The major histocompatibility complex (MHC) is a group of genes critical to immune function in vertebrates (Doherty and Zinkernagel 1975; Klein 1986). Due to its central role against infections from pathogens, the study of MHC genes has been the subject of many ecological and evolutionary studies (Sommer 2005; Spurgin and Richardson 2010), especially those studying host-parasite interactions (Westerdahl 2007; Dionne 2009). The characterization of MHC genes is constrained by its multigene nature, a result from gene duplications during evolutionary time and a turnover of new and old genes, the so-called birth-and-death model (Klein and Figueroa 1986; Nei et al. 1997; Edwards and Hedrick 1998; Nei and Rooney 2005). Thus, vertebrates have their own particularities on MHC evolution, with orthologous and paralogous genes not only maintained mainly by natural selection from parasites but also by sexual selection (Hughes and Yeager 1998). As a consequence, some MHC alleles have a long persistence time, even exceeding the species evolutionary time (i.e., trans-species polymorphism) (Klein and O'Huigin 1994).
The MHC genes constitute the most polymorphic genes among vertebrates (Piertney and Oliver 2006) and its study has been challenging, especially in non-model organisms (Babik 2010). In passerines, the study of this genetic region is also complicated, due to the existence of concerted evolution among genes (Edwards et al. 1999; Hess and Edwards 2002). Different molecular methods have been used to characterize MHC genes in birds, including PCR and non-PCR-based methods (Babik 2010). These studies involved the analysis of whole genetic region or partial regions, including complete or incomplete introns/exons. In this manner, several orders of passerine and non-passerine species have been studied to date (see Bollmer et al. 2010; Li et al. 2011; Miller et al. 2011). The first species with a completely sequenced MHC was a non-passerine, the chicken (Gallus gallus) and its MHC appeared simple and compact (Kaufman et al. 1999). Since then more species have been studied and the general conclusion is that the genetic organization of the passerines MHC seems to be more complex than that of non-passerines (Balakrishnan et al. 2010; Ekblom et al. 2011). This finding is supported by the derived phylogenetic position of passerines from non-passerines, where a minimal MHC seems to be the ancestral condition for birds, at least for class II genes (Hughes et al. 2008; Balakrishnan et al. 2010). Although a ratite species was observed to have at least five MHC class II loci (Miller et al. 2011). Thus, passerines have a higher number of genes, larger class I and II genes (longer introns) and also nonfunctional genes (pseudogenes) (Edwards et al. 1998; Kaufman et al. 1999; Miller and Lambert 2004a; Westerdahl et al. 2004b; Westerdahl 2007; Bollmer et al. 2010). Both MHC class I and II genes have been studied in birds involving host-parasites implications. Having an elevated/optimal number of alleles (heterozygote advantage) and/or rare advantageous alleles (negative frequency-dependent selection) would be favored by natural selection for parasite detection and elimination (Bodmer 1972; Doherty and Zinkernagel 1975; Wegner et al. 2003).
Different groups of MHC genes have been detected in different species. In the galliform birds there are two similar, but independent MHC complexes, both with class I and class II genes (Briles et al. 1993; Strand et al. 2007). The B-complex has high polymorphic and expressed genes and has been associated with disease resistance (Kaufman 2000) and the Y-complex (MHC-Y), a separate group of genes, less polymorphic, and expressed genes to a lower extent. The Y-complex has been suggested to be involved in the innate immunity (Miller et al. 2004) and controversial associations among the Y-complex and Marek's disease have been reported (Wakenell et al. 1996; Vallejo et al. 1997). In some passerines, low polymorphic genes seem reminiscent from the MHC-Y-complex (Edwards et al. 2000; Gasper et al. 2001; Bonneaud et al. 2004; Jarvi et al. 2004), therefore a phylogenetic approach has been employed to classify groups of genes (Edwards et al. 1999).
In this study, we investigate MHC class II B genes in the blue tit (Cyanistes caeruleus) for the first time. The blue tit is established as a model species in different ecology studies where parasite prevalence, ecological factors, and their effects on their host have been studied in depth (Hurtrez-Boussèz et al. 1997; Tripet and Richner 1997; Fargallo and Merino 1999; Merilä and Andersson 1999; Wiles et al. 2000; Merino et al. 2006; Martínez-de la Puente et al. 2010). The blue tit MHC class I genes have recently been studied (Schut et al. 2011; Wutzler et al. 2012), therefore a preliminary characterization of the MHC class II B would be a determinant for a later in-depth molecular characterization, as it gives an idea of the complexity of the system (Wittzell et al. 1994; Babik 2010). We used sequencing and restriction methods to investigate the exon 2 that codes from the most variable peptide-binding region (PBR) of the MHC class II molecule.