Martinez-Barnetche J , Godoy-Lozano EE , Saint Remy-Hernández S , Pacheco-Olvera DL , Téllez-Sosa J , Valdovinos-Torres H , Pastelin-Palacios R , Mena H , Zambrano L , López-Macías C .

	FIGURE 1 The heavy chain locus is located in the centromeric portion of chr13q (483 Mbp). (A) Gene density plot of chr13q where the IGH locus is encoded (box). Dark blue colors indicate low gene density. (B) Zoom of the whole IGH locus (35.967 – 48.91 Mbp). Non-Ig genes (black) in proximal flank OXA1L gene and distal flank ABHD4, IGHC genes (blue), IGHV genes (red), and IGHD and IGHJ (yellow). (C) Zoomed view (36.9 – 38.09 Mbp) of the IGHJ-IGHC gene cluster. The lower panel shows AID hotspots density per 2.5 Kb along the IGHJ-IGHC cluster to map Switch regions. (D) Close-up of the IGHJ cluster and the IGHM gene. The bottom panel shows the spleen RNA-seq coverage histogram of the IGHJ-IGHM region. (E) Schematic representation of IGH locus in A. mexicanum genome (v6) and the corresponding locus in X. tropicalis. Color code as in A X. tropicalis displays the canonical architecture, in which the V, J and C clusters have the same orientation. Note that in A. mexicanum the JH-CH cluster and 3 IGHV segments appear to be inverted, which seems implausible due to the mechanism of V(D)J recombination. Moreover, IGHV segment orientation in axolotl are intercalated, which is atypical. Interestingly, in axolotl, the TRA/TRD locus (Yellow) is not linked to the IGH locus as in X. tropicalis and is coded across the centromere (black circle) in chr13p: 3.3-7.7 Mbp). Not on scale. The IGHD cluster is not shown for simplicity.
	FIGURE 2 The lambda light chain locus is located in the centromeric portion of chr10p (116.7 - 125.8 Mbp). (A) Gene density plot of chr10p where the IGL locus is encoded (box). Dark blue colors indicate low gene density. (B) Zoom of the whole IGL locus (114.5-128.8 Mbp). Non-Ig genes (black) in proximal flank DGCR2 gene, and distal flank GST. Zoomed view of IGLC genes (blue), IGLV genes (red), and IGLJ (yellow). (C) Zoomed view (116.76-116.88 Mbp) of the IGHC gene cluster. (D) Spleen RNA-seq coverage histogram. Note that there is no expression in Cλ3. (E) Schematic representation of immunoglobulin lambda locus in A. mexicanum genome (chr10p:116-124 Mbp) and X. tropicalis (chr1:144.7-144.9 Mbp). Color code as in A X. tropicalis displays the canonical architecture, in which the V, J, and C clusters are in the same orientation. Note that in A. mexicanum, IGLV genes are in at least two different clusters (IGLV1 and IGLV2), separated by the MMP11, SMARCB11, and OTUB genes, which in Xenopus are located in the distal flank. Also, note that the OTUB gene in axolotl is inverted. Moreover, as in the IGHV locus, IGLV segment orientation in axolotl is intercalated, which is atypical. VJ recombination involving the more distal IGLV segments would delete the OTUB, SMARCB11, MMP11, and the SLC2A11 gene cluster. Not on a scale.
	FIGURE 3 The sigma light chain locus is located in the centromeric portion of chr1p (609.1 - 609.2 Mbp). (A) Gene density plot of chr1p where the IGS and the putative surrogate light chain (SLC) loci are encoded (box). Dark blue colors indicate low gene density. (B) Zoom of the whole putative SLC locus (67.5 - 68.6 Mbp). Non-Ig genes (black) in proximal flank DNAJC18 and distal flank TM4ESF5. Zoomed view of IGLC genes (blue). (C) Zoomed view (68.08 - 68.29 Mbp) of the putative SLC gene. (D) Spleen RNA-seq coverage histogram. (E) Zoom of the whole IGS locus (601 - 616 Mbp). Non-Ig genes (black) in proximal flank RAB11B gene and distal flank PRPF3. (F) Zoomed view of IGSC genes (blue), IGSV genes (red) and IGSJ (yellow) (609.15 - 610 Mbp) (G) Spleen RNA-seq coverage histogram. (H) Schematic representation of Immunoglobulin sigma locus in A. mexicanum genome (v6) (chr1p:609 - 610 Mbp), and X. tropicalis (chr1: 82.1 - 82.22 Mbp). Color code as in A. X. tropicalis displays the canonical architecture, in which the V, J and C clusters are in the same orientation. Note that in A. mexicanum, IGSV gene orientation in axolotl is intercalated, which is atypical. Non-Ig genes in the downstream flank are different in A. mexicanum and at least the PRPF3, RPRD2 and TARS3 genes are located in chr8 106.9 – 107.1 Mbp in X. tropicalis. Also, non Ig genes in the upstream flank correspond to the downstream flank in X. tropicalis. It is possible that this chromosomal configuration is correct, however it is also possible that the IGS locus may be inverted so that the downstream flank is syntenic with X. tropicalis. Not on scale.
	FIGURE 4 Relative immunoglobulin gene transcription (Log10 relative abundance). Publicly available RNA-seq data (SRA: SRP101842) (17) was used to estimate relative gene transcription in 13 adult tissues (spleen, liver, heart, lung, testes, ovary, brain, head, 9 and 15 day post - amputation forelimb blastemas, tail blastema, intact, 1 and 6 days post-injured spinal cord), limb and tail buds. A hierarchical clustering by column (tissue) was performed. (A) Immunoglobulin C region relative transcription. (B) IGHV relative transcription. Each IGHV gene is grouped by IGHV family (rows). (C) IGLV and IGSV relative transcription. Each IGLV segment is grouped according family.
	FIGURE 5 IGHM gene length in Ambystoma mexicanum is evolutionarily constrained. A. mexicanum genome (orange), IGH (purple), IGHC (light blue), IGHV cluster (light green) and individual IGHC gene length size ratios are plotted (from left to right). Comparisons include four placental mammals (human, mouse, dog and a bat), two non-placental mammals (Tasmanian devil and platypus), two bird species (chicken and duck), and one reptile, amphibian and teleost fish. Only the IGHM A. mexicanum: species size ratio was smaller to the respective genome size ratio (Kruskal-Walllis test, Dunnet multiple comparison correction (p = 0.0003).
	FIGURE 6 Distribution of V intron length in tetrapods. (A) The heavy chain and (B) lambda light chain locus in A. mexicanum according to their functionality class (functional, ORF or pseudogene). In A. mexicanum, intron length in functional IGHV genes was shorter than in IGHV pseudogenes. No differences were observed in the lambda locus and (C) D. rerio, (D) H. sapiens, and (E) X. tropicalis. V introns in functional IGHV A. mexicanum were not larger than functional A. mexicanum IGLV genes, or (F) functional IGHV of other tetrapods, however, V introns in (G) non-functional A. mexicanum IGHV genes were larger than other tetrapods. Statistical analysis for multiple comparisons (A, B, F, G) was performed with the robust ANOVA One-Way Trimmed Means Comparisons, with a trimming level of 5% (tr = 0.05), followed by the post hoc Lincon test (30). For two sample comparisons (C-F), Yuen’s robust Tests for Two Independent Groups were performed with a trimming level of 5%. The violin area is scaled for comparability. The median is shown as a black dot.
	FIGURE 7 Intergenic distances by species. Violin plots depicting overall coding to coding gene intergenic distance distribution across the whole genome (A), between IGHV segments (B), and between cytochrome p450 family gene clusters (C) in (D) A. mexicanum (red), (E) X. tropicalis (purple), (F) D. rerio (blue), and (G) H. sapiens (green). Kruskal-Wallis rank sum test followed by Dunn’s multiple comparison tests with Benjamini – Hochberg p adjustment.
	Figure 1. The heavy chain locus is located in the centromeric portion of chr13q (483 Mbp). (A) Gene density plot of chr13q where the IGH locus is encoded (box). Dark blue colors indicate low gene density. (B) Zoom of the whole IGH locus (35.967 – 48.91 Mbp). Non-Ig genes (black) in proximal flank OXA1L gene and distal flank ABHD4, IGHC genes (blue), IGHV genes (red), and IGHD and IGHJ (yellow). (C) Zoomed view (36.9 – 38.09 Mbp) of the IGHJ-IGHC gene cluster. The lower panel shows AID hotspots density per 2.5 Kb along the IGHJ-IGHC cluster to map Switch regions. (D) Close-up of the IGHJ cluster and the IGHM gene. The bottom panel shows the spleen RNA-seq coverage histogram of the IGHJ-IGHM region. (E) Schematic representation of IGH locus in A. mexicanum genome (v6) and the corresponding locus in X. tropicalis. Color code as in A X. tropicalis displays the canonical architecture, in which the V, J and C clusters have the same orientation. Note that in A. mexicanum the JH-CH cluster and 3 IGHV segments appear to be inverted, which seems implausible due to the mechanism of V(D)J recombination. Moreover, IGHV segment orientation in axolotl are intercalated, which is atypical. Interestingly, in axolotl, the TRA/TRD locus (Yellow) is not linked to the IGH locus as in X. tropicalis and is coded across the centromere (black circle) in chr13p: 3.3-7.7 Mbp). Not on scale. The IGHD cluster is not shown for simplicity.
	Figure 2. The lambda light chain locus is located in the centromeric portion of chr10p (116.7 - 125.8 Mbp). (A) Gene density plot of chr10p where the IGL locus is encoded (box). Dark blue colors indicate low gene density. (B) Zoom of the whole IGL locus (114.5-128.8 Mbp). Non-Ig genes (black) in proximal flank DGCR2 gene, and distal flank GST. Zoomed view of IGLC genes (blue), IGLV genes (red), and IGLJ (yellow). (C) Zoomed view (116.76-116.88 Mbp) of the IGHC gene cluster. (D) Spleen RNA-seq coverage histogram. Note that there is no expression in Cλ3. (E) Schematic representation of immunoglobulin lambda locus in A. mexicanum genome (chr10p:116-124 Mbp) and X. tropicalis (chr1:144.7-144.9 Mbp). Color code as in A X. tropicalis displays the canonical architecture, in which the V, J, and C clusters are in the same orientation. Note that in A. mexicanum, IGLV genes are in at least two different clusters (IGLV1 and IGLV2), separated by the MMP11, SMARCB11, and OTUB genes, which in Xenopus are located in the distal flank. Also, note that the OTUB gene in axolotl is inverted. Moreover, as in the IGHV locus, IGLV segment orientation in axolotl is intercalated, which is atypical. VJ recombination involving the more distal IGLV segments would delete the OTUB, SMARCB11, MMP11, and the SLC2A11 gene cluster. Not on a scale.
	Figure 3. The sigma light chain locus is located in the centromeric portion of chr1p (609.1 - 609.2 Mbp). (A) Gene density plot of chr1p where the IGS and the putative surrogate light chain (SLC) loci are encoded (box). Dark blue colors indicate low gene density. (B) Zoom of the whole putative SLC locus (67.5 - 68.6 Mbp). Non-Ig genes (black) in proximal flank DNAJC18 and distal flank TM4ESF5. Zoomed view of IGLC genes (blue). (C) Zoomed view (68.08 - 68.29 Mbp) of the putative SLC gene. (D) Spleen RNA-seq coverage histogram. (E) Zoom of the whole IGS locus (601 - 616 Mbp). Non-Ig genes (black) in proximal flank RAB11B gene and distal flank PRPF3. (F) Zoomed view of IGSC genes (blue), IGSV genes (red) and IGSJ (yellow) (609.15 - 610 Mbp) (G) Spleen RNA-seq coverage histogram. (H) Schematic representation of Immunoglobulin sigma locus in A. mexicanum genome (v6) (chr1p:609 - 610 Mbp), and X. tropicalis (chr1: 82.1 - 82.22 Mbp). Color code as in A. X. tropicalis displays the canonical architecture, in which the V, J and C clusters are in the same orientation. Note that in A. mexicanum, IGSV gene orientation in axolotl is intercalated, which is atypical. Non-Ig genes in the downstream flank are different in A. mexicanum and at least the PRPF3, RPRD2 and TARS3 genes are located in chr8 106.9 – 107.1 Mbp in X. tropicalis. Also, non Ig genes in the upstream flank correspond to the downstream flank in X. tropicalis. It is possible that this chromosomal configuration is correct, however it is also possible that the IGS locus may be inverted so that the downstream flank is syntenic with X. tropicalis. Not on scale.
	Figure 4. Relative immunoglobulin gene transcription (Log10 relative abundance). Publicly available RNA-seq data (SRA: SRP101842) (17) was used to estimate relative gene transcription in 13 adult tissues (spleen, liver, heart, lung, testes, ovary, brain, head, 9 and 15 day post - amputation forelimb blastemas, tail blastema, intact, 1 and 6 days post-injured spinal cord), limb and tail buds. A hierarchical clustering by column (tissue) was performed. (A) Immunoglobulin C region relative transcription. (B) IGHV relative transcription. Each IGHV gene is grouped by IGHV family (rows). (C) IGLV and IGSV relative transcription. Each IGLV segment is grouped according family.
	Figure 5. IGHM gene length in Ambystoma mexicanum is evolutionarily constrained. A. mexicanum genome (orange), IGH (purple), IGHC (light blue), IGHV cluster (light green) and individual IGHC gene length size ratios are plotted (from left to right). Comparisons include four placental mammals (human, mouse, dog and a bat), two non-placental mammals (Tasmanian devil and platypus), two bird species (chicken and duck), and one reptile, amphibian and teleost fish. Only the IGHM A. mexicanum: species size ratio was smaller to the respective genome size ratio (Kruskal-Walllis test, Dunnet multiple comparison correction (p = 0.0003).
	Figure 6. Distribution of V intron length in tetrapods. (A) The heavy chain and (B) lambda light chain locus in A. mexicanum according to their functionality class (functional, ORF or pseudogene). In A. mexicanum, intron length in functional IGHV genes was shorter than in IGHV pseudogenes. No differences were observed in the lambda locus and (C) D. rerio, (D) H. sapiens, and (E) X. tropicalis. V introns in functional IGHV A. mexicanum were not larger than functional A. mexicanum IGLV genes, or (F) functional IGHV of other tetrapods, however, V introns in (G) non-functional A. mexicanum IGHV genes were larger than other tetrapods. Statistical analysis for multiple comparisons (A, B, F, G) was performed with the robust ANOVA One-Way Trimmed Means Comparisons, with a trimming level of 5% (tr = 0.05), followed by the post hoc Lincon test (30). For two sample comparisons (C-F), Yuen’s robust Tests for Two Independent Groups were performed with a trimming level of 5%. The violin area is scaled for comparability. The median is shown as a black dot.
	Figure 7. Intergenic distances by species. Violin plots depicting overall coding to coding gene intergenic distance distribution across the whole genome (A), between IGHV segments (B), and between cytochrome p450 family gene clusters (C) in (D) A. mexicanum (red), (E) X. tropicalis (purple), (F) D. rerio (blue), and (G) H. sapiens (green). Kruskal-Wallis rank sum test followed by Dunn’s multiple comparison tests with Benjamini – Hochberg p adjustment.

André, Structure and diversity of Mexican axolotl lambda light chains. 2000, Pubmed, Xenbase

André, Structure and diversity of Mexican axolotl lambda light chains. 2000, Pubmed , Xenbase
Barbosa-Morais, The evolutionary landscape of alternative splicing in vertebrate species. 2012, Pubmed , Xenbase
Charlemagne, Thymus independent anti-horse erythrocyte antibody response and suppressor T cells in the Mexican axolotl (Amphibia, Urodela, ambystoma mexicanum). 1979, Pubmed
Cotter, Transcriptional response of Mexican axolotls to Ambystoma tigrinum virus (ATV) infection. 2008, Pubmed , Xenbase
Cunningham, Ensembl 2022. 2022, Pubmed
Das, Evolutionary dynamics of the immunoglobulin heavy chain variable region genes in vertebrates. 2008, Pubmed
Das, Evolutionary redefinition of immunoglobulin light chain isotypes in tetrapods using molecular markers. 2008, Pubmed
Dobin, Mapping RNA-seq Reads with STAR. 2015, Pubmed
Edgar, MUSCLE: multiple sequence alignment with high accuracy and high throughput. 2004, Pubmed
Fellah, Characterization of an IgY-like low molecular weight immunoglobulin class in the Mexican axolotl. 1988, Pubmed
Fellah, Characterization of immunoglobulin heavy chain variable regions in the Mexican axolotl. 1994, Pubmed
Flajnik, A cold-blooded view of adaptive immunity. 2018, Pubmed
Fonte, The urodele amphibian Pleurodeles waltl has a diverse repertoire of immunoglobulin heavy chains with polyreactive and species-specific features. 2015, Pubmed
Frías-Alvarez, Chytridiomycosis survey in wild and captive mexican amphibians. 2008, Pubmed
Gelfman, Changes in exon-intron structure during vertebrate evolution affect the splicing pattern of exons. 2012, Pubmed
Golub, Structure and diversity of the heavy chain VDJ junctions in the developing Mexican axolotl. 1997, Pubmed
Golub, Structure, diversity, and repertoire of VH families in the Mexican axolotl. 1998, Pubmed , Xenbase
Gu, Complex heatmaps reveal patterns and correlations in multidimensional genomic data. 2016, Pubmed
Haire, A third Ig light chain gene isotype in Xenopus laevis consists of six distinct VL families and is related to mammalian lambda genes. 1996, Pubmed , Xenbase
Hollander, How Are Short Exons Flanked by Long Introns Defined and Committed to Splicing? 2016, Pubmed
Kenter, Igh Locus Polymorphism May Dictate Topological Chromatin Conformation and V Gene Usage in the Ig Repertoire. 2021, Pubmed
Lam, B Cell Activation and Response Regulation During Viral Infections. 2020, Pubmed
Larson, Genomic features of humoral immunity support tolerance model in Egyptian rousette bats. 2021, Pubmed
Lefranc, Antibody Informatics: IMGT, the International ImMunoGeneTics Information System. 2014, Pubmed
Liao, featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. 2014, Pubmed
Liu, Immunological responses and protection in Chinese giant salamander Andrias davidianus immunized with inactivated iridovirus. 2014, Pubmed
Lopez, Mapping hematopoiesis in a fully regenerative vertebrate: the axolotl. 2014, Pubmed
Mair, Robust statistical methods in R using the WRS2 package. 2020, Pubmed
Manso, IMGT® databases, related tools and web resources through three main axes of research and development. 2022, Pubmed
Medina-Rivera, RSAT 2015: Regulatory Sequence Analysis Tools. 2015, Pubmed
Melchers, Fit for life in the immune system? Surrogate L chain tests H chains that test L chains. 1999, Pubmed
Nei, Evolution by the birth-and-death process in multigene families of the vertebrate immune system. 1997, Pubmed
Nowoshilow, The axolotl genome and the evolution of key tissue formation regulators. 2018, Pubmed , Xenbase
Parra, The dynamic TCRδ: TCRδ chains in the amphibian Xenopus tropicalis utilize antibody-like V genes. 2010, Pubmed , Xenbase
Patel, Abbreviated junctional sequences impoverish antibody diversity in urodele amphibians. 1997, Pubmed , Xenbase
Pyron, Divergence time estimation using fossils as terminal taxa and the origins of Lissamphibia. 2011, Pubmed
Qin, Genomic organization of the immunoglobulin light chain gene loci in Xenopus tropicalis: evolutionary implications. 2008, Pubmed , Xenbase
Robert, Comparative and developmental study of the immune system in Xenopus. 2009, Pubmed , Xenbase
Schaerlinger, IgX antibodies in the urodele amphibian Ambystoma mexicanum. 2008, Pubmed , Xenbase
Schatz, Recombination centres and the orchestration of V(D)J recombination. 2011, Pubmed
Schloissnig, The giant axolotl genome uncovers the evolution, scaling, and transcriptional control of complex gene loci. 2021, Pubmed
Singh, Rates of in situ transcription and splicing in large human genes. 2009, Pubmed
Smith, A chromosome-scale assembly of the axolotl genome. 2019, Pubmed
Sun, Slow DNA loss in the gigantic genomes of salamanders. 2012, Pubmed , Xenbase
Sun, A comparative overview of immunoglobulin genes and the generation of their diversity in tetrapods. 2013, Pubmed , Xenbase
Thorvaldsdóttir, Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. 2013, Pubmed
Voss, A Tale of Two Axolotls. 2015, Pubmed
Wake, Amphibians. 2018, Pubmed
Waterhouse, Jalview Version 2--a multiple sequence alignment editor and analysis workbench. 2009, Pubmed
Xu, Immunoglobulin class-switch DNA recombination: induction, targeting and beyond. 2012, Pubmed
Ye, Construction of the axolotl cell landscape using combinatorial hybridization sequencing at single-cell resolution. 2022, Pubmed
Yoshinaga, Four immunoglobulin isotypes and IgD splice variants in urodele amphibians. 2021, Pubmed
Zhang, The role of chromatin loop extrusion in antibody diversification. 2022, Pubmed
Zhao, Identification of IgF, a hinge-region-containing Ig class, and IgD in Xenopus tropicalis. 2006, Pubmed , Xenbase
Zinkernagel, Immunology taught by viruses. 1996, Pubmed