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Immunoglobulin T genes in Neopterygii
Serafin Mirete-Bachiller1, David N. Olivieri2 and Francisco Gambón-Deza 1
1Unidad de Inmunologı́a Hospital do Meixoeiro, Vigo, Spain,2Computer Science Department, School of Informatics (ESEI), Universidade de Vigo. As Lagoas s/n, Ourense, 32004
Abstract
In teleost fishes there are three immunoglobulin isotypes named immunoglobulin M (IgM), D(IgD) and T (IgT). IgT has been the last to be described and is considered a teleosts-fish spe-cific isotype. From the recent availability of genome sequences of fishes, an in-depth analysis ofActinopterygii immunoglobulin heavy chain genes was undertaken. With the aid of a bioinformat-ics pipeline, a machine learning software, CHfinder, was developed that identifies the coding exonsof the CH domains of fish immunoglobulins. Using this pipeline, a high number of such sequenceswere obtained from teleosts and holostean fishes. IgT was found in teleost and holostean fishesthat had not been previously described. A phylogenetic analysis reveals that IgT CH1 exons aresimilar to the IgM CH1. This analysis also demonstrates that the other three domains (CH2, CH3and CH4) were not generated by recent duplication processes of IgM in Actinopterygii, indicatingit is an immunoglobulin with an earlier origin.
Keywords: IgT, Neopterygii, Holostei
1. Introduction
Some 550 million years ago the adaptive immune system arose coinciding with the appearanceof the non-jawed vertebrates (Agnathans) (Cooper & Alder, 2006). The adaptive response beganwhen antigens were recognized by specific receptors on lymphocytes. In Agnathans, such recep-tors are encoded by a family of genes first identified in the lamprey variable lymphocyte receptors(VLRs), while in jawed vertebrates (gnathostomes), these were the B and T lymphocyte receptors(BCR and TCR, respectively). Many features of the VLR system are analogous to BCR and TCR,although a notable difference is that the VLR system is made up of highly diverse leucine-richrepeats (LRR), whereas in gnathostomes the antigenic receptors have immunoglobulin domains(Pancer et al., 2004).
Immunoglobulin genes emerged with the gnathostomes (Flajnik & Kasahara, 2010). Thesegenes are made up of multiple exons that code for domains of approximately 100 amino acids andwith a sequence and structure that is called the immunoglobulin domain (Peterson et al., 1972;Edelman et al., 1969). These domains were already found in proteins of invertebrate species,Preprint submitted to Elsevier May 21, 2020
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted May 24, 2020. ; https://doi.org/10.1101/2020.05.21.108993doi: bioRxiv preprint
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encoding for membrane proteins with cellular communication functions and some of them partici-pate in the innate immune response (Sun et al., 1990; Zhang et al., 2004). An organization of thesegenes occurred in jawed vertebrates, which gave rise simultaneously to the immunoglobulin genes(both for the heavy and light chain), the genes for T lymphocyte receptors (for both TCR α/β asfor the TCR γ/δ), and the genes for the major histocompatibility complex.
Today’s cartilaginous fish have what is believed to be the oldest gene structure of all im-munoglobulins (IgLs). Genes in these animals that code for the variable (V) and constant (C)regions that appear in multiple repeated V-C tandem arrangements. Bony fishes have a differenti-ated structure with separate V and C gene groups. These structures evolved such that in order tohave a valid mRNA coding for an immunoglobulin chain, V and C genes must be joined together.Thereafter, this process was maintained throughout all posterior vertebrate lines.
Different immunoglobulin isotypes have been described in gnathostomes. With only someexceptions, IgM and IgD are present in all jawed vertebrates, appearing approximately 500 millionyears ago. IgM has been stable throughout evolution, while IgD undergone great plasticity withrespect to the number of CH domains and the amount of splice variants found (Ohta & Flajnik,2006). Apart from these two Igs, other isotypes have been described in vertebrates. In amphibiansand reptiles, two new isotypes were found that are not present in fishes (IgY and IgA(X)) and inmammals are been found 5 isotypes (IgM, IgD, IgG, IgE and IgA) (Gambon-Deza et al., 2009;Zhao et al., 2009).
In the 1990s, IgM was cloned and characterized in Ictalurus punctatus and Gadus morhua(Ghaffari & Lobb, 1989; Bengtén et al., 1991). Amongst all the immunoglobulins, IgM in bonyfishes has the highest concentration in serum. In these species, it has some peculiarities withrespect to its mammalian orthologs. First, it is tetrameric and does not possess a J chain, sopolymerization occurs by interchain disulfide bonds (Castro & Flajnik, 2014). Also, by meansof an alternative splicing mechanism, the membrane form of IgM consists of only three domains,however this does not prevent it from interacting with CD79 (Ross et al., 1998; Sahoo et al.,2008). IgM can be formed as monomers, dimers, and trimers; it was suggested that the degree ofpolymerization of IgM is related to maturation affinity (Ye et al., 2010).
IgD in Teleostei was first described in Ictalurus punctatus (Wilson et al., 1997). BetweenTeleostei species, there is a high variability in the number of constant domains. The Cµ1 exonthat contains the region coding the cysteine residue which binds to the light chains, is splicedwith the first Cδ exon. This immunoglobulin has been described in several teleost fish species(Hordvik et al., 1999; Stenvik & Jørgensen, 2000). The secreted form of IgD has been identifiedin Ictalurus punctatus and Takifugu rubripes (Hikima et al., 2011). In Ictalurus punctatus, thesecreted form lacks the VH regions and can bind to basophils, mediated by an Fc receptor thatinduces pro-inflammatory cytokines production (Chen et al., 2009).
In 2005, two independent groups described a new immunoglobulin isotype in teleosts, withfour constant domains in Danio rerio and Oncorhynchus mykiss, which they named IgZ and IgTrespectively. In the same year, an isotype with two domains was described in Takifugu rubripesthat they called IgH (Danilova et al., 2005; Hansen et al., 2005; Savan et al., 2005b). However,these immunoglobulins were shown to be orthologs to each other and whose consensus was latercalled IgT (Gambón-Deza et al., 2010). Later, IgT was described in more teleosts specie and foundto have a variable number of constant domains, ranging from 2 to 4. Also, fishes can have more
2
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than one gene for IgT (See Table S2). However, IgT is not found in all teleosts; for example, catfishand medaka lack IgT (Bengtén et al., 2002; Magadán-Mompó et al., 2011). This isotype occurs asmonomers in serum and as tetramers in mucous (Zhang et al., 2011). Studies of Cyprinus carpiosuggested an important role of IgT in mucosal immunity (Ryo et al., 2010).
Because previous work has only found IgT in Teleostei, a widely accepted conclusion wasthat it was specific to species of this order. Here, we provide evidence that, in fact, IgT has itsorigins prior to the appearance of Teleostei. Specifically, we describe its presence in Holostei,indicating that the origin of this Ig isotype dates back to the late Permian with the appearance ofthe Neopterygii subclass.
2. Methods
2.1. Sequence DataWe studied the presence of immunoglobulin genes of bony fishes present from genome assem-
blies available from the https://vgp.github.io/ and NCBI repositories. The gene finding programCHfinder, developed by our group, was used to search and annotate immunoglobulin exons fromchromosomal regions in these assemblies.
2.2. Bioinformatics ToolsCHfinder is based on a multiclass neural network machine learning classifier that was trained
to identify exons coding for the CH domains of fish immunoglobulins. For the training process,domain sequences of IgM (CH1, CH2, CH3 and CH4), IgD (CH1, CH1B, CH2, CH3, CH4, CH5and CH6) and IgT (CH1, CH2, CH3 and CH4), together with random background sequences con-stituted the training set. The resulting neural network has an accuracy of 99.99 % for identifyingIG domains.
The gene identification workflow is as follows. First, a BLAST (tblastn) is used as a coarsegrained filter by using a query consisting of an artificially constructed sequence, concatenatingrepresentative IgT, IgD, and IgM into a single sequence. The Tblastn query was performed againstthe target genome, producing a hit table - those sequences in the genome, together with their lo-cations, having similarity to the query sequence. The coordinates of the hits are used to extracta nucleotide region, with extra padding of 1000 nucleotides at both ends. From these extractednucleotide regions, the CHfinder identifies candidate exon reading frames, defined by the AG andGT start/stop motifs and having a min/max nucleotide length of 240nt and 450nt, respectively. Ad-ditionally, the reading frames were restricted to those that were a multiple of 3 and do not containstop codons within the reading frame. Even with these restrictions, a large number of candidateexon sequences are obtained result that are then subjected to the machine learning prediction stepof CHfinder.
Exon prediction and classification in CHfinder is done by first transforming the amino acid(AA) sequences into an integer feature vector that spatially preserves the AA ordering. Also, thetransformation acts to direct training on extreme ends of each exon. In this way, each AA sequenceis first converted into a sequence of 80 amino acids, by extracting and concatenating the 40 aminoacids from front/back ends of the sequence coding from a hypothetical exon. These sequences arethen converted to numerical “one-hot” based feature vectors. In this one-hot scheme, each amino
3
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acid in the 80aa-long exon is converted to 20 integers, all 0 except 1 for the AA in question; thus, asingle exon will have a feature vector of 80 x 20 = 1600 integers. For supervised machine learning,the training set consists of known sequences with their respective IG domain label, and a set ofrandom background sequences for null discrimination, in a ratio of 3:1 to labelled sequences.
While genome assemblies exist for many fish species, there are still several species that do notyet have published genomes. One such example is Amia calva. To explore the presence or absenceof IgT in this species where a genome is not available, we used raw sequence records (SRA) ofbowfin gills (SRX661027) from the NCBI repository. To construct transcripts, Trinity (a de novoRNA-seq transcript assembler) was used (Grabherr et al., 2011). Once assembled, the nucleotidesequences were transformed into amino acids using TransDecoder that identifies candidate cod-ing regions within transcript sequences, such as those generated by de novo RNA-Seq transcriptassembly using Trinity (Haas et al., 2013). These bioinformatics steps were performed in Galaxy(Afgan et al., 2018).
2.3. Phylogenetic studiesTo align the IgM and IgT sequences obtained with CHfinder, the SEAVIEW (Gouy et al., 2009)
and Jalview (Waterhouse et al., 2009) programs were used together with the clustalω algorithm(Sievers & Higgins, 2014). Due to the heterogeneity of the genomic sources, we manually deletedthe V and stem regions, in order to only retain the immunoglobulin constant domains. From thiscuration process, a Fasta file with all the sequences was generated (included in the SupplementaryMaterial). Using Galaxy, sequences were aligned with MAFFT, a multiple alignment program foramino acid or nucleotide sequences (Galaxy Version 7.221.3) (Katoh & Standley, 2013). Align-ments were done using the BLOSUM62 matrix. Maximum likelihood phylogenetic trees wereconstructed from the amino acid sequences by FastTree (Galaxy Version 2.1.10 + galaxy1) (Priceet al., 2010) using the LG protein evolution model (Le & Gascuel, 2008) + CAT. Finally, thegraphical representations of the trees were made using the online program iTOL (Letunic & Bork,2019).
3. Results
IgT has been described in Teleostei. Indeed, this immunoglobulin is observed in several ofthese species, but in others it is absent. This lack of consistency throughout Teleostei raises ques-tions about the origins and functions of IgT. Given the availability of genomes and transcriptomesfrom a broad sampling of species from this order, we can finally obtain a more complete cataloguedthe IgT prevalent in this order.
The three immunoglobulin classes found in fish are IgM, IgD, and IgT. The exons coding forthe CH domains and the deduced immunoglobulin genes identified by the CHfinder program aregiven in Table S1. With few exceptions, all fish have genes for IgM, IgD, and most for IgT. Rep-resentative sequences for the IgT were used to search the Fish-T1K (Sun et al., 2016) repositorythat contains transcriptome sequences for 124 fish species, shown in Table S3. Additionally, a bib-liographic search was carried out since 2005 to identify the different IgT that have been publishedin teleosts (see Table S2). These results indicate that the IG constant chain locus is quite unstable,having undergo duplications in some species and having large variability in the number of genes
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between species. For example, one species has up to 35 constant region genes (Labrus bergylta),while another (Guania wildenowi) has no genes at all.
By studying the genomes of teleost fishes, we have confirmed previous observations - mostspecies in this order possess genes for IgT, while some do not. Nonetheless, a pattern that explainthe absence of this gene remains elusive. In addition, we found genes for IgT in the Holostei cladethat have not been described previously.
3.1. IgT in HolosteiHolostei is one of the two infraclasses of the Neopterygii subclass (Teleostei, is the other), and
it comprises two orders, the Amiiformes with a single representative and which is considered a liv-ing fossil Amia calva and the Lepisosteiformes with two genera Atractosteus and Lepisosteus, with7 extant species between both. The Lepisosteus oculatus (spotted gar) genome is available fromthe NCBI with a chromosomal assembly level (accession number GCA 000242695.1). A searchfor the immunoglobulin constant regions was performed using CHfinder. Three scaffolds contain-ing Ig exons coding for CH domains were identified: NC 023183.1 (LG5 chromosome) containsfour exons coding for CHs domains of IgM and 12 exons for the IgD domains, NW 006270078.1has a gene with four exons for the CH domains (first identified as CH1M (similar to CH1 ofIgM), while the rest of the domains were identified as CH2T, CH3T and CH4T). The scaffoldNW 006270215.1 has another gene with 3 exons coding for domains: the first exon, is identifiedas the CH1 of IgM (CH1M), while the others were identified as CH3 of IgT (CH3T) and CH4 ofIgT (CH4T) (See Figure 1).
exon
1_M
-0.9
85
exon
1_M
-0.9
85
exon
1_M
-0.6
24
exon
2_M
-0.9
88
exon
3_M
-0.9
81
exon
3_T-
0.63
7
exon
3_T-
0.65
4
exon
2_T-
0.61
6
exon
4_M
-0.5
6
exon
4_T-
0.66
exon
4_T-
0.68
9
exon
4_D-0
.991
exon
5_D-0
.992
exon
3_D-0
.991
exon
4_D-0
.993
exon
5_D-0
.986
exon
3_D-0
.993
exon
4_D-0
.995
exon
5_D-0
.99
exon
3_D-0
.983
exon
4_D-0
.994
exon
5_D-0
.998
exon
6_D-0
.989
IgT_4 IgT_3D IgDIgM
NW_006270215.1 NW_006270078.1 NC_023183.1 Chromosome LG5
D
LOC102685290 LOC102687547 LOC107077209LOC102694914
Lepisosteus oculatus
Figure 1: A schematic representation of the immunoglobulin genes found in the Lepisosteus oculatusgenome. NC 023183.1 corresponds to chromosome LG5 containing the genes for IgM (red) and IgD (green).NW 006270215.1 and NW 006270078.1 are not yet assigned to chromosome and contain the exons for two differentIgTs, one for 4 domains and the other for 3 domains, respectively.
Candidate IgT sequences in the spotted gar genome (IgT4D, an IgT consisting of four domains,and IgT3D, an IgT consisting of three domains) were used to search for orthologs in other Holosteispecies. Because the genome of Amia calva (bowfin) is not available, we searched for the presenceof these exons in the RNA-seq transcript (SRX661027). Sequences of four CH domain ortholo-gous to the spotted gar IgT4D were found. In the Fish-T1K repository, we identified anotherIgT4D (Atractosteus spatula) in the scaffold WHYR15010142 A C1141006. Additionally, thesegenes were searched in the genomes and transcripts of Polypteriformes and Acipenseriformes
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(firsts Actinopterygii) without finding IgT orthologues. In the amino acid sequences of theseIgT in Holostei found cysteine residues are important in the formation of intra-domain disulfidebridges. Additionally, the CH1 domain contains the necessary Cys residue to form the disulfidebridge between IgH chain and IgL chain (see Fig 2).
19 29 39 49 59 69 79 89 99 109 119 129IgT4D_Amia_calvaIgT4D_Lepisosteus_oculatusIgT4D_Atractosteus_spatulaIgT3D_Lepisosteus_oculatusIgM_Lepisosteus_osseusIgM_Amia_calvaIgM_Lepisosteus_oculatus
- - - - - - - - - - T V T HA P E V A P HV Y V L L - P C E E DL E T AG K V T I G C L A K E F S P S F A TMTWY K NV S E I T A D I VMY P S A E L - NS R Y NQV S T I T V S E S E RQ E - DT S Y T C K A S T S AQ SMA T - - S S PQ K F P KG I P PF E YWG KG TMV T V T S AQ I NP P L A S V L W - P CG VNL E T T S P V T L G C L V E E S Y SGQA T V YWS K DGQK L T T D I KMY P S V E D - SG K Y K K T S T L T V T K Y - - - - - - DS Y T C V A NDR S S NE NS T A S S P K P - Q A PQ A P- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - DGQ K L T T D I QMY P S V K D - SG K Y K K T S T L T V T K Y - - - - - - DS Y T C V A NDR S S NE NS S A S S P K P - Q A PQ A PF DYWG KG TQ V P V T SG S P T K P T L F P L V A S CG SG - T SGG V V S VG C L A TG F L P DS L T F KWT DS T NK E L T P F E K Y P S V L S - GG T Y S S T SMV S L S T S DWNS - G K S F Y C E A K HPQGDV K T DP I K K I D - P P K I Q VF DYWG KG TMV T V T S A S P T K P T L F P L V A S CG SG - T SG DV V A L G C L A TG F L P DS L T F KWT DS T DK E L T P F R K Y P S V L N - G E T Y S S T SQ L S L P T S DWNS - G K A F F C E A K HPQGDV K L HL L - T P P V P P - - - -- - - - - - - - - V T V T S A A A T A P T L F P L V - P CG SG - A S V DP V S VG C L A TG F S P DS L T F GWS EG - S K E L T NF Q K Y P S V L G SGG L Y SMT SQ L S I P K A DWE NK DKMF Y C K A T NP SG T A S A E L K P P T P T P P P I P VP S SGG RG R L EQG T A A S P T K P T L F P L V A S CG SG - T SG DV V A L G C L A TG F L P DS L T F KWT DS T E K E L T P F R K Y P S V L N - G E T Y S S T SQ L S L P T S DWNS - G K A F F C E A K HPQGDV K V T F AG T P P V P P - - - -
147 157 167 177 187 197 207 217 227 237IgT4D_Amia_calvaIgT4D_Lepisosteus_oculatusIgT4D_Atractosteus_spatulaIgT3D_Lepisosteus_oculatusIgM_Lepisosteus_osseusIgM_Amia_calvaIgM_Lepisosteus_oculatus
- - Q V S I L P L S K - - - QQ T DT L I L V C V V S E Y Y P K T V N I SWT RDDT S V T - - - TG F S TMA P VQNP S NS L FWTMS HL S V T T DSWNK N - NV F S C I V E HK A S T T R - KQ T S K AQ E E DP- - NV S I L - - T S - - - P NMS P P NL V CV V S DF Y PQ T V T I SWK KG K T S L DAQA AG I I E R S P V K K P S T E L F T AMS L L P V T A Y DWDK D - T I Y T C S V HHA K TG TQ - K E L S T S K S R - V- - NV S I L - - T S - - - A NMS P P NL V CV V S DF Y PQ T V T I SWK KG K T S L DAQA AG I I E K S P V K K P S T E L F T AMS L L P V T A Y DWDK D - T I Y T C S V HHA K TG TQ - K E L S T S K S R A V- - - - - - - - - - - - - - - - - - - - - - - - - - - G Y - - - - - - - - - YQ S T E - - - - - - - - - - - - - P - - T L T S P L Y S - - - - - - - - - - - - - - S F MV - - - - - - - - - - - - - - - - - - F E A - - - -- A S V L L NP P S L E E F AQNHT A T L V CV R SG F S P K T HE F KWWRGNT K L DEG V - - - - T N I P A T V DE K K L Y S A S S L L T V T E K DWK S S - A E F A C E F V HK TG S V L K N I T Y T S R E CQ -HP T V Y V V P P S A E DF K S DK T A K I I C L A I G F S P L DV S F KWWNNQG L V T EG V - - - - E S HG S V K DE K E K Y S A C S S L R V T E K EWRNP F NS F A C E V HHT EGWS K L K NT S F I G E CDP- A S V L L NP P S L E E L AQNHT A T L V CV A SG F S P K T HE F KWWRGNT K L DEG V - - - - T N I P A T E DE K K L Y S A S S L L T V T E K DWK S S - A E F A C E F V HK TG S V L K N I T Y T S P E CQ -
257 267 277 287 297 307 317 327 337 347 357IgT4D_Amia_calvaIgT4D_Lepisosteus_oculatusIgT4D_Atractosteus_spatulaIgT3D_Lepisosteus_oculatusIgM_Lepisosteus_osseusIgM_Amia_calvaIgM_Lepisosteus_oculatus
EMP C L E V V L R E PG I R E L F T Y NRAML DC E V V A A N - - - - S DV K I FWT I DG I NV T K DS K K TG P - - - HT T NNK V SMK S T L NA S HVDWF NNK R F E C T VQHNSG - - S V V K S - - T R V S DV VG S T S I F V T L HP P K V K E L F L NNL A V L V C E V NG K E A - - - A NV E I SWF VDSQ KQ SQNV K T SG D - - - N - - - - - NQ R T S T L T T PQ E NWF SG K K F E C K V S HS S I - - Q P I I K HAQ V L I DK NG S T S I F V T L HP P K V K E L F L NNL A V L V C E V NG K E A - - - A NV E I SWF VDSQ KQ SQHV K T SG D - - - N - - - - - NQ R T S T L T T PQ E NWF SG K K F E C K V S HS S I - - Q P I I K HAQ V L I DK N- - P C L Q V NL K DADQ K E L F I S NR A V L S C E V EG DNL - - - I G AQ V SWT - - EGG K P A TGQ L - - - - - - - - NL TQ T R A V S NL T I DA S KWY SG E V F E C I VQ L - K SQQDP I K RQ I Q V NNRG L- - E T V K V V I E P P T NE EQ F V K K T A T L T CR R I A L V S T S D - - V SMTWS - - SGG K P L A AG A P E F - - - G HEGG K I V A V S R V S V V L E KWRTG T E Y K C I V S HL DS F P T P I T K T Y K RQ I A T -L T T DV T V T I QG P E A K E V F L Q K RG T L T C I A RG L A DE NE K T I A I TWY K DNG K K P L A S V T P T P E L Q Y T DDG R I Q A T S R V I I S K E EWS S NS T Y K CV V A HP A S F P S P K E E T Y R RDNGHV- - E T V K V V I E P P T NE EQ F V K NT A T L T C R A I G L V S T S D - - V SMTWS - - SGG K P L A AG A P E F - - - G HEGG K I V A V S R V S V V L E KWRTG T E Y K C I V S HL DS F P T P I T K T Y K RQ I A T -
371 381 391 401 411 421 431 441 451 461 471IgT4D_Amia_calvaIgT4D_Lepisosteus_oculatusIgT4D_Atractosteus_spatulaIgT3D_Lepisosteus_oculatusIgM_Lepisosteus_osseusIgM_Amia_calvaIgM_Lepisosteus_oculatus
QQR P T V Y T L P P S E E E I - N I NK T A TMMCY V YG F K P RD I Y V FWK HH I L DG S DL P A - - - - I T G E P VM - - KG NS Y S V I S HL T V S K A DWE T NS - Y L C A V K HS SMGNN I H I P L T S HV K K NGL K K P V V N I Q K P NDNRV - G NS NT V NL T CV V SG F Y P DD I Y I MWK E S DKQG S T T S DR E DDL T S K P V KQ P NDE S Y T A F SQ L T V R K E EWNE K K - Y T C A V K HA S L G NNSQ S P E L DS I T K DDL K K P V V N I Q K P NDNRV - G NS NT V NL T CV V SG F Y P DD I Y I MWK E S DKQG S T T S DR E DDL T S K P V KQ P NDE S Y T A F SQ L T V R K E EWNE K K - Y T C A V K HV S L G NNSQ S P E L DS I T K DDKQ K P T L Y I L P P S E A E I - R E KG S A T L V C L V TG F F P E N I Y I MWG E NGNL K E EG I - - - - - - - - T S R P V K NNG L Y S AMS Y L T I A K DQWE K NE - Y L C A V K HP S L G DNK T A P RMK T I K K E DK I R P S V F L L A P S T E E NS S T RDE V T L T C F V K DF S P K D I Y I SWL AQDS I V DK T HY - - - - T V T DL I P S HD - G A Y S V Y S K Y T I S S S DWNSG TMY S CA V HHE T A P L P V S V I - T R T T DS S TE R T P S V F L L P P S S EQNSG I R E E L P L T C F I K DF Y P K DV Y V SWL ADDV P V A E S K F - - - - HT T S L I E E E AG K S Y S V Y S K L S V R T S DWNSG V F Y T CV V HHE S T P DS V PM I - T R T I DS S SK I R P S V F L L A P S T E E S S S T RDE V T L T C F V K DF S P K D I Y I SWL AQDS V VDK T HY - - - - T V T DL I P S HD - G T Y S V Y S K Y T I S S S DWNSG TMY S CA V HHE T A P L P V S V I - T R T T DS S T
CH1
CH2
CH3
CH4
Figure 2: Alignment of the IgT and IgM immunoglobulin domains sequences of Holosteos. The graph was ob-tained with Jalview. The IgT sequences from Lepisosteus oculatus were deduced from the sequenced genome usingCHfinder, while those of Amia calva and Atractosteus spatula were obtained from RNA-seq transcriptomes. The IgMsequences of Lepisosteus oculatus was obtained from CHfinder, while the IgM of Amia calva and Lepisosteus osseuswere obtained from GenBANK with accession number AAC59687 and AAC59688, respectively.
3.2. IgT in TeleosteiCHfinder allowed us to identify 136 candidate IgT genes from the 73 Actinopterygii (see Table
S1). In the Fish-T1K repository, 57 IgT sequences from 57 fish species were identified. Addition-ally, a bibliographical search identified 24 articles that made reference to IgT sequences in 24teleost fishes. The obtained data set was processed manually in order to eliminate those sequencesthat were incomplete or repeated. Finally, we obtained 142 complete IgT sequences and 88 IgMsequences from 88 Actinopterygii species. With these sequences, a comparative phylogeneticstudy was undertaken.
Evolutionary relationships between the IgT and the other immunoglobulins IgM, IgW, andIgNAR can be observed from comparative phylogenetic trees of these sequences. Additionally,the sequences from Elasmobranchii were also included since their origin dates prior to that ofActinopterygii. Thus, using the appearance of the Elasmobranchii as a reference, three cladescan be be observed in the resulting tree: one for Neopterygii IgT, one for Actinopterygii IgM,and a third clade encompassing the Elasmobranchii immunoglobulins. Within the IgM clade, theappearance of two Neopterygii IgT subclades is observed, one is for the 2-domain IgT found in
6
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Cichliform fish, and the other is for the IgT3D found in Lepisosteus oculatus. These two anomaliesmay be explained by the high homology of its CH1 with CH1M (both domains have quasi-identicalsequences), together with the decrease in the number of domains present.
The clade corresponding to the majority of IgT sequences is unique and does not share acommon node with IgM from Actinopterygii. This suggests an early origin, thereby discountingthe notion of an IgM duplication in the evolution towards Actinopterygii (see Fig 3).
A study of the IgT domains in holosteos is of particular interest, since until now, the oldestIgTs described in the scientific literature correspond to those of Cypriniformes. In addition, theseobservations of IgT in other teleost species whose origin is prior to that of Cypriniformes, havenot been previously described. As such, the large number of these IgT sequences provides anopportunity to study the evolutionary events that may underlie the origin of IgT (Figure 4). In 53IgT sequences, exon coding CH1T is very similar to that of CH1M (taxa are in the same clade),while 80 other sequences are within a clade related to CH1M . These results suggest that the exoncoding CH1 of the Neopterygii IgT shows a tendency to be exchanged for the exon coding CH1of the IgM of the same species; in some cases, this occurred quite recently, since the CH1M andCH1T sequences have similarities of more than 98%. Such phenomena is already observed earlyin the IgT3D of Lepisosteus oculatus.
The CH2 and CH3 domains of IgT show a greater evolutionary proximity to CH2 and CH3 ofIgW and IgNAR than to those of Actinopterygii IgM. Finally, the CH4 of IgT generates a cladethat is evolutionary distant from the rest of the immunoglobulins. This may be because its origincoincides with the rise of immunoglobulins, or because of rapid evolutionary processes of thisdomain driven by selective pressure.
3.3. IgT absence in TeleosteiFrom the analysis with CHfinder, genes were not found for the IgT in 25 species out of the 73
Actinopterygii studied (see Table S1). The absence in two of these species (Epinephelus lanceo-latus and Datnioides undecimradiatus) can be explained by assemble problems (presence of gapsin the locus that contain the immunoglobulins). In other cases, one species of Siluriform (channelcatfish) and another of Beloniformes (medaka) had already been described as lacking the genefor the IgT (Magadán-Mompó et al., 2011; Bengtén et al., 2006). Here, we also did not find thisgene in three other Siluriformes species as well as another from the Beloniformes. In all other fishspecies studied, the loss of IgT is new information that has not been previously described. For acomplete listing, refer to Table S4 and Figure 5.
7
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IgT1 4
D Asty
anax
mexic
anus
IgT
3D C
ynog
loss
us s
emila
evis
IgT 3D Cebidichthys violaceus
IgM C
yprinus carpio
IgM
Dat
nioi
des
mic
role
pis
IgM Huso huso
IgT 4D
Cten
opha
ryng
odon
idell
a
IgT1 2D Maylandia zebra
IgW Scyliorhinus canicula
IgT 4D Atractosteus spat
ula
IgT 4D Xiphophorus maculatus
IgT1 4D Salmo salar
IgM Papyrocranus afer
IgT5 2D Oreochrom
is niloticus
IgM Lepisosteus OculatusIg
M L
utja
nus
seba
e
IgT 4D B
ovichtus diacanthus
IgT5 4D Planiliza haematocheilus
IgT1 3D Planiliza haematocheilus
IgM
Eso
x lu
cius
IgT3 3D Ang
uilla japonica
IgT2 4D
Astyan
ax mexic
anus
IgT4 4D Myripristis murdjan
IgM
Pla
niliz
a ha
emat
oche
ilus
IgM An
abas
testu
dineu
s
IgM
Sal
mo
sala
r
IgT1
3D
Trip
loph
ysa
tibet
ana
IgT 2D
Astya
nax me
xicanu
s
IgT 2D Astatotilapia calliptera
IgM Acipenser baerii
IgT1 3D Perca flavescens
IgM
Lab
rus
berg
ylta
IgNAR Scyliorhinus canicula
IgM
Aulo
stom
us m
acul
atus
IgT1 4D Esox lucius
IgT 3D Lepisosteus oculatus
IgT2 4D Lepisosteus oculatus
IgT1 4D Oncorhynchus kisutch
IgM
Ter
apon
jarb
ua
IgT
4D D
icen
trarc
hus
labr
ax
IgW Triakis scyllium
IgM Astyanax m
exicanus
IgNAR Squalus acanthias
IgM Triakis scyllium
IgT1 4D P
aramormy
rops king
sleyae
IgM Arc
hocent
rus cen
trarchu
s
IgT1 4D Salarias fasciatusIg
M M
yrip
ristis
mur
djan
IgT1 4D Mastacembelus arm
atus
IgM Chiloscyllium punctatum
IgM C
ollich
thys l
ucidu
s
IgT2 2D Maylandia zebra
IgT4 2D Oreochromis niloticus
IgM Lepisosteus osseus
IgM A
nguilla japonica
IgT
4D D
repa
ne p
unct
ata
IgM P
erca
flave
scen
s
IgT1 3D Parambassis ranga
IgT 4D Ang
uilla japon
ica
IgT1
4D
Lat
eola
brax
mac
ulat
us
IgT 4D Thunnus orientalisIgT3 4D Planiliza haem
atocheilus
IgM
Tak
ifugu
rubr
ipes
IgM
Sin
iper
ca c
huat
si
IgT 4D Terapon jarbua
IgM Cy
noglo
ssus
semi
laevis
IgT 4D Epinephelus coioides
IgT6
3D
Chan
osch
anos
IgT1
4D D
anio
rerio
IgM Acipenser ruthenus
IgM Ginglymostoma cirratum
IgT 4D C
entropomus sp
IgM Sa
larias
fasci
atus
IgT 3D P
apyrocr
anus af
er
IgT 2D Mastacembelus armatus
IgT 4D Amia Calv
a
IgM D
anio rerio
IgT2 4D Planiliza haematocheilus
IgT1 4D Myripristis murdjan
IgT 2D
Cyp
rinus
carp
io
IgM Pa
rambas
sis ran
ga
IgT 2D Takifugu rubripes
IgM Scyliorhinus canicula
IgM Maylan
dia zebra
IgT 4D
Ambly
gaste
r clup
eoide
s
IgT1 2D Ana
bas testudin
eusIgT1 2D Archoc
entrus centrar
chus
IgT
4D S
paru
s au
rata
IgM Squalus acanthias
IgM Asta
totilapia
callipter
a
IgT
4D S
coph
thal
mus
max
imus
/1-6
05
IgM Erpetoichthys calabaricus
IgM Amia calvaIgM
Amblygaster clupeoides
IgNAR Triakis scyllium
IgM Ore
ochrom
is niloti
cus
IgM Chanos chanos
IgW Squalus acanthias
IgT1
3D C
hano
s cha
nos
IgT 3D
Astya
nax me
xicanu
s
IgT1 4D Planiliza haematocheilus
IgT
4DLa
brus
ber
gylta
IgT1 4D Oncorhynchus mykiss
Figure 3: The phylogenetic tree consisting of 105 immunoglobulin sequences. The identified clades are as follows:IgT of Neopterygii (green) , IgM of Actinopterygii (red), and the immunoglobulins of Elasmobranchii (blue). IgT4D, IgT 3D, and IgT 2D refer to the number of immunoglobulin domains that IgT exhibits. Gray circles mark thedivergence points of immunoglobulins, taking as reference their appearance in Elasmobranchii.
4. Discussion
At present, there is an increasing availability of high-quality genomes and transcriptomes offish species. By using machine learning, the CHfinder program has allowed us to study fish Igs ina fast and agile manner. The program identifies the majority of the CH exons in available genomes
8
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-
CH4IgTActinopterygii(142)
CH2IgTActinopterygii(99)
CH3IgTActinopterygii(142)
CH1IgTTeleostei (80)
CH1IgT/CH1IgMTeleostei (53/70)
CH1IgTAnguilla japonica(5)
CH3/CH5 IgW/IgNARElasmobranchii(13)
CH3IgMElasmobranchii(5)
CH3IgMActinopterygii(85)
CH6 IgW/IgNARElasmobranchii(6)
CH4IgMElasmobranchii(5)
CH4IgMActinopterygii(82)
CH2IgM
Elasmobr
anchii(5
)
CH2/CH4 IgW/IgNARElasmobranchii(14)
CH1IgM
Actinopterygii(7)
CH2IgMActinopterygii(85)
CH1IgW
Elasmobranchii(3)CH1IgNAR
Elasmobranchii(3)
CH1IgMTeleostei(5)
CH1IgM
Elasmobranchii(5)
12
3
45
6
Figure 4: The phylogenetic tree of the domain study of the 252 immunoglobulin sequences used in this work. Thenumber of domains per clade is provided in brackets. The immunoglobulin domains are labelled as follows: IgM ofActinopterygii and Elasmobranchii (red), the Neopterygii IgT (green), those of IgW and IgNAR of Elasmobranchii(orange), and a mixed clade with IgM and IgT CH1 domains (blue). Asterisks are used to indicate the presence ofa domain for the IgT of holosteos. In particular, these are as follows: (asterisk 1) the CH1 IgT of the IgT3D ofLepisosteus oculatus, (asterisk 2) the CH1 IgT domain of the Holosteos IgT residue, (asterisk 3) the CH2 IgT fromHolosteos, (asterisk 4) the CH3 IgT of IgT3D, (asterisk 5) the CH3 IgT domain of the Holosteos IgT residue, and(asterisk 6) the CH4 IgT from Holosteos.
with high accuracy. The absolute number of CH exons may be an underestimate in some casesdue to the early stages of sequencing projects and the presence of gaps. For genome searches, itdoes not attempt to differentiate whether an exon sequence belongs to a pseudogene or a viablegene. The program provides information about the presence of homologous exon sequences tohigh precision. As such, it is valid for the identification of genes and obtaining sequences forevolutionary studies.
Previous publications suggest that IgT had its origin through duplication of the IgM gene.
9
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Ictaluridae
Pangasiidae
Loricariidae
Percophidae
Denticipitidae
Salmonidae
Congridae
Batrachoididae
Mormyridae
Characidae
Osphron
emidae
Sinipercidae
Siluridae
Nemacheilidae
Pomacentridae
Gasteropelecidae
Muraenesocidae
Callionymidae
Bovichtidae
Cirrhitidae
Amiidae
Echeneidae
Nothobranchiidae
Amblycipitidae
Lutjanidae
Esocidae
Adrianichthyidae
Ostraciidae
Syngnathidae
Moronidae
Botiidae
Mullidae
Aulostomid
ae
Gadidae
Cynoglossidae
Haemulidae
Terapontidae
Paralichthyidae
Diodontidae
Serranid
ae
PolypteridaeSisoridae
Percichthyidae
Notopteridae
Poeciliidae
Oplegnathidae
Channid
ae
Mugilidae
Callichthyidae
Datnioididae
SciaenidaeTetraodontidae
GobiesocidaeChaenopsidae
Cichlidae
Cyprinidae
Eleotridae
Lateolabracidae
Scophthalmidae
Gymnotidae Pl
otosidae
Pomacanthidae Gerreidae
Caproidae
Ambassidae
Apogonidae
Percidae
Chanidae
Centrarchidae
Engraulidae
Anguillidae
Labridae
Tripterygiidae
Muraenidae
Stichaeidae
Acanthuridae
Pleuronectidae
Carangidae
Lepisosteidae
Gobiidae
Holocentridae
Osteoglossidae
Mastacem
belidae
Phallostethidae
Acipenseridae
Pantodontidae
Cobitidae
Blenniidae
Gasterosteida
e
Anabantid
ae
Balistidae
Clupeidae
Bagridae
Gyrinocheilidae
Peristediidae
Nototheniidae
Erythrinidae
Melanotaeniidae
Rivulidae
Epigonidae
Scorpaenid
ae
Sparidae
Ictaluridae
Pangasiidae
Loricariidae
Percophidae
Denticipitidae
Salmonidae
Congridae
Batrachoididae
Mormyridae
Characidae
Osphron
emidae
Sinipercidae
Siluridae
Nemacheilidae
Pomacentridae
Gasteropelecidae
Muraenesocidae
Callionymidae
Bovichtidae
Cirrhitidae
Amiidae
Echeneidae
Nothobranchiidae
Amblycipitidae
Lutjanidae
Esocidae
Adrianichthyidae
Ostraciidae
Syngnathidae
Moronidae
Botiidae
Mullidae
Aulostomid
ae
Gadidae
Cynoglossidae
Haemulidae
Terapontidae
Paralichthyidae
Diodontidae
Serranid
ae
PolypteridaeSisoridae
Percichthyidae
Notopteridae
Poeciliidae
Oplegnathidae
Channid
ae
Mugilidae
Callichthyidae
Datnioididae
SciaenidaeTetraodontidae
GobiesocidaeChaenopsidae
Cichlidae
Cyprinidae
Eleotridae
Lateolabracidae
Scophthalmidae
Gymnotidae Pl
otosidae
Pomacanthidae Gerreidae
Caproidae
Ambassidae
Apogonidae
Percidae
Chanidae
Centrarchidae
Engraulidae
Anguillidae
Labridae
Tripterygiidae
Muraenidae
Stichaeidae
Acanthuridae
Pleuronectidae
Carangidae
Lepisosteidae
Gobiidae
Holocentridae
Osteoglossidae
Mastacem
belidae
Phallostethidae
Acipenseridae
Pantodontidae
Cobitidae
Blenniidae
Gasterosteida
e
Anabantid
ae
Balistidae
Clupeidae
Bagridae
Gyrinocheilidae
Peristediidae
Nototheniidae
Erythrinidae
Melanotaeniidae
Rivulidae
Epigonidae
Scorpaenid
ae
Sparidae
No IgT
IgT
No Evidence for IgT
Siluriform
es
Osteoglossiformes
Atheri
nomorp
hae
Gobiaria
Figure 5: Cladogram from the data obtained in Table ??. Clades in green indicate the presence of IgT in at least onemember of that family. The red clades indicate the loss of IgT in the members analyzed for that family. Those cladeswith insufficient data to determine the absence of IgT are colored orange.
However, this work indicate a more complex origin. CH1T is evolutionarily related to CH1Mand there are numerous species with identical sequences in these two domains. Probably the needto receive the VDJ complex is the basis of its similarities and the cause of its integration intoIgT. The CH2T and CH3T domains by evolutionary proximity are closer to the CH2 and CH3domains of IgW and IgNAR of Elasmobranchii than to those of IgM. Finally, the CH4 domainraises doubts as to its origin due to the high divergence that it presents against its IgM and ofthe IgW and IgNAR counterparts, suggesting the possibility that this domain is so ancient that itdirectly connects with the appearance of the immunoglobulins themselves. In 2016, the genome
10
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of Lepisosteus oculatus was sequenced and the presence of two Immunoglobulin loci was noted(Braasch et al., 2016). In this work, we describe two new IgTs, one with 4 domains located in allHolostei fishes and one with 3 domains in spotted gar (IgT3D). Holostei have approximately 320million years of independent evolution with respect to Teleostei. The CH2 and CH3 domains of theHolostei IgT are more similar to IgW domains, suggestive of their probable origin. IgT3D presentstwo peculiarities: it does not present the CH2 domain and the CH1 domain has a high similaritywith the CH1 of the IgM, indicating a recent exchange. Both observations are in accordancewith previous works in Teleosts. The presence of IgT in Holostei show that it is not a specificimmunoglobulin of Teleostei.
Here, we found that both Holostei and Telostei possess IgT, contrary to the belief that it is aspecific teleost immunoglobulin. Its origin would be approximately 300 Mya, coinciding with thelate Permian and the appearance of the Neopterygii. This is supported by the fact that we havenot found IgT in Acipenseriformes or in Polypteriformes. However, the Ig domain tree shows thatCH2, CH3, and CH4 of the IgT in Neopterygii diverged prior to the occurrence of Actinopterygii,suggesting a recent loss of IgT in Acipenseriformes and Polypteriformes. This loss in fish ofwhat seem to be key elements of the adaptive immune systems, without seeming to condition theviability of these species, is seen in several species. Examples include the loss of MHC-II in theAtlantic cod, pipefish y anglerfish (Star et al., 2011; Haase et al., 2013; Dubin et al., 2019), theloss of IgT in the medaka and channel catfish, or the loss of immunoglobulin genes in Gouaniawilldenowii.
Our work corroborates previous studies that IgT exists throughout the Neopterygii clade. Untilnow however, IgT loss has been documented in only two species of Teleostei. Here we describethat the extent of this loss is greater than previously observed. Moreover, this appears to be at thelevel of order or family. Nonetheless, the causes and consequences of the loss of this Ig isotyperemains unclear. In this work we have sought to clarify the origin of IgT and described its presencein Holostei. However, several questions remain, including whether IgT arose as an IgM-IgW/NARchimera with the emergence of the Neopterygii.
11
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5. References
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Supplementary Material
Immunoglobulins genes in ActinopterygiiSpecie IGT IGM IGD Order FamilyCLADISTIAErpetoichthys calabaricus 0 1 1 Polypteriformes PolypteridaeACTINOPTERI->ChondrosteiAcipenser ruthenus 0 2 3 Acipenseriformes Acipenseridae->Neopterygii–>HolosteiLepisosteus oculatus 2 1 1 Lepisosteiformes Lepisosteidae–>Teleostei—>ElopocephalaAnguilla japonica 5 1 1 Anguilliormes Anguillidae—>OsteoglossocephalaScleropages formosus 0 2 2 Osteoglossiformes OsteoglossidaeParamormyrops kingsleyae 2 1 1 Osteoglossiformes Mormyridae—>Clupeocephala—->OtomorphaClupea harengus 1 2 2 Clupeiformes ClupeidaeCoilia nasus 2 1 1 Clupeiformes EngraulidaeDenticeps clupeoides 0 1 1 Clupeiformes DenticipitidaeChanos chanos 6 2 2 Gonorynchiformes ChanidaeCarassius auratus 4 2 2 Cypriniformes CyprinidaeDanio rerio 1 1 1 Cypriniformes CyprinidaeCyprinus carpio 2 2 3 Cypriniformes CyprinidaeTriplophysa tibetana 2 2 2 Cypriniformes NemacheilidaeElectrophorus electricus 1 1 1 Gymnotiformes GymnotidaeBagarius yarrelli 0 1 1 Siluriformes SisoridaeIctalurus punctatus 0 1 2 Siluriformes IctaluridaeTachysurus fulvidraco 0 1 1 Siluriformes BagridaePangasianodon hypophthalmus 0 Gap 1 Siluriformes PangasiidaeAstyanax mexicanus 5 1 3 Characiformes Characidae—->Euteleosteomorpha—–>ProtacanthopterygiiSalmo trutta 3 2 2 Salmoniformes SalmonidaeOncorhynchus mykiss 3 2 4 Salmoniformes SalmonidaeOncorhynchus tshawytscha 1 2 2 Salmoniformes SalmonidaeOncorhynchus kisutch 3 1 2 Salmoniformes SalmonidaeCoregonus 3 1 3 Salmoniformes SalmonidaeEsox lucius 2 2 2 Esociformes Esocidae
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-
Specie IGT IGM IGD Order Family—->Neoteleostei—–>ParacanthomorphaceaGadus morhua 0 3 3 Gadiformes Gadidae——>HolocentrimorphaceaeMyripristis murdjan 5 8 8 Holocentriformes Holocentridae——>Percomorphaceae——->BatrachoidiariaThalassophryne amazonica 1 2 0 Batrachoidiformes Batrachoididae——>Percomorphaceae——->EupercariaLabrus bergylta 7 12 16 Labriformes LabridaeNotolabrus celidotus 2 1 1 Labriformes LabridaeTakifugu rubripes 1 1 1 Tetraodontiformes TetraodontidaeLarimichthys crocea 1 2 1 Acanthuriformes SciaenidaeSparus aurata 5 2 2 Spariformes SparidaeCebidichthys violaceus 1 2 2 Perciformes StichaeidaeCottoperca gobio 1 1 1 Perciformes BovichtidaePerca flavescens 3 1 1 Perciformes PercidaePlectropomus leopardus 1 1 1 Perciformes SerranidaeEpinephelus lanceolatus Gap 2 1 Perciformes SerranidaeEpinephelus moara 0 1 1 Perciformes SerranidaeOplegnathus fasciatus 2 1 1 Centrarchiformes OplegnathidaeLateolabrax maculatus 2 2 2 Pempheriformes LateolabracidaeCollichthys lucidus 1 4 3 I. s. in Eupercaria SciaenidaeDatnioides undecimradiatus Gap 1 1 Lobotiformes Datnioididae——>Percomorphaceae——->GobiariaPeriophthalmus magnuspinnatus 0 1 1 Gobiiformes GobiidaeNeogobius melanostomus 0 2 2 Gobiiformes GobiidaeSphaeramia orbicularis 0 1 2 Kurtiformes Apogonidae——>Percomorphaceae——->SyngnathariaSyngnathus acus 0 1 1 Syngnathiformes Syngnathidae——>Percomorphaceae——->AnabantariaAnabas testudineus 3 1 3 Anabantiformes AnabantidaeBetta splendens 0 1 1 Anabantiformes OsphronemidaeChanna argus 1 1 4 Anabantiformes ChannidaeMastacembelus armatus 4 1 1 Synbranchiformes Mastacembelidae
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Specie IGT IGM IGD Order Family——>Percomorphaceae——->CarangariaCynoglossus semilaevis 1 1 1 Pleuronectiformes CynoglossidaeScophthalmus maximus 1 1 1 Pleuronectiformes ScophthalmidaeReinhardtius hippoglossoides 0 1 1 Pleuronectiformes PleuronectidaeHippoglossus hippoglossus 0 1 1 Pleuronectiformes PleuronectidaeEcheneis naucrates 1 3 3 Carangiformes Echeneidae——>Percomorphaceae——->OvalentariaPlaniliza haematocheilus 20 4 4 Mugiliformes MugilidaeArchocentrus centrarchus 4 4 4 Cichliformes CichlidaeOreochromis niloticus 5 6 6 Cichliformes CichlidaeAstatotilapia calliptera 1 2 3 Cichliformes CichlidaeMaylandia zebra 4 2 3 Cichliformes CichlidaeNeostethus bicornis 0 1 1 Atheriniformes PhallostethidaePoecilia reticulata 2 1 1 Cyprinodontiformes PoeciliidaeXiphophorus maculatus 2 1 1 Cyprinodontiformes PoeciliidaeNothobranchius furzeri 0 1 2 Cyprinodontiformes NothobranchiidaeKryptolebias hermaphroditus 2 2 2 Cyprinodontiformes RivulidaeOryzias latipes 0 6 6 Beloniformes AdrianichthyidaeOryzias javanicus 0 3 3 Beloniformes AdrianichthyidaeSalarias fasciatus 5 9 11 Blenniiformes BlenniidaeGouania willdenowi 0 0 0 Blenniiformes GobiesocidaeParambassis ranga 4 1 2 I. s. in Ovalentaria AmbassidaeAmphiprion percula 0 4 6 I. s. in Ovalentaria Pomacentridae
Table S1: Results of gene/exon identification with CHfinder
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IgT 2D M
astacembelus arm
atus
IgM
Larim
ichth
ys cro
cea
IgM Megalobrama amblycephala
IgT3 4D M
astacembelus arm
atus
IgM Esox lucius
IgT 4
D Th
alass
oma b
ifasc
iatum
IgT2 4D Astyanax m
exicanus
IgT3 3D Chanos chanos
IgT2 2D Maylandia zebra
IgM Aul
ostomus
macula
tus
IgT3 2D Archocentrus centrarchus
IgT2 4D Coregonus sp
IgM
Das
cyllu
s tr
imac
ulay
us
IgT1 4D Paramormyrops kingsleyae
IgM Scleropages form
osus
IgM
Lat
eola
brax
mac
ulat
us
IgT3
4D
Pla
niliz
a ha
emat
oche
ilus
IgT3 2D M
aylandia zebra
IgM Danio rerio
IgT 2D Astyanax
mexicanus
IgT4
4D
Pla
niliz
a ha
emat
oche
ilus
IgM
Par
amba
ssis
ran
ga
IgT 4D Epi
nephelus c
oioides
IgM M
aylandia zebra
IgT 4D Oncorhynchus tshawytscha
IgM Scyliorhinus canicula
IgT 4D
Ople
gnat
hus f
ascia
tus
IgT2 3D Param
bassis ranga
IgM
Ceb
idic
hthy
s vi
olac
eus
IgT 2D Echeneis naucrates
IgT3 3D Param
bassis ranga
IgM Ec
heneis
naucr
ates
IgT 2D Cyprinus carpio
IgT2 4D Anguilla japonica
IgT
4D T
rach
inot
us o
vatu
s
IgT2 4D Paramormyrops kingsleyae
IgM
Poe
cilia
ret
icul
ata
IgM Ctenopharyngodon idella
IgM Param
ormyrops kingsleyae
IgM
Xip
hoph
orus
mac
ulat
us
IgT3 3D Planiliza
haematocheilus
IgT1 4D Myripristis m
urdjan
IgT 4D Polynem
us dubius
IgM-A Salm
o/1-448
IgT 4D Thunnus orientalis
IgT 3D Thalassophryne am
azonica
IgT
2D O
plegn
athu
s pun
ctat
us
IgM
Lutja
nus s
ebae
IgM
Tak
ifugu
rubr
ipes
IgT1 4D Esox lucius
IgT 3D Trematomus bernacchii
IgM Erpetoichthys calabaricus
IgT1 4D
Sparus
aurata
IgM
Sal
aria
s fa
scia
tusIgM
Archocentrus centrarchus
IgT2 4D Myripristis m
urdjan
IgT2 3D Planiliz
a haematocheil
us
IgT1
4D
Pla
niliz
a ha
emat
oche
ilus
IgM O
ncorhynchus mykiss
IgT
4D P
aral
ichth
ys o
livac
eus
IgM
Par
aper
cis
xant
hozo
na
IgT 3D
Lutjan
us seba
e
IgT 4D Pro
notogramm
us martinice
nsis
IgT 4D
Labru
s berg
ylta
IgT 3D Gaste
ropelecus sp
IgM Ch
anna m
icropel
tes
IgM Erythrinus erythrinus
IgW Scyliorhinus canicula
IgT 4D
Zanc
lus co
rnutus
IgT3 3D Anguilla japonica
IgT1 3D Plan
iliza haematoc
heilus
IgT
4D S
inip
erca
chu
atsi
IgM
Zanc
lus co
rnut
us
IgT 4D
Diodo
n holo
canthu
s
IgT4 3D Anguilla japonica
IgT
4D A
ulos
tom
us m
acul
atus
IgM Amia calva
IgT2 4D Oncorhynchus mykiss
IgT2 3D Triplophysa tibetana
IgM
Tha
lass
oma
bifa
scia
tum
IgT 4D P
oecilia r
eticulata
IgM Scleropages form
osus
IgT 3D Param
bassis pulcinella
IgM
Par
amba
ssis
pul
cine
lla
IgM Lepisosteus osseus
IgT 3D Papyrocranus afer
IgM K
ryptolebias hermaphroditus
IgT 4D Cottoperca g
obio
IgM
Pom
acan
thus
par
u
IgT 4D Anguilla japonica
IgM O
reochromis niloticus
IgT 2D
Lutjan
us fulv
iflamm
a
IgT5 4D Anguilla japonica
IgM Squalus acanthias
IgT
4D C
hann
a ga
chua
IgT6
4D
Pla
niliz
a ha
emat
oche
ilus
IgT2 2D Oreochromis niloticus
IgT 3D Esox lucius
IgM
Sin
iper
ca c
huat
si
IgT 3D Lepisosteus oculatus
IgNAR Squalus acanthiasIg
T 2D
Kry
ptol
ebia
s he
rmap
hrod
itus
IgM Sc
ophth
almus
maxim
us
IgM Acipenser ruthenus
IgT 3D Plecoglossus altivelis
IgM
Datn
ioide
s micr
olepis
IgM
Tha
lass
ophr
yne
amaz
onic
a
IgM
Dice
ntra
rchus
labr
ax
IgT5 2D O
reochromis niloticus
IgT2 4D Danio rerio
IgT2 4D Salarias fasciatus
IgM Ginglymostoma cirratum
IgT 4D K
ryptolebias hermaphroditus
IgT1 2D M
aylandia zebra
IgW Squalus acanthias
IgT
4D T
erap
on ja
rbua
IgM
Anab
as te
studin
eus
IgT1 4D Salarias fasciatus
IgNAR Scyliorhinus canicula
IgM Cy
noglo
ssus s
emila
evis
IgT 2D
Chae
todon
aurig
a
IgT3 4D Myripristis m
urdjan
IgT 3D
Labru
s berg
ylta
IgT 4D Megalobrama amblycephala
IgM Chiloscyllium punctatum
IgNAR Triakis scyllium
IgT 3D Astyana
x mexicanus
IgM
Per
ca fl
aves
cens
IgM
Collic
hthy
s luc
idus
IgM Carassius auratus
IgT 4D A
mblyga
ster clup
eoides
IgM Cyprinus carpio
IgT 3D Notothenia coriiceps
IgM Plecoglossus altivelis
IgT 4D
Sparu
s aurata
IgM A
statotilapia calliptera
IgT 4D Ele
ctrophorus
electricus
IgT 3D Cebid
ichthys viola
ceus
IgT1 4D Coregonus sp
IgM Ma
stacem
belus
armatu
s
IgM Huso huso
IgMAstyanax m
exicanus
IgT4 3D Chanos chanos
IgM
Ter
apon
jarb
ua
IgM
Myr
iprist
is m
urdj
an
IgM
Ple
ctro
pom
us le
opar
dus
IgM Anguilla anguilla
IgM
Cot
tope
rca
gobi
o
IgM Electrophorus electricus
IgM
Bov
icht
us d
iaca
nthu
s
IgT3 4D Oncorhynchus mykiss
IgM Tr
achino
tus ov
atus
IgT 4D La
rimichthy
s crocea
IgT1 4D Danio rerio
IgT
4D G
erre
s fil
amen
tosu
s
IgW Triakis scyllium
IgT2 4D Lepisosteus oculatus
IgT4 4D Myripristis m
urdjan
IgT1 3D Chanos chanos
IgT 4D Bovichtus diacant
hus
IgT1 4D M
astacembelus arm
atus
IgT2 4D M
astacembelus arm
atus
IgT3 4D Salarias fasciatus
IgM Lepisosteus Oculatus
IgM Anguilla japonica
IgT2 2D Archocentrus centrarchus
IgM Amblygaster clupeoides
IgT2 4D Oncorhynchus kisutch
IgM
Not
othe
nia
corii
ceps
IgT6 3D Chanos chanos
IgT1 3D Triplophysa tibetana
IgM Salm
o salar
IgM Chanos chanos
IgT3 4D Carassius auratus
IgT 4D Hemig
rammus blehe
ri
IgM Acipenser baerii
IgT1 3D
Coilia na
sus
IgT2 3D
Coilia na
sus
IgM Pa
ralich
thys o
livaceu
s
IgT7
4D
Pla
niliz
a ha
emat
oche
ilus
IgT2
2D A
naba
s tes
tudin
eus
IgM Ch
anna
argu
s
IgT 4D C
entropomus sp
IgT1 4D Salmo salar
IgT1 4D Carassius auratus
IgM C
haet
odon
aur
iga
IgT
4D X
ipho
phor
us m
acul
atus
IgM
Epi
neph
elus
coi
oide
s
IgT 2D Astatotilapia calliptera
IgT4 4D Salarias fasciatus
IgT5 3D Chanos chanos
IgT2 4D Carassius auratus
IgT
3D C
ynog
loss
us s
emila
evis
IgT3 2D Oreochromis niloticus
IgT 4D
Dice
ntra
rchus
labr
ax
IgT4 2D Archocentrus centrarchus
IgM
Lab
rus
berg
ylta
IgT 2D Takifugu rubripes
IgT1
2D
Anab
as te
studin
eus
IgT 4D Ery
thrinus eryt
hrinus
IgM
Pla
niliz
a ha
emat
oche
ilus
IgT9
4D
Pla
niliz
a ha
emat
oche
ilus
IgM Rhamphichthys rostratus
IgT1 3D Param
bassis ranga
IgM Th
unnus o
rientalis
IgT2 3D Perca flavescens
IgM Po
lynemu
s dubiu
s
IgT 4D C
hanna argus
IgM S
paru
s au
rata
IgM G
erre
s fil
amen
tosu
s
IgT 4D
Drepa
ne pun
ctata
IgT2 3D Chanos chanos
IgT1
4D
Late
olab
rax
mac
ulat
us
IgT
4D P
arap
ercis
xan
thoz
ona
IgT2
4D
Late
olab
rax
mac
ulat
us
IgT
4D C
hann
a m
icro
pelte
s
IgT1 3D Perca flavescens
IgT 4D Ctenopharyngodon idella
IgM-B Salm
o/1-448
IgT2 4D
Sparus
aurata
IgT8
4D
Pla
niliz
a ha
emat
oche
ilus
IgM Hemigram
mus bleheri
IgM Gyrinocheilus aymonieri
IgT1 4D Oncorhynchus mykiss
IgM
Lutja
nus f
ulvifla
mma
IgT 4D Amia Calva
IgT1
1 4D
Pla
niliz
a ha
emat
oche
ilus
IgT
2D O
pleg
nath
us fa
sciat
us
IgT 4D Atractosteus spatula
IgT2
4D
Pla
niliz
a ha
emat
oche
ilus
IgT1 4D A
styanax m
exicanus
IgT 2D
Datni
oides
micro
lepis/
1-300
IgT1
0 4D
Pla
niliz
a ha
emat
oche
ilus
IgT2 4D Salmo salar
IgT5
4D
Pla
niliz
a ha
emat
oche
ilus
IgT1 2D Archocentrus centrarchus
IgT
4D D
ascy
llus
trim
acul
ayus
IgT3 4D Salmo salar
IgM O
reochromis niloticus/1-397
IgM
IgT3 4D Oncorhynchus kisutch
IgT1 4D Oncorhynchus kisutch
IgM Gasteropelecus sp IgM
Chan
na ga
chua
IgT 4D Gyrinocheilus aymonieri
IgT4 2D Oreochromis niloticus
IgT1
2 4D
Pla
niliz
a ha
emat
oche
ilus
IgT 4D Co
llichthys
lucidus
IgT 4D
Poma
canthu
s paru
IgM Papyrocranus afer
IgT
4D S
coph
thal
mus
max
imus
IgM Triakis scyllium
Figure S1: The phylogenetic tree that includes the 252 immunoglobulin sequences used in this work. The clades arelabelled as follows: IgT of Neopterygii (green), the IgM of Actinopterygii (red), Ig of Elasmobranchii (blue). Thelabels: IgT 4D, IgT 3D, and IgT 2D refer to the number of immunoglobulin domains that IgT exhibits.
19
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Summary TablesOrder Family Genome Fish T1K BibliographyPolypteriformes (3)
Polypteridae (3) NO (1) NO (3) NOAcipenseriformes (2)
Acipenseridae NO (1) No sample NOPolyodontidae No Genome NO (1) NO
Lepisosteiformes (3)Lepisosteidae NO (1) NO (2) NO
Anguilliormes (4)Anguillidae YES (1) YES (1) NOMuraenidae No Genome NO (1) NOMuraenesocidae No Genome NO (1) NOCongridae No Genome NO (1) NO
Osteoglossiformes (5)Osteoglossidae NO (1) NO (1) NONotopteridae No Genome NO (1) NOPantodontidae No Genome NO (1) NOMormyridae YES (1) YES (1) NO
Clupeiformes (4)Clupeidae YES (1) YES (1) NOEngraulidae YES (1) No sample NODenticipitidae NO (1) No sample NO
Gonorynchiformes (1)Chanidae YES (1) No sample NO
Cypriniformes (13)Cyprinidae YES (3) YES (1) YES (5)Nemacheilidae YES (1) NO (1) NOBotiidae No Genome YES (1) NOGyrinocheilidae No Genome YES (1) NOCobitidae No Genome YES (1) YES (1)
Gymnotiformes (2)Gymnotidae YES (1) YES (2) No
Siluriformes (11)Sisoridae NO (1) NO (1) NOIctaluridae NO (1) NO (1) NOBagridae NO (1) NO (2) NOPangasiidae NO (1) No sample NOSiluridae No Genome NO (1) NOLoricariidae No Genome NO (1) NOPlotosidae No Genome NO (1) NOAmblycipitidae No Genome NO (1) NO
20
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https://doi.org/10.1101/2020.05.21.108993
-
Order Family Genome Fish T1K BibliographyCallichthyidae No Genome NO (1) NO
Characiformes (4)Characidae YES (1) YES (1) NOGasteropelecidae No Genome YES (1) NOErythrinidae No Genome YES (1) NO
Salmoniformes (6)Salmonidae YES (5) No sample YES(2)
Esociformes (1)Esocidae YES (1) No sample NO
Gadiformes (2)Gadidae NO (1) NO (1) NO
Holocentriformes (4)Holocentridae YES (1) YES (3) NO
Batrachoidiformes(2)Batrachoididae YES (1) YES (1) NO
Labriformes (4)Labridae YES (2) YES (2) YES (1)
Tetraodontiformes (5)Tetraodontidae YES (1) No sample YES (1)Balistidae No Genome YES (1) NODiodontidae No Genome YES (1) NOOstraciidae No Genome NO (2) NO
Acanthuriformes (3)Sciaenidae YES (1) NOZanclidae No Genome YES (1) NOAcanthuridae No Genome NO (1) NO
Spariformes (1)Sparidae YES (1) No sample YES (1)
Perciformes (16)Stichaeidae YES (1) No sample NOBovichtidae YES (1) No sample YES (1)Percidae YES (1) No sample NOSerranidae YES/NO (1/2) YES (1) YES (1)Nototheniidae No Genome No sample YES (2)Gasterosteidae No Genome No sample YES (1)Scorpaenidae No Genome YES/NO (2/2) NOPeristediidae No Genome NO (1) NO
Centrarchiformes (8)Oplegnathidae YES (1) YES (1) NOSinipercidae No Genome YES (1) YES (1)Terapontidae No Genome YES (1) NO
21
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Order Family Genome Fish T1K BibliographyCentrarchidae No Genome YES (1) NOCirrhitidae No Genome NO (1) NOPercichthyidae No Genome NO (1) NO
Pempheriformes (3)Lateolabracidae YES (1) No sample NOEpigonidae No Genome NO (1) NOPercophidae No Genome NO (1) NO
I. s. in Eupercaria (10)Sciaenidae YES (1) YES (1) NOLutjanidae No Genome YES (2) NOPomacanthidae No Genome YES (1) NOGerreidae No Genome YES (2) NOHaemulidae No Genome YES (1) NOCaproidae No Genome YES (1) NOMoronidae No Genome No sample YES (1)
Lobotiformes (2)Datnioididae NO (1) YES (1) NO
Gobiiformes (5)Gobiidae NO (2) NO (2) NOEleotridae No Genome NO (1) NO
Kurtiformes (3)Apogonidae NO (1) NO (2) NO
Syngnathiformes (2)Syngnathidae NO (1) NO (1) NOMullidae No Genome YES (1) NOCallionymidae No Genome NO (1) NOAulostomidae No Genome YES (1) NO
Anabantiformes (6)Anabantidae YES (1) NO (1) NOOsphronemidae NO (1) YES (1) NOChannidae YES (1) YES (2) NO
Synbranchiformes (2)Mastacembelidae YES (1) NO (2) NO
Pleuronectiformes (5)Cynoglossidae YES (1) No sample NOScophthalmidae NO (1) NO (1) YES (1)Pleuronectidae NO (2) No sample NOParalichthyidae No Genome No sample YES (1)
Carangiformes (3)Echeneidae YES (1) No sample NOCarangidae No Genome YES (2) NO
22
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Order Family Genome Fish T1K BibliographyMugiliformes
Mugilidae YES (1) YES/NO (1/1) NOCichliformes (6)
Cichlidae YES (4) NO (2) YESAtheriniformes
Phallostethidae NO (1) No sample NOMelanotaeniidae No Genome NO (2) NO
CyprinodontiformesPoeciliidae YES (2) YES (2) NONothobranchiidae NO (1) No sample NORivulidae YES No sample NO
Beloniformes (3)Adrianichthyidae NO (2) NO (1) NO
BlenniiformesBlenniidae YES (1) No sample NOGobiesocidae NO (1) NO (2) NOTripterygiidae No Genome NO (1) NOChaenopsidae No Genome NO (1) NO
I. s. in Ovalentaria (5)Ambassidae YES (1) (1) NOPomacentridae NO (1) YES (2) NO
Table S4: Data for the IgT cladogram.
23
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IgT of different Teleostei published in scientific articlesSpecie IGT Domain Order Family Protein NCBI Bibliography
Megalabrama amblycephala 1 4 Cypriniformes Cyprinidae AGR34024.1 (Xia et al., 2016)
Ctenopharyngodon idella 1 4 Cypriniformes Cyprinidae ADD82655.1 (Xiao et al., 2010)
Cyprinus carpio 1 2(1,4) Cypriniformes Cyprinidae BAD69715.1 (Savan et al., 2005a)Cyprinus carpio 1 4 Cypriniformes Cyprinidae BAJ41037.1 (Ryo et al., 2010)
Danio rerio 1 4 Cypriniformes Cyprinidae AAT67444.1 (Danilova et al., 2005)Danio rerio 1 4 Cypriniformes Cyprinidae ACH92959.1 (Hu et al., 2010)
Oncorhynchus mykiss 3 4 Salmoniformes Salmonidae AAW66978.1 (Zhang et al., 2017)Oncorhynchus mykiss 3 4 Salmoniformes Salmonidae AAV48553.1 (Zhang et al., 2017)Oncorhynchus mykiss 3 4 Salmoniformes Salmonidae ANW11927.1 (Zhang et al., 2017)
Salmo salar 3 4 Salmoniformes Salmonidae ADD59873.1 (Yasuike et al., 2010)Salmo salar 3 4 Salmoniformes Salmonidae ADD59858.1 (Yasuike et al., 2010)Salmo salar 3 4 Salmoniformes Salmonidae ADD59859.1 (Yasuike et al., 2010)
Plecoglossus altivelis 1 3(1,2,4) Osmeriformes Plecoglossidae BAP75403.1 (Kato et al., 2015)
Takifugu rubripes 1 2(1,4) Tetraodontiformes Tetraodontidae BAD69712.1 (Savan et al., 2005b)
Sparus aurata 1 4 Spariformes Sparidae ASK39431.1 (Piazzon et al., 2016)
Bovichtus diacanthus 1 4 Perciformes Bovichtidae AKA09828.1 (Giacomelli et al., 2015)
Trematomus bernacchii 3 3(1,3,4) Perciformes Nototheniidae AKA09825.1 (Giacomelli et al., 2015)
Notothenia coriiceps ? 3(1,3,4) Perciformes Nototheniidae AKA09827.1 (Giacomelli et al., 2015)
Epinephelus coioides 1 4 Perciformes Serranidae ACZ54909.1 (Du et al., 2016)
Siniperca chuatsi 1 4 Centrarchiformes Sinipercidae AAY42141 (Tian et al., 2009)
Thunnus orientalis 1 4 Scombriformes Scombridae AHC31432 (Mashoof et al., 2014)
Dicentrarchus labrax 1 4 Eupercaria I.S. Moronidae AKK32388.1 (Buonocore et al., 2017)
Scophthalm