Molecular characterization of MHC class IIB genes of sympatric Neotropical cichlids

Abstract

Background: The Major Histocompatibility Complex (MHC) is a key component of the adaptive immune system of all vertebrates and consists of the most polymorphic genes known to date. Due to this complexity, however, MHC remains to be characterized in many species including any Neotropical cichlid fish. Neotropical crater lake cichlids are ideal models to study evolutionary processes as they display one of the most convincing examples of sympatric and repeated parallel radiation events within and among isolated crater lakes.

Results: Here, we characterized the genes of MHC class IIB chain of the Midas cichlid species complex (Amphilophus cf. citrinellus) including fish from five lakes in Nicaragua. We designed 19 new specific primers anchored in a stepwise fashion in order to detect all alleles present. We obtained 866 genomic DNA (gDNA) sequences from thirteen individuals and 756 additional sequences from complementary DNA (cDNA) of seven of those individuals. We identified 69 distinct alleles with up to 25 alleles per individual. We also found considerable intron length variation and mismatches of alleles detected in cDNA and gDNA suggesting that some loci have undergone pseudogenization. Lastly, we created a model of protein structure homology for each allele and identified their key structural components.

Conclusions: Overall, the Midas cichlid has one of the most diverse repertoires of MHC class IIB genes known, which could serve as a powerful tool to elucidate the process of divergent radiations, colonization and speciation in sympatry.

Keywords: Amphilophus; Major Histocompatibility Complex; Midas cichlid fish; Neotropical; Sympatric.

Fig. 1

Map of the Pacific coast of Nicaragua (Central America) with the great lakes and several crater lakes where samples were collected (source http://photojournal.jpl.nasa.gov/catalog/PIA03364; wikimedia.org). Samples belonged to the four species Amphilophus citrinellus, A. labiatus, A. amarillo and A. xiloaensis

Fig. 2

Alignment of amino acid sequences of MHC IIB. A majority consensus sequence was made for comparison. ‘•’ represent identical amino acids, ‘-’ represent gaps or introns that were not sequenced. Cysteine residues are outlined in red boxes

Fig. 3

Representation of intron-exon organization of MHC IIB sequences showing length of fragments and position of primers. The gel shows variation in the length of intron 2 (showing exon 2 / intron 2 / exon 3) for three individuals. * indicates lengths observed in sequences of the Amphilophus citrinellus draft genome shotgun sequencing project and ** indicate the exon that we found to be 12 bp longer in allele DXB*060101 and DXB*07

Fig. 4

Neighbor-net network based on exons 2 and 3 of MHC IIB allele relationships. Groups of alleles found with estimates of evolutionary divergence between sequences are shown

Fig. 5

Phylogenetic tree showing trans-species polymorphisim of exons 2 and 3 of all MHC IIB alleles found in the Midas cichlid (in black) and 20 alleles from other species (in blue), with posterior probabilities. Alleles are grouped according to how they cluster in Fig. 4

Fig. 6

Models of tertiary structure of MHC IIB sequences, where red boxes represent cysteine amino acids forming the disulfide bond in the β₁ domain, and black boxes represent cysteine residuals that form the disulfide bonds of the β₂ domain. a Model of allele DXB*2202; b Model of allele DXB*060101, with an unpaired cysteine at position 47 (indicated with a black arrow). The graphs show how each model (red star) compares to a non-redundant set of Protein data bank (PDB) structures, indicating the quality of the model compared to molecules of the same size as a value equivalent to a z-score test