Introduction

Reptiles represent the evolutionary lineage of vertebrates that have adapted to live on land. There are four living orders currently identified in reptiles: Crocodilia that includes crocodiles, gavials, caimans, and alligators; Sphenodontia that only includes the tuatara from New Zealand; Squamata, where we find lizards, snakes, and worm lizards; and the Testudines which includes turtles, terrapins, and tortoises (Janes et al. 2010; Shedlock et al. 2007; Modesto and Anderson 2004).

Immunoglobulins are proteins that are important for defending the body against infections. Many studies have elucidated the immunoglobulin structure and genomic organization in mammals, amphibians, and fish (Paul 2008), yet little is known in reptiles. All reptiles studied, until now, have at least three classes of immunoglobulins: immunoglobulin M (IgM), also found in all vertebrates; immunoglobulin D (IgD) with 11 domains in the constant region (IgD in mammals only have two or three domains); and immunoglobulin Y (IgY) that is already present in amphibians and birds, from which immunoglobulin G and immunoglobulin E in mammals are derived (Wei et al. 2009; Gambón Deza et al. 2009, 2012).

Recent studies performed in reptilian species of the Squamata order indicate that the lizard Anolis carolinensis only possesses three Ig isotypes (Wei et al. 2009; Gambón Deza et al. 2009). Nevertheless, other immunoglobulin types in the gecko Eublepharis macularius were described that may have been produced by recombination processes of the IGHM, IGHD, and/or IGHY genes. One of these immunoglobulins is similar to immunoglobulin A (named IgA-like) and was generated during evolution by a recombination between IgM and IgY (Deza et al. 2007). The other, denominated IgD2, is a variant of IgD and seems to have been generated by an exon shuffling between IgD and the IgA-like immunoglobulin (Gambón-Deza and Espinel 2008). This IgD2 antibody is highly expressed and may have an evolutionary significance since there is a pseudogene with a similar structure in the genome of the monotreme Ornithorhynchus anatinus (Gambón-Deza et al. 2009). Finally, in snakes, which represent a highly evolved group of reptiles, IgM, IgD, and new forms of IgY, called IgYb and IgYc, have been described—the latter with only three domains in its immunoglobulin constant region (Gambón-Deza et al. 2012).

Reptiles are nonmammalian, nonavian amniotes. Birds have evolved from these species (Erickson et al. 2009; Steiger et al. 2009), and their immunoglobulin genes have been studied in ducks and chickens. IgM and IgY (the first animals to be described) were found in both cases. Surprisingly, while no IgD was found, an immunoglobulin gene with opposite orientation and sequence homology to that of IgA of mammals was encountered (Magor et al. 1994; Lundqvist et al. 2001; Zhao et al. 2000).

Studies of immunoglobulins in turtles, or even in the reptilia Testudines order, are scarce. Only a messenger RNA has been described for IgM in red-eared slide turtle (Trachemys scripta elegans) (Turchin and Hsu 1996), and sequences of messenger RNA for IgM, IgD, and IgY in the Chinese softshell turtle (Pelodiscus sinensis) have recently been published (Xu et al. 2009). Recently, however, the International Painted Turtle Genome Sequencing Consortium has published and made available the genome of painted turtle (Chrysemys picta bellii), and the RIKEN institute in Japan has made available the Chinese softshell turtle (P. sinensis) genome. This is a great opportunity to provide a clear picture of the immunoglobulins in turtles and to understand their preservation or modification during the reptilian evolutionary history, which is paralleled to that of mammalians. In addition, present phylogenetic studies are not yet clear about Testudines (turtles, terrapins, and tortoises) evolutionary location (Lukoschek et al. 2012). The painted turtle immunoglobulin sequences have placed the turtles in the same evolutionary lineage that gave rise to crocodiles and birds. Therefore, their study can provide answers to the origin of immunoglobulins that are currently found in birds and mammals.

Materials and methods

Identification of the immunoglobulin heavy chain loci

The complete genome of C. picta bellii build 3.0.1 (AHGY00000000.1) and P. sinensis PelSin_1.0 (AGCU00000000.1) deposited in NCBI (www.ncbi.nlm.nih.gov) was examined to locate antibody genes. Published immunoglobulin sequences of A. carolinensis and E. macularius were used to search for painted turtle immunoglobulin sequences. We studied A. carolinensis and E. macularius sequences and C. picta bellii and P. sinensis genomes from FASTA files using the Galaxy website (http://main.g2.bx.psu.edu/) (Giardine et al. 2005; Blankenberg et al. 2010; Goecks et al. 2010). Different scaffolds and contigs that contain immunoglobulin genes were identified: scaffold group 175 (JH584564.1) and scaffold 2278 (JH586043.1) for immunoglobulin heavy chain (IGH) genes in C. picta bellii and scaffolds JH210939 and JH205552 in P. sinensis. All sequences were retrieved and analyzed in detail using Vector NTI (Invitrogen).

Identification of the exons coding for the constant heavy (CH) domains was performed using the software FGENESH (www.softberry.com) (Solovyev et al. 2006; Yao et al. 2005) and Augustus (http://augustus.gobics.de/submission) (Stanke et al. 2004). Protein products, predicted from previous searches, were compared with reptile sequences and found to be evolutionarily close because there are no RNA studies to date for these genes.

Phylogenetic studies

Comparative phylogenetic studies were carried out using MEGA5 (Tamura et al. 2011) and sequence alignments using SeaView (Gouy et al. 2010) with the ClustalW and MUSCLE algorithms. Subsequently, the neighbor-joining and maximum likelihood methods were used to produce phylogenetic trees (pairwise deletion, and JTT or WGA matrix). The veracity of our results was tested with 500 replicate bootstrapping execution runs.

GenBank sequences used

The following IgM accession numbers were used: AAO37747 O. anatinus, EU287910 and EU287911 E. macularius, FJ605150 Pelodiscus sinensis, CAA30613.1 Heterodontus francisci, AAB03838 Trachemys scripta elegans, ABV66128 A. carolinensis, PO1875 Gallus gallus, CAC43061 Anas platyrhynchos, MGC69066 X. laevis, AAH89670 Xenopus tropicalis, A46532 Axolotl mexicanum, CAE02685 Pleurodeles waltl, AAC48834 Monodelphis domestica, CAB37838 Homo sapiens.

The following IgD accession numbers were used: EU312156, EU327165, and EU327166 E. macularius and ABV66130 A. carolinensis. The following IgY accession numbers were used: EF690361 A. carolinensis, EU827594 and EU827595 E. macularius, CAA46322 A. platyrhynchos, S00390 G. gallus, and AJ575800 P. waltl.

The kappa light chain accession numbers that were used are XP 003222451.1 A. carolinensis, AAA49880.1 X. laevis, AAI58339.1 X. tropicalis, ABV02006.1 Macropus eugenii, AAO84652.1 Tachyglossus aculeatus, CAA53284.1 Equus caballus, and P01834.1 H. sapiens.

Results

With the recent publication of the C. picta bellii and P. sinensis genomes, made available by the International Painted Turtle Genome Sequencing Consortium and RIKEN Institute, we searched the immunoglobulin sequences of A. carolinensis and E. macularius that we had identified previously (Deza et al. 2007; Gambón-Deza and Espinel 2008; Gambón Deza et al. 2009). The turtle genes for the immunoglobulin heavy chain constant regions were found as described in “Materials and methods.” With these data, we were able to perform a detailed study of the immunoglobulins in the evolutionary line that emerged from the first reptiles.

In the IGHC locus, there are 13 sequences coding for different immunoglobulin isotypes in C. picta belli and 17 in P. sinensis (Fig. 1). The IGHM and IGHD loci are first placed and correspond to the IgM and the reptilian IgD (with 11 exons for CH domains), respectively. Downstream of IgD, there is a region, which includes an IGHD2 gene and an IGHY gene, and that is repeated five times in C. picta bellii and seven times in P. sinensis. While IGHYs are in the same direction to the IGHM-IGHD, IGHD2 are in the opposite directions. Finally, another sequence coding for IgY was located in each genome without the IGHD2 mate.

Fig. 1
figure 1

Genomic organization of the IGH locus in turtles. The organization of the immunoglobulin genes identified in C. picta bellii and P. sinensis genomes is shown. The transcriptional orientation of genes is indicated by the direction of the arrows. In both turtles, the genes were found in two scaffolds whose lengths in base pairs are annotated

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Immunoglobulin M

The locus IGHM contains four exons for CH domains plus TM1 and TM2 exons for transmembrane and cytoplasmic regions, respectively (Fig. 2). Two IgMs have been previously described in turtles, namely, the IgM of T. scripta elegans (Turchin and Hsu 1996) and the IgM of P. sinensis (Xu et al. 2009). The deduced amino acid sequence in C. picta bellii is similar (Fig. 3 and Additional File 1 of Electronic Supplementary Material (ESM)). Unlike those described for the anole, gecko, and snakes (Wei et al. 2009; Gambón-Deza et al. 2009, 2012), turtles possess a cysteine at the beginning of the CH1 domain, which is required in order to join the light chain. In the CH2 domain, there are two noncanonical cysteines, where one is present in turtles studied (position 118) while the other (position 153) is also present in snakes but absent in the lizard A. carolinensis and gecko E. macularius. At the beginning of the CH3 domain (position 239), there is a cysteine that is specific for reptiles and is found in lizards, snakes, and turtles. Moreover, the cysteine at position 319 is maintained in most vertebrates. Finally, a cysteine is also present in the secretor tail (position 486), suggesting the possibility of a J-chain bonding to form IgM multimers.

Fig. 2
figure 2

Schematic representation and distribution of exons in each of the immunoglobulin heavy chain genes of turtles. The lines show the direction in the genome sequence. The boxes indicate exons. The exon type was assigned as indicated in the results. Those exons with an altered reading frame or with the appearance of stop codons are marked with the psi symbol

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Fig. 3
figure 3

Analysis of IgM. On the left, a phylogenetic tree was constructed using IgM amino acid sequences obtained from different reptiles, amphibians, birds, mammals, and shark. On the right, a schematic representation of the different IgMs identified in the turtles C. picta bellii, P. sinensis, and T. scripta elegans is displayed. The domains are represented as hexagons with the intracatenary bonds. The putative cysteines that establish bonds with heavy chains (H) and those that form multimers (J) are shown. Potential glycosylation sites are indicated with pentagons

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A phylogenetic tree was created with IgM sequences from turtles and other vertebrates (Fig. 3) that supports the claim that turtles shared a common ancestor with birds.

Immunoglobulin D

The 11 exons for the IgD immunoglobulin domains are located behind the IGHM (Fig. 1). This confirms the generation of a three-immunoglobulin core (Cμ-Cδ-Cυ) in the ancestor of all reptiles and its preservation up until the present. The turtle IgD has features that are similar to those described in other reptiles and the platypus IgD. Most noncanonical cysteines and the high glycosylation rate in domains CH7 and CH10 are maintained (Fig. 4 and Additional File 2 of ESM). In P. sinensis, the exon that codes for the CH10 domain may not be viable due to the absence in the sequence of the second canonical cysteine and presence of stop codon. The existence of several types of differential splicing in IgD indicates that messenger RNA which is created without the exon could synthesize a functional protein.

Fig. 4
figure 4

Shown at the top of the figure is a schematic representation of the IgD identified in the turtles C. picta bellii and P. sinensis. The domains are represented as hexagons with the intracatenary bonds. The putative cysteines that establish bonds with heavy chains (H) are shown. Potential glycosylation sites are indicated with pentagons. The CH10 P. sinensis is shaded to emphasize important alterations in the sequence due to the absence of the second canonical cysteine. In the lower part of the figure, a phylogenetic tree constructed using the amino acid IgD domain sequences identified in turtles, other reptiles (snakes, lizard, and gecko), and in the platypus is shown. It indicates the IgD domain-to-domain orthology between these species. The tree was constructed using the evolution algorithm, JTT matrix, and differences by sites activated with gamma parameter 2.5. The GenBank accession numbers are indicated in “Materials and methods”

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The phylogenetic tree supports the domain-to-domain orthology with lizard, gecko, snakes, and platypus. Each group of orthologous domains comes from a corresponding exon. The exonic numbering of this tree will be maintained in subsequent studies in the article (Fig. 4).

Immunoglobulin Y

We found six IgY gene isotypes in C. picta belli and eight in P. sinensis (Fig. 1). In C. picta belli, five isotypes were found in the contig group 175, while the gene for IgY6 was located in contig 2278. All these genes have four Cυ exons, except in IGHY-5 that has the Cυ2 duplicated, generating an IgY with five domains (Fig. 2). The second CH2 domain has a sequence that is identical to the CH2 domain of IgY1 (data not shown).

The gene classes for the immunoglobulin Y in P. sinensis have a more complex structure. The genes IGHY1, IGHY4, IGHY7, and IGHY8 have the four upsilon exons for the four immunoglobulin domains. Both the IGHY2 and IGHY3 genes have upsilon exons duplicated. In the case of IGHY2, the Cυ3 is duplicated since the two exons are nonviable (due to the presence of a stop codon) and also lack an Cυ2 exon, suggesting that the gene is a pseudogene. The IGHY3 gene also lacks the Cυ2 exon and has duplications of the Cυ3 and Cυ4 exons, thereby generating an immunoglobulin with five domains in the constant region. Of great interest is the structure of the IGHY6 gene, which has three upsilon domains with a structure very similar to the mammalian IGHG (with a missing Cυ2 exon).

The amino acid sequence alignments of the turtle IgYs with those found in other vertebrates indicate that turtle IgYs do not contain the cysteine that would establish the second intrachain bridge between CH1 and CH2. The noncanonical cysteines for interchain bridging are located in the CH2 domain (Fig. 5 and Additional File 3 of ESM).

Fig. 5
figure 5

Analysis of IgYs. The left figure shows the phylogenetic tree constructed from amino acid sequences coding for all Cυ exons. The tree identifies the relationship between the exon sequences and assigns the correct numbering. The right figure shows the phylogenetic tree constructed using the IgY amino acid sequences obtained from different reptiles, amphibians, and birds. The tree branches represent all turtle IgY sequences that emerged from the evolutionary line of birds

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All the IgY isotypes are very similar to each other, indicating a recent duplication processes in their genesis (Fig. 5 and Additional File 3 of ESM). As described in the case of the IgM sequences analysis, the phylogenetic tree constructed with IgY indicates that turtles shared a common ancestor with birds (Fig. 5). The high sequence identity between the IgYs within each species and the tree phylogeny can be explained by the diversification of the IgY isotypes that took place in the evolutionary lineage of turtles, or perhaps through a process of concerted gene evolution.

Immunoglobulin D2

In a previous study, we described the immunoglobulin IgD2 in E. macularius (Gambón-Deza and Espinel 2008). This immunoglobulin is a variant of IgD, with the first four CH domains similar to IgD and the last two CH domains similar to the CH3 and CH4 domains of IgA-like immunoglobulin (Fig. 6). One year later, an immunoglobulin with the same characteristics was described in the turtle P. sinensis (Xu et al. 2009). As shown in Fig. 6, this immunoglobulin corresponds structurally to the IgD2 of E. macularius, having the same number and order of CH domains.

Fig. 6
figure 6

The left of the figure represents the phylogenetic tree which is used to study the relationship between IgD2 and IgD. The tree was constructed after the alignment of the amino acid immunoglobulin domain sequences of IgD from C. picta bellii and IgD2 from C. picta bellii and P. sinensis. The exons coding for each domain group are also indicated. The maximum likelihood, WGA matrix, was used for obtaining this tree. The IgD domains are marked in red while the IgD2-1 domains are drawn in brown to demonstrate an earlier origin to other IgD2 domains. To the right of the figure, the structure of IgD2 is identified in turtles (P. sinensis and C. picta bellii). The domains are displayed as hexagons with the intracatenary bonds. The noncanonical cysteines are drawn with a line and the points of N-glycosylation are represented with pentagons. The exons that code each domain are indicated at the top of the figure. The graphics of IgD2-1 and IgD2-4 from C. picta bellii have been adapted so that all domains are placed with the corresponding exon

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Several genes encoding for this kind of immunoglobulin are found in the genome of both turtles. As shown in Fig. 1, we found five in C. picta bellii and seven in P. sinensis that are oriented in an opposite direction to IGHM-IGHD and IGHY genes. All of these genes contain Cδ and Cα exons. The assignment of domain type that codes for each exon was made from the phylogenetic tree that emerges from the alignment of all domains (Fig. 6 and Additional File 4 of ESM).

The analysis of their sequences indicates that in C. picta bellii, only three of the five genes can be functional and two may be pseudogenes (IGHD2-3 has an altered sequence in the Cδ2 and Cδ3 domains, and IGHD2-5 contains a stop codon in the Cα3 domain). IGHD2-1 has eight exons (Cδ1-Cδ2-Cδ3-Cδ2-Cδ3-Cδ4-Cα3-Cα4), and the transmembrane exons have not been detected, so it should only be expressed as a secreted form. The other functional immunoglobulin genes, IGHD2-2 and IGHD2-4, have four (Cδ3-Cδ4-Cα3-Cα4) and five (Cδ1-Cδ2-Cδ4-Cα3-Cα4) immunoglobulin heavy chain exons and can be expressed as transmembrane forms due to the presence of transmembrane exons (TM1 and TM2) (Figs. 2 and 6).

Of the seven sequences in P. sinensis, two could be pseudogenes, only two exons are detected in gene IGHD2-5, and, in the gene IGHD2-2, a stop codon in the Cδ2 exon is found. The remaining five IGHD2 genes have viable structures. Four of them have the same number of exons with the structure Cδ1-Cδ2-Cδ3-Cδ4-Cα3-Cα4 while IGHD2-7 has the structure Cδ1-Cδ2-Cδ3-Cδ4-Cδ1-Cδ2-Cδ3-Cα3-Cα4.

A phylogenetic tree was created with the amino acid sequences obtained from the C. picta bellii functional IGHD2 domains, the IgD2 immunoglobulin sequences from P. sinensis, and IgD sequences from C. picta bellii (Fig. 6). The results suggest that the IGHD2-1 gene was created before the rest of the IGHD2 genes. In C. picta bellii, the evolutionary distance between IgD domains from the IGHD2-1 is higher than the distance obtained when we compared the same domains from IGHD2-2 and IGHD2-4 (Additional File 5 of ESM). However, the evolutionary distance between the IgA domains deduced from the IGHD2-1, IGHD2-2, and IGHD2-4 genes is very low. These results suggest that these two latter immunoglobulin domains arose by a recombination between IGHD and IGHD2-1. The IGHD provided the Cδ exons, and the IGHD2-1, the Cα exons.

Relationship of the IgD2 with bird IgA

It is worth noting that the IGHD2 location (between the IGHM-IGHD and IGHY genes) and the direction (laying in the opposite direction to the IGHM-IGHD and the IGHY genes) are very similar to that found in avian IGHA, suggesting an evolutionary relationship. To determine whether the IGHA found in birds and IGHD2 evolved from a common ancestor, we aligned the domain amino acid sequences obtained from A. platyrhynchos and G. gallus IgA, P. sinensis IgD2 domains, and C. picta bellii IgD2-1 and IgD domains. As shown in Fig. 7, the resulting phylogenetic tree constructed from these sequences suggests a clear evolutionary relationship between the last two domains of IgD2 and the CH3 and CH4 domains from the bird IgA. This analysis also indicates that CH1 and CH2 of the IgD2 arise from the duplication of Cδ exons. This duplication happened after the bird Cα1 and Cα2 divergence, suggesting that these IgA domains do not derive from the IGHD2 gene.

Fig. 7
figure 7

The evolutionary relationship between IgD2 and bird IgA. The amino acid sequences obtained from the A. platyrhynchos and G. gallus IgA domains, P. sinensis IgD2 domains, and C. picta bellii IgD2-1 and IgD domains were aligned, and then, an unrooted phylogenetic tree was constructed. The constant heavy chain domains (CH) are grouped according to the exon which originated from them. Red arrows indicate the origin of the CH1 and CH2 domains of the IgD2-1. They arose from the duplication of IGHD. For phylogenetic tree, the algorithm used was maximum likelihood, WGA matrix, and bootstrapping of 200 replicates

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Therefore, we can conclude that the two terminal domains of the turtle IgD2 and the two terminal domains of the bird IgA are orthologues; however, any orthologue exon to the bird Cα1 and Cα2 has been detected in the turtles’ IGH locus.

Discussion

Reptile evolution may be divided into two lineages: one line gave rise to lizards and snakes (Squamata) while the other to turtles, crocodiles, and birds. With respect to immunoglobulins, there are significant studies in birds (Lundqvist et al. 2006; 2001; Higgins and Warr 1993), and recently, these genes have also been described in several Squamata (Wei et al. 2009; Gambón-Deza et al. 2012; 2009; Gambón-Deza and Espinel 2008). From this information, we can affirm that the common ancestor of Squamata and birds must have had IGHM and IGHY genes. In this work, we describe the immunoglobulin genes in turtles, providing new insights into the origin of the IGHD2 and IGHA genes.

The scanning of the painted turtle genomic sequences revealed the presence of IGHM, IGHD, and IGHY genes, which are similar to those previously found in Squamata (lizards and snakes). So, we may deduce that these immunoglobulins were present in early reptiles 300 million years ago. These three immunoglobulins were already present in amphibians and, although the structure of IgM and IgY has remained similar to those found today, the amphibian IgD has been modified through evolution. The IGHD sequence itself described in the frog X. tropicalis (Qin et al. 2008; Zhao et al. 2006), coding for an IgD with eight CH domains, proves the presence of duplications. On the other hand, the IGHD gene described in reptiles gives rise to an immunoglobulin with 11 CH domains that has survived for at least 300 million years without modifying its basic structure. It suggests essential immune functions that remain to be discovered. The obtained immunoglobulin sequences support the conclusions provided by others about the origin of turtles as a sister group to birds and crocodilians, contradicting the long-held view that they were basal amniotes (Chiari et al. 2012).

The IGHY genes found in turtle indicate the presence of a process of duplication of these genes in the evolutionary line. Probably, once they have made the duplications, a process of concerted evolution can explain the phylogenetic tree that emerges from the study (Fig. 5). The turtle ancestor must have already had several IGHY and nonhomologous recombination processes between different genes should have given the homogenization of the sequences and the presence of genes with duplicate upsilon domains. Considering that turtles diverged from other reptiles in a recent time to the divergence of mammals, this process of duplication and concerted evolution may be a remnant of the process that must have occurred to generate the IGHE and IGHG genes of mammals. The presence of the IGHY-6 gene with three domains is similar to the IGHG mammalian gene.

We recently described a new isotype in E. macularius (Gambón-Deza and Espinel 2008), the IgD2, which is defined as an immunoglobulin that has arisen by duplication of the IgD and the IgA domains. In turtles, immunoglobulins with these characteristics also exist, as well as in different evolutionary lines as compared with Squamata. Different IgD2 with a variable number of domains exist in turtles; however, all have their first domains from the IgD and two terminal domains that are similar to the CH3 and CH4 of IgA. Between E. macularius and the turtles’ (P. sinensis and C. picta bellii) IgD2, there is no full-domain orthology. All have the same structural characteristics yet do not share a common ancestor, since the evolutionary relationship between the IgD and the IgD2 from the same species demonstrates that these IgD2 were created independently in their own evolutionary line.

The results obtained regarding the IgD2 in turtles indicate that the original gene was IGHD2-1. The other IGHD2 genes have recently been generated by combination between IGHD gene (providing the Cδ exons) and IGHD2-1 (providing the Cα exons). This may explain why some of these new IGHD2 are pseudogenes. Similar to the IGHY genes, a concerted evolution process must have occurred in order to explain the identity between the sequences.

The presence of IgA domains in the turtle IGH locus and the current avian IgA indicate that the first reptiles must have had other immunoglobulin apart from IgM, IgD, and IgY. Our results suggest that it could be the IgA that probably arose from the evolution of the amphibian immunoglobulin X (Zhao et al. 2006). This IgA was most likely lost after recombining (in an inverted transcriptional direction) the IGHA and the IGHD genes to generate the turtle IGHD2-1 gene. Within the evolutionary line from which birds originated, other changes took place that also prompted the loss of IGHD. Further genomic studies in other reptiles, belonging to the bird evolutionary line, are needed to verify this explanation of the data. As a result, we can firmly establish that the remnant IgA in reptiles corresponds to the IgA found in birds. The analysis of the American alligator (Alligator mississippiensis) and saltwater crocodile (Crocodylus porosus) genomes, recently published by the International Crocodilian Genomes Working Group (www.crocgenomes.org), shows the presence of IGHA (summit to publication) and supports this hypothesis.

All these results, maintenance of a common reptile core (Cμ-Cδ-Cυ) and emergence of new immunoglobulin isotypes (different IgY and IgD2) or loss (IgA), suggest that immunoglobulin loci are shaped by a complex model of birth-and-death evolution (Nei and Rooney 2005). In this process, new genes are created by duplication, some of them are maintained, and others are deleted or diversified by accumulation of mutations or recombination process. All these processes seem to have acted to create the current turtle immunoglobulin heavy chains loci, and probably, they must have been the cause of the emergence of the mammalian isotypes.

In conclusion, the increasing availability of animal genome information is very useful for understanding the genetic basis of immunoglobulin diversity and the evolutionary divergence of the immunoglobulin loci in vertebrates.