Immunoglobulin T genes in Neopterygii - bioRxiv.org · 2020. 5. 21. · Immunoglobulin T genes in...

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

[email protected]

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

https://doi.org/10.1101/2020.05.21.108993

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

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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

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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

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https://doi.org/10.1101/2020.05.21.108993

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.

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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


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


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


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


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


https://doi.org/10.1101/2020.05.21.108993

CH4IgTActinopterygii(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)


CH2IgM

Elasmobr

anchii(5

)

CH2/CH4 IgW/IgNARElasmobranchii(14)

CH1IgM

Actinopterygii(7)


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.

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https://doi.org/10.1101/2020.05.21.108993

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


https://doi.org/10.1101/2020.05.21.108993

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.

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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

18


https://doi.org/10.1101/2020.05.21.108993

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


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


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


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


IgT

4D A

ulos

tom

us m

acul

atus

IgM Amia calva


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


IgM Squalus acanthias

IgT

4D C

hann

a ga

chua

IgT6

4D

Pla

niliz

a ha

emat

oche

ilus


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


IgNAR Scyliorhinus canicula

IgM Cy

noglo

ssus s

emila

evis

IgT 2D

Chae

todon

aurig

a


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


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


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


urdjan


IgT 4D Bovichtus diacant

hus

IgT1 4D M

astacembelus arm

atus

IgT2 4D M

astacembelus arm

atus


IgM Lepisosteus Oculatus

IgM Anguilla japonica


IgM Amblygaster clupeoides


IgM

Not

othe

nia

corii

ceps


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


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




IgT

3D C

ynog

loss

us s

emila

evis


IgT 4D

Dice

ntra

rchus

labr

ax


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


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


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


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


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


IgT

4D D

ascy

llus

trim

acul

ayus

IgT3 4D Salmo salar

IgM O

reochromis niloticus/1-397

IgM



IgM Gasteropelecus sp IgM

Chan

na ga

chua

IgT 4D Gyrinocheilus aymonieri


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.

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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

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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

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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

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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.

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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)

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