according to extant present day species: A Mesoamerican goldfinch radiation, a North American siskin radiation and a South American siskin radiation [11Arnaiz-Villena A, Areces C, Rey D, et al. Three different North American Siskin/Goldfinch Evolutionary Radiations (Genus Carduelis): Pine Siskin Green Morphs and European siskins in America. Open Ornithol J 2012; 5: 73-81.
[http://dx.doi.org/10.2174/1874453201205010073] ]. In this work, species from South American and North American groups have been studied. The age of appearance on Earth of the extant ancestor of North American group, Eurasian siskin (C. spinus) is approximately 5 million years ago [8Arnaiz-Villena A, Alvarez-Tejado M, Ruíz-del-Valle V, et al. Phylogeny and rapid northern and southern hemisphere speciation of goldfinches during the Miocene and Pliocene epochs. Cell Mol Life Sci 1998; 54(9): 1031-41.
[http://dx.doi.org/10.1007/s000180050230] [PMID: 9791543] , 11Arnaiz-Villena A, Areces C, Rey D, et al. Three different North American Siskin/Goldfinch Evolutionary Radiations (Genus Carduelis): Pine Siskin Green Morphs and European siskins in America. Open Ornithol J 2012; 5: 73-81.
[http://dx.doi.org/10.2174/1874453201205010073] , 12Arnaiz-Villena A, Ruiz-del-Valle V, Reguera R, Gomez-Prieto P, Serrano-Vela JI. What might have been the ancestor of New World siskins? Open Ornithol J 2008; 1: 46-7.
[http://dx.doi.org/10.2174/1874453200801010046] ] (Figs. 1 and 2). It is suggested that this bird passed to America through Beringia/Aleutian Islands [38McLaren IA, Morlan J, Smith P, Gosselin M, Bailey SF. Eurasian siskins in North America - distinguishing females from green-morp pine siskins. American Birds 1989; 43: 1268-74.], since there have been sightings of these birds in areas near these islands. However, it is possible that C. spinus entering to America came also through the East Coast (Greenland, Iceland, Newfoundland): both East and West entering may have possible [11Arnaiz-Villena A, Areces C, Rey D, et al. Three different North American Siskin/Goldfinch Evolutionary Radiations (Genus Carduelis): Pine Siskin Green Morphs and European siskins in America. Open Ornithol J 2012; 5: 73-81.
[http://dx.doi.org/10.2174/1874453201205010073] ]. Eurasian siskin (or extinct close relative) evolves to Antillean siskin (C. dominicensis) during the Pliocene Epoch due to a geographical isolation after reaching Antillean area. Pine siskin (C. pinus) seems to be the descendent of Antillean siskin (Figs. 1 and 2). Regarding the South American siskin group, the Black-headed siskin (C. notata) is suggested to be as extant ancestor of this group. South American radiation occurred probably after 3 million years ago, and C. notata or an extinct ancestor passed to South America from Mexican mountains, probably after Isthmus of Panama emerged [11Arnaiz-Villena A, Areces C, Rey D, et al. Three different North American Siskin/Goldfinch Evolutionary Radiations (Genus Carduelis): Pine Siskin Green Morphs and European siskins in America. Open Ornithol J 2012; 5: 73-81.
[http://dx.doi.org/10.2174/1874453201205010073] ]. This fact favoured the invasion of mesothermal plants from the Rocky Mountains to Andean Spine causing the expansion of this species Carduelis / Spinus genus and triggering the radiation [11Arnaiz-Villena A, Areces C, Rey D, et al. Three different North American Siskin/Goldfinch Evolutionary Radiations (Genus Carduelis): Pine Siskin Green Morphs and European siskins in America. Open Ornithol J 2012; 5: 73-81.
[http://dx.doi.org/10.2174/1874453201205010073] ].

North American siskin group and South American siskin group are closely related. Both were separated almost 5 million years ago (Fig. 1). However, the common precursor of South and North American radiations, namely, the link between Eurasian siskin (C. spinus) and Black-headed siskin (C. notata) is missing, as well as the common ancestor of all three groups (North, Meso and South radiations) [11Arnaiz-Villena A, Areces C, Rey D, et al. Three different North American Siskin/Goldfinch Evolutionary Radiations (Genus Carduelis): Pine Siskin Green Morphs and European siskins in America. Open Ornithol J 2012; 5: 73-81.
[http://dx.doi.org/10.2174/1874453201205010073] ], unless that C. spinus was the American siskin ancestor and several links are missed [12Arnaiz-Villena A, Ruiz-del-Valle V, Reguera R, Gomez-Prieto P, Serrano-Vela JI. What might have been the ancestor of New World siskins? Open Ornithol J 2008; 1: 46-7.
[http://dx.doi.org/10.2174/1874453200801010046] ].

Fig. (1) Linearized Maximum likelihood dendrogram based on mitochondrial cytochrome b DNA sequences. Fringilla coelebs was chosen as outgroup.

Fig. (2) Linearized Bayesian dendrogram based on mitochondrial cytochrome b DNA sequences. Fringilla coelebs was chosen as outgroup.

Once collected all MHC DNA sequences, they were classified according to DNA coding groups of similar or identical proteins. Then, a new provisional nomenclature of alleles was carried out according to the proposal of J. Klein [39Klein J, Bontrop RE, Dawkins RL, et al. Nomenclature for the major histocompatibility complexes of different species: A proposal. Immunogenetics 1990; 31(4): 217-9.
[http://dx.doi.org/10.1007/BF00204890] [PMID: 2329006] ], (Fig. (3) footnote). The MHC locus was generically identified with the letter F, referring to chicken class I genes (BF complex), since found alleles have been compared to human and other species class I molecules and found to belong to class I type of MHC molecules [26Arnaiz-Villena A, Ruiz-del-Valle V, Reche P, et al. Songbirds conserved sites and intron size of MHC Class I molecules reveal a unique evolution in vertebrates. Open Ornithol J 2010; 3: 156-65.
[http://dx.doi.org/10.2174/1874453201003010156] ]. MHC class I molecules often different genus Carduelis species have been studied in the present work. Details of appearance on Earth and habitat of South American siskins are accounted in Table 2 footnote (low case letter).

Eurasian siskin (C. spinus) thrives in Paleartic and Oriental forests and conifer woodland in summer, whereas in winter it is observed in common weedy areas, plantations and gardens [4Clement P, Harris A, Davis J. Finches and Sparrows 1999.]. Eurasian siskin appeared on Earth 5 million years ago: this is observed in the linearized Maximum Likelihood dendrogram (Fig. 1). It is the extant ancestor of the North American goldfinch group [1Arnaiz-Villena A, Guillén J, Ruiz-del-Valle V, et al. Phylogeography of crossbills, bullfinches, grosbeaks, and rosefinches. Cell Mol Life Sci 2001; 58(8): 1159-66.
[http://dx.doi.org/10.1007/PL00000930] [PMID: 11529508] , 7Arnaiz-Villena A, Gomez-Prieto P, Ruiz-del-Valle V. Phylogeography of finches and sparrows 2009; 1-54., 8Arnaiz-Villena A, Alvarez-Tejado M, Ruíz-del-Valle V, et al. Phylogeny and rapid northern and southern hemisphere speciation of goldfinches during the Miocene and Pliocene epochs. Cell Mol Life Sci 1998; 54(9): 1031-41.
[http://dx.doi.org/10.1007/s000180050230] [PMID: 9791543] , 10Arnaiz-Villena A, Ruiz-del-Valle V, Moscoso J, Serrano-Vela JI, Zamora J. mtDNA phylogeny of North American Carduelis pinus group. Ardeola 2007; 54: 1-14., 12Arnaiz-Villena A, Ruiz-del-Valle V, Reguera R, Gomez-Prieto P, Serrano-Vela JI. What might have been the ancestor of New World siskins? Open Ornithol J 2008; 1: 46-7.
[http://dx.doi.org/10.2174/1874453200801010046] ]. In this study, twenty different alleles of C. spinus were found in 14 individuals (Table 2). This species may have been ancestor of all North American siskins [1Arnaiz-Villena A, Guillén J, Ruiz-del-Valle V, et al. Phylogeography of crossbills, bullfinches, grosbeaks, and rosefinches. Cell Mol Life Sci 2001; 58(8): 1159-66.
[http://dx.doi.org/10.1007/PL00000930] [PMID: 11529508] , 7Arnaiz-Villena A, Gomez-Prieto P, Ruiz-del-Valle V. Phylogeography of finches and sparrows 2009; 1-54., 8Arnaiz-Villena A, Alvarez-Tejado M, Ruíz-del-Valle V, et al. Phylogeny and rapid northern and southern hemisphere speciation of goldfinches during the Miocene and Pliocene epochs. Cell Mol Life Sci 1998; 54(9): 1031-41.
[http://dx.doi.org/10.1007/s000180050230] [PMID: 9791543] , 10Arnaiz-Villena A, Ruiz-del-Valle V, Moscoso J, Serrano-Vela JI, Zamora J. mtDNA phylogeny of North American Carduelis pinus group. Ardeola 2007; 54: 1-14., 12Arnaiz-Villena A, Ruiz-del-Valle V, Reguera R, Gomez-Prieto P, Serrano-Vela JI. What might have been the ancestor of New World siskins? Open Ornithol J 2008; 1: 46-7.
[http://dx.doi.org/10.2174/1874453200801010046] ]. Its close relative Pine siskin (C. pinus) inhabits in all North America and goes South to Guatemala. It may be observed in conifer forests, plantations, thickets and shrubs [4Clement P, Harris A, Davis J. Finches and Sparrows 1999.]. This bird is living on Earth since 200,000 years ago, as it is deduced from Fig. (1). Eight different alleles of pine siskin were found in 6 individuals in this present study (Table 2).

DNA alleles were compared with each other (Table 2). Similarities were found: Caat-F*0101 C. atrata allele also appears in C. olivacea (Caol -F*0201) and Caat-F*0401 C. atrata allele is present in other 6 species of South American siskins, such as C. spinescens (Caspe-F*0101), C. xanthogastra (Caxa-F*0101), C. olivacea (Caol-F*0101), C. notata (Cano-F*0101), C. magellanica (Cama-F*0101) and C. cucullata (Cacu-F*0102).

Fig. (3) Map which shows the geographic location of species which share MHC-I proteins. Proteins of different species are named as follows: Carduelis spinus (Casp-F*); Carduelis pinus (Capi-F*); Carduelis notata (Cano-F*); Carduelis spinescens (Caspe-F*); Carduelis olivacea (Caol-F*); Carduelis atrata (Caat-F*); Carduelis magellanica (Cama-F*); Carduelis yarrellii (Caya-F*); Carduelis xanthogastra (Caxa-F*) and Carduelis cucullata (Cacu-F*). Color of the circles indicates the three different proteins found and number inside each circle indicates species that share each protein (Fig. 4).

Fig. (4) Carduelis spinus (left side) and Carduelis atrata (right side). Carduelis spinus appeared on Earth 5 MYA and Carduelis atrata appeared on Earth 0.5-1 MYA [8Arnaiz-Villena A, Alvarez-Tejado M, Ruíz-del-Valle V, et al. Phylogeny and rapid northern and southern hemisphere speciation of goldfinches during the Miocene and Pliocene epochs. Cell Mol Life Sci 1998; 54(9): 1031-41. [http://dx.doi.org/10.1007/s000180050230] [PMID: 9791543] , 10Arnaiz-Villena A, Ruiz-del-Valle V, Moscoso J, Serrano-Vela JI, Zamora J. mtDNA phylogeny of North American Carduelis pinus group. Ardeola 2007; 54: 1-14.-12Arnaiz-Villena A, Ruiz-del-Valle V, Reguera R, Gomez-Prieto P, Serrano-Vela JI. What might have been the ancestor of New World siskins? Open Ornithol J 2008; 1: 46-7. [http://dx.doi.org/10.2174/1874453200801010046] ].

When MHC protein alleles are compared, it is observed that C. spinus shared a allele with C. pinus (Casp-F*07 = Capi-F*08) and another one with C. atrata (Casp-F*01 = Caat-F*01) (Fig. 3). Furthermore, if South American siskins proteins are considered, it is observed that Casp-F*01 protein is the same one than Caol-F*02 (C. olivacea) and Caya-F*01 (C. yarrellii). Fig. (3) shows one the most shared protein (blue) that is present in 8 species of South American siskins; the most worldwide extended protein (red) appears both in Europe and in South America. And finally, there are 3 species which share 2 proteins (C. olivacea, C. atrata y C. yarrellii, blue and red).

Therefore a total of 2 DNA allele sequences present in 7 different species were found: (C. atrata (Caat-F*0401), C. spinescens (Caspe-F*0101), C. xanthogastra (Caxa-F*0101), C. olivacea (Caol-F*0101), C. notata (Cano-F*0101), C. magellanica (Cama-F*0101), C. cucullata (Cacu-F*0102)) and in 2 (C. atrata (Caat-F*0101) and C. olivacea (Caol -F*0201)) and 3 protein allele sequences are showed in 8 different species (C. notata (Cano-F*01), C. olivacea (Caol-F*01), C. xanthogastra (Caxa-F*01), C. yarrellii (Caya-F*02); C. magellanica (Cama-F*01), C. spinus (Casp-F*01); C. cucullata (Cacu-F*01); C. atrata (Caat-F*04)), 4 (C. spinus (Casp-F*01), C. olivacea (Caol-F*02), C. yarrellii (Caya-F*01), C. atrata (Caat-F*01)) and 2 (C. spinus (Casp-F*07), C. pinus (Capi-F*08)). South American siskins are the species that most sequences have in common, and the Eurasian siskin (C. spinus) shares proteins with the pine siskin (C. pinus) and even South American siskins C. atrata, C. olivacea and C. yarrellii (Fig. 3). A preliminary comparison of these DNA sequences with those found in Passer domesticus class I sequences [40Bonneaud C, Sorci G, Morin V, et al. Diversity of Mhc class I and IIB genes in house sparrows (Passer domesticus). Immunogenetics 2004; 55(12): 855-65.
[http://dx.doi.org/10.1007/s00251-004-0648-3] [PMID: 14963619] ] do not show close relatedness with this other species.

Eurasian siskin migratory behaviour is unpredictable each year: its North to South migrations do not always follows the same longitudinal patterns (it is “irruptive”) [4Clement P, Harris A, Davis J. Finches and Sparrows 1999.]. Nowadays, this bird does not thrive in America, but it lives in easternmost and westernmost Eurasia, being a gap between Central Russia and its easternmost range. It is possible that Eurasian siskin was thriving in Eurasia and also in North America about Pliocene / Pleistocene Epoch limits, approximately 2 million years ago. Later, the Eurasian siskin might have advanced to Caribbean Islands and to Mexican mountains and Guatemalan-Mexican altiplano. About 200,000 years ago, the Eurasian siskin might have given rise to pine siskin (C. pinus) in Mexican sierras and to Antillean siskin (C. dominicensis), nowadays isolated in Hispaniola Island [11Arnaiz-Villena A, Areces C, Rey D, et al. Three different North American Siskin/Goldfinch Evolutionary Radiations (Genus Carduelis): Pine Siskin Green Morphs and European siskins in America. Open Ornithol J 2012; 5: 73-81.
[http://dx.doi.org/10.2174/1874453201205010073] ,41Lomolino MV, Riddle BR, Brown JH. Biogeography 3^rd ed. 2006.]; documented rainfall variations in the Caribbean during the Pleistocene, however, could have also affected distribution of these birds [42Pregill GK, Olson SL. Zoogeography of west indian vertebrates in relation to pleistocene climatic cycles. Annu Rev Ecol Syst 1981; 12: 75-98.
[http://dx.doi.org/10.1146/annurev.es.12.110181.000451] ]. This may be a typical example of adaptive radiation originated by a North to South migration barrier and provincialism that drove evolution to create these new finch species. Last Wisconsin Glaciation ended and North American ice melted about 12,000 years ago: pine siskin would have followed the ancestral North to South migrations observed today and covered all North America. It might occupy American niches of Eurasian siskin which could not reach America from Asia during the last 2 million years because of an extant thick ice shield. Neither could it later because of species competition by ecologic niche with its descent pine siskin [12Arnaiz-Villena A, Ruiz-del-Valle V, Reguera R, Gomez-Prieto P, Serrano-Vela JI. What might have been the ancestor of New World siskins? Open Ornithol J 2008; 1: 46-7.
[http://dx.doi.org/10.2174/1874453200801010046] ].

A detailed genetic map of the MHC in songbirds has not been obtained, so nothing can be said about the precise number of genes that composes it and its proximity on the chromosome. In spite of this, some gene numbers have been postulated [43Westerdhal H. Passerine MHC: Genetic variation and disease resistance in the wild. J Ornithol 2007; 148: 469-77.
[http://dx.doi.org/10.1007/s10336-007-0230-5] ]. In this work we have not analyzed such a characteristic and we have found no more than two different MHC alleles per single individual of the studied species of these particular genes. Therefore, our findings fit with detection of one paternal and one maternal gene belonging to a single locus.

In species such as Coturnix japonica at least four classic class I genes have been found with a high variety of alleles [44Shiina T, Ando A, Imanishi T, et al. Isolation and characterization of cDNA clones for Japanese quail (Coturnix japonica) major histocompatibility complex (MhcCoja) class I molecules. Immunogenetics 1995; 42(3): 213-6.
[http://dx.doi.org/10.1007/BF00191227] [PMID: 7642233] -46Shiina T, Shimizu S, Hosomichi K, et al. Comparative genomic analysis of two avian (quail and chicken) MHC regions. J Immunol 2004; 172(11): 6751-63.
[http://dx.doi.org/10.4049/jimmunol.172.11.6751] [PMID: 15153492] ]; this also is the case of goose [47Xia C, Hu T, Yang T, et al. cDNA cloning, genomic structure and expression analysis of the goose (Anser cygnoides) MHC class I gene. Vet Immunol Immunopathol 2005; 107(3-4): 291-302.
[http://dx.doi.org/10.1016/j.vetimm.2005.05.005] [PMID: 16005079] ]. Duck has five classic class I genes although only two of them seem to actively work [48Moon 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(10): 6702-12.
[http://dx.doi.org/10.4049/jimmunol.175.10.6702] [PMID: 16272326] ]. In songbirds, class I sequences have been studied in very few species. In the great reed warbler [49Westerdahl H, Wittzell H, von Schantz T. Polymorphism and transcription of Mhc class I genes in a passerine bird, the great reed warbler. Immunogenetics 1999; 49(3): 158-70.
[http://dx.doi.org/10.1007/s002510050477] [PMID: 9914330] , 50Wittzell H, Bernot A, Auffray C, Zoorob R. Concerted evolution of two Mhc class II B loci in pheasants and domestic chickens. Mol Biol Evol 1999; 16(4): 479-90.
[http://dx.doi.org/10.1093/oxfordjournals.molbev.a026130] [PMID: 10331274] ] a high genetic MHC variability has been described compared with chicken (Gallus gallus) while in South American siskins [51Ferre S. Evolución de los genes de histocompatibilidad de clase I en la radiación de jilgueros (lúganos) sudamericanos [Doctoral Thesis] 2001.] and in canaries [52Lowy E. Evolución y Sistema Principal de Histocompatibilidad en canarios (Género Serinus) [Doctoral Thesis] 2003., 53Arnaiz-Villena A, Lowy E, Ruiz-del-Valle V, et al. Evolution of the major histocompatibility complex class I genes in Serinus canaria from the Canary Islands is different from that of Asian and African continental Serinus species. J Ornithol 2007; 148: 479-84.
[http://dx.doi.org/10.1007/s10336-007-0146-0] ] only one gene with a low variability has been found.

A transpecific gene existance occurs in several mammals MHC, like apes [25Suárez B, Morales P, Castro MJ, et al. Mhc-E polymorphism in Pongidae primates: The same allele is found in two different species. Tissue Antigens 1997; 50(6): 695-8.
[http://dx.doi.org/10.1111/j.1399-0039.1997.tb02938.x] [PMID: 9458133] , 51Ferre S. Evolución de los genes de histocompatibilidad de clase I en la radiación de jilgueros (lúganos) sudamericanos [Doctoral Thesis] 2001.]. This phenomenon usually occurs when speciation happens quickly, while gene differentiation has not yet taken place. It could also mean that the MHC naturally adapts to habitat of species and select alleles to combat characteristic antigens / pathogens thriving in the area, and does not need to generate an unlimited polymorphism as in the case of “artificial” MHC, where there are a high number of alleles and numerous immunological disorders that appear to be associated with the HLA system, such as autoimmune processes [51Ferre S. Evolución de los genes de histocompatibilidad de clase I en la radiación de jilgueros (lúganos) sudamericanos [Doctoral Thesis] 2001., 52Lowy E. Evolución y Sistema Principal de Histocompatibilidad en canarios (Género Serinus) [Doctoral Thesis] 2003.]. Human, laboratory mouse and chicken are considered “artificial” vertebrate models because all have originated through a bottleneck and subsequent relatively high inbreeding, which enhances crossover at meiosis and thus to excessive MHC diversity [22Klein J, Sato A, Nagl S. O’hUigin C. Molecular trans-species polymorphism. Annu Rev Ecol Syst 1998; 29: 1-21.
[http://dx.doi.org/10.1146/annurev.ecolsys.29.1.1] , 51Ferre S. Evolución de los genes de histocompatibilidad de clase I en la radiación de jilgueros (lúganos) sudamericanos [Doctoral Thesis] 2001., 52Lowy E. Evolución y Sistema Principal de Histocompatibilidad en canarios (Género Serinus) [Doctoral Thesis] 2003.].

Phylogenetic analysis of MHC sequences from the species studied in the present work allowed to visualizing more clearly the phenomenon of trans-specificity since, two matches between MHC-I alleles from different South American siskin species were found at DNA level. It was also found that the Eurasian siskin shares a MHC protein allele with the pine siskin (North-Central America range) and another protein allele with three South American siskin species. Eight South American siskins species, including parental C. notata, also share another MHC protein among themselves (Fig. 3). All MHC genes transmit their alleles to the descendant species, but the most common fact is that the allelic identity between the ancestral species and their descendants get lost due to balancing selection diversification. The antiquity of studied MHC alleles goes back no longer than four - five millions years, when South American siskins and North American goldfinches species were separated (Figs. 1 and 2) [11Arnaiz-Villena A, Areces C, Rey D, et al. Three different North American Siskin/Goldfinch Evolutionary Radiations (Genus Carduelis): Pine Siskin Green Morphs and European siskins in America. Open Ornithol J 2012; 5: 73-81.
[http://dx.doi.org/10.2174/1874453201205010073] ].

A closest relationship between the Eurasian siskin and the Pine siskin had been found [8Arnaiz-Villena A, Alvarez-Tejado M, Ruíz-del-Valle V, et al. Phylogeny and rapid northern and southern hemisphere speciation of goldfinches during the Miocene and Pliocene epochs. Cell Mol Life Sci 1998; 54(9): 1031-41.
[http://dx.doi.org/10.1007/s000180050230] [PMID: 9791543] , 10Arnaiz-Villena A, Ruiz-del-Valle V, Moscoso J, Serrano-Vela JI, Zamora J. mtDNA phylogeny of North American Carduelis pinus group. Ardeola 2007; 54: 1-14., 11Arnaiz-Villena A, Areces C, Rey D, et al. Three different North American Siskin/Goldfinch Evolutionary Radiations (Genus Carduelis): Pine Siskin Green Morphs and European siskins in America. Open Ornithol J 2012; 5: 73-81.
[http://dx.doi.org/10.2174/1874453201205010073] ]. This is further supported by the fact of sharing one MHC protein allele. Eurasian siskin (o extant relative) could have been a species widely spread around Europe, Asia and America that could be have led to both North American goldfinches and South American siskins radiations. Otherwise, it could have only originated North American goldfinches radiations and these could have originated the South American siskin radiation [11Arnaiz-Villena A, Areces C, Rey D, et al. Three different North American Siskin/Goldfinch Evolutionary Radiations (Genus Carduelis): Pine Siskin Green Morphs and European siskins in America. Open Ornithol J 2012; 5: 73-81.
[http://dx.doi.org/10.2174/1874453201205010073] , 12Arnaiz-Villena A, Ruiz-del-Valle V, Reguera R, Gomez-Prieto P, Serrano-Vela JI. What might have been the ancestor of New World siskins? Open Ornithol J 2008; 1: 46-7.
[http://dx.doi.org/10.2174/1874453200801010046] ].

In conclusion, our data on mitochondrial cytochrome-b combined with the first evidence of trans-species MHC evolution so far described in birds, suggests that the Eurasian siskin is the extant ancestor of all North and South American Carduelis species [11Arnaiz-Villena A, Areces C, Rey D, et al. Three different North American Siskin/Goldfinch Evolutionary Radiations (Genus Carduelis): Pine Siskin Green Morphs and European siskins in America. Open Ornithol J 2012; 5: 73-81.
[http://dx.doi.org/10.2174/1874453201205010073] , 12Arnaiz-Villena A, Ruiz-del-Valle V, Reguera R, Gomez-Prieto P, Serrano-Vela JI. What might have been the ancestor of New World siskins? Open Ornithol J 2008; 1: 46-7.
[http://dx.doi.org/10.2174/1874453200801010046] ].

This set of wild bird species studied (Table 1 [26Arnaiz-Villena A, Ruiz-del-Valle V, Reche P, et al. Songbirds conserved sites and intron size of MHC Class I molecules reveal a unique evolution in vertebrates. Open Ornithol J 2010; 3: 156-65.
[http://dx.doi.org/10.2174/1874453201003010156] ],) has given the first direct evidence that one of the main characters of “Minimal Essential Bird MHC” postulated for birds [54Kaufman J, Milne S, Göbel TW, et al. The chicken B locus is a minimal essential major histocompatibility complex. Nature 1999; 401(6756): 923-5.
[http://dx.doi.org/10.1038/44856] [PMID: 10553909] ] is in fact not universal for birds.

MHC class I introns from presently and previously [26Arnaiz-Villena A, Ruiz-del-Valle V, Reche P, et al. Songbirds conserved sites and intron size of MHC Class I molecules reveal a unique evolution in vertebrates. Open Ornithol J 2010; 3: 156-65.
[http://dx.doi.org/10.2174/1874453201003010156] ] studied songbirds are longer than humans. Chicken introns are the shortest ones [26Arnaiz-Villena A, Ruiz-del-Valle V, Reche P, et al. Songbirds conserved sites and intron size of MHC Class I molecules reveal a unique evolution in vertebrates. Open Ornithol J 2010; 3: 156-65.
[http://dx.doi.org/10.2174/1874453201003010156] ]. In addition, MHC class I genes introns 2 were homologous on 38.3% to human class I MHC and 35% to Gallus gallus one. MHC class I intron 2 had an average of 304 bp in songbirds, 238 bp in human and 288 bp in Gallus gallus [26Arnaiz-Villena A, Ruiz-del-Valle V, Reche P, et al. Songbirds conserved sites and intron size of MHC Class I molecules reveal a unique evolution in vertebrates. Open Ornithol J 2010; 3: 156-65.
[http://dx.doi.org/10.2174/1874453201003010156] ]. This is also a direct evidence that the main postulate of “Minimal Essential MHC” for birds only applies to Gallus gallus, and not to Passerines [54Kaufman J, Milne S, Göbel TW, et al. The chicken B locus is a minimal essential major histocompatibility complex. Nature 1999; 401(6756): 923-5.
[http://dx.doi.org/10.1038/44856] [PMID: 10553909] ]. Additional information may be found in references [55Arnaiz-Villena A, Ruiz-del-Valle V, Gomez-Prieto P, et al. Carduelini New Sistematics: Crimson-winged Finch (Rhodopechys sanguineus) is Included in “Arid-Zone” Carduelini Finches by Mitochondrial DNA Phylogeny. Open Ornithol J 2014; 7: 55-62.
[http://dx.doi.org/10.2174/1874453201407010055] , 56Karlsson 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(1-2): 8-21.
[http://dx.doi.org/10.1007/s00239-013-9575-y] [PMID: 23877344] ].The fact that songbirds other than Carduelinae family (i.e.: Acrocephalus arundinaceus and Taeniopygia guttata, zebra finch [26Arnaiz-Villena A, Ruiz-del-Valle V, Reche P, et al. Songbirds conserved sites and intron size of MHC Class I molecules reveal a unique evolution in vertebrates. Open Ornithol J 2010; 3: 156-65.
[http://dx.doi.org/10.2174/1874453201003010156] ]) are also different at residues 10 and 96 at MHC class I proteins than all other vertebrates (including Gallus gallus) may indicate that our observations of on large intron size and different conserved 10 and 96 residues on MHC proteins could be extended to songbird family (i.e.: about half of the about 10,000 avian extant species [3Sibley CG, Monroe BL. Distribution and Taxonomy of Birds of the World 1990.]). Passerine evolutionary pathway may be altogether different from that of other birds [26Arnaiz-Villena A, Ruiz-del-Valle V, Reche P, et al. Songbirds conserved sites and intron size of MHC Class I molecules reveal a unique evolution in vertebrates. Open Ornithol J 2010; 3: 156-65.
[http://dx.doi.org/10.2174/1874453201003010156] ].