Convergent gene losses and pseudogenizations in multiple lineages of stomachless fishes

ABSTRACT The regressive evolution of independent lineages often results in convergent phenotypes. Several teleost groups display secondary loss of the stomach, and four gastric genes,

_atp4a_, _atp4b_, _pgc_, and _pga2_ have been co-deleted in agastric (stomachless) fish. Analyses of genotypic convergence among agastric fishes showed that four genes, _slc26a9_, _kcne2_,

_cldn18a_, and _vsig1_, were co-deleted or pseudogenized in most agastric fishes of the four major groups. _kcne2_ and _vsig1_ were also deleted or pseudogenized in the agastric monotreme

echidna and platypus, respectively. In the stomachs of sticklebacks, these genes are expressed in gastric gland cells or surface epithelial cells. An ohnolog of _cldn18_ was retained in some

agastric teleosts but exhibited an increased non-synonymous substitution when compared with gastric species. These results revealed novel convergent gene losses at multiple loci among the

four major groups of agastric fish, as well as a single gene loss in the echidna and platypus. SIMILAR CONTENT BEING VIEWED BY OTHERS EVOLUTIONARY DIVERGENCE OF NOVEL OPEN READING FRAMES IN

CICHLIDS SPECIATION Article Open access 09 December 2020 THE MACROEVOLUTIONARY DYNAMICS OF PHARYNGOGNATHY IN FISHES FAIL TO SUPPORT THE KEY INNOVATION HYPOTHESIS Article Open access 28

November 2024 THE HAGFISH GENOME AND THE EVOLUTION OF VERTEBRATES Article Open access 23 January 2024 INTRODUCTION Actinopterygii (ray-finned fishes) consists of 44 orders, 453 families, and

approximately 30,000 species, thereby constituting the largest class of fishes, as well as greater than half of all extant vertebrates1,2,3. The stomach is absent from the gastrointestinal

tract in certain Actinopterygii orders, while others have true stomachs that secrete gastric acid and pepsinogen from the gastric gland. Cypriniformes (~3,200 species; e.g., minnows),

Beloniformes and Cyprinodontiformes (~1200 species; e.g., medaka and killifish, respectively), Tetraodontiformes (~3500 species; e.g., pufferfishes), and Labriformes (~600 species; e.g.,

wrasse) are the main predominantly agastric orders of this class4,5. These groups are phylogenetically scattered, showing that the Actinopterygii originally possessed a stomach, but this

organ disappeared in the ancestors of each agastric lineage individually. In the most recent review of stomach loss in fishes, Wilson and Castro5 estimated that 7% of families and 20–27% of

fish species are agastric, and at least 15 individual stomach loss events have occurred in fishes during evolution. Secondary loss of an organ or tissue is a type of regressive evolution

that has received considerable attention as a model of evolution, development, and physiology. These losses are convergent phenotypes, suggesting the presence of a specific benefit and

selection in each lineage. For example, secondary eye and pigment losses are observed in cave animals such as cavefishes (_Astyanax mexicanus_, _Amblyopsis rosae_, and _Typhlichthys

subterraneus_) and cave salamanders6,7, with eye loss suggested to relate to the conservation of metabolic energy8. Other examples are the loss of the swim bladder in Pleuronectiformes,

Gobiiformes, and Scorpaenidae9, and the disappearance of scales in some lineages of Actinopterygii10. Most stenohaline marine fishes lack a distal tubule from the nephron of the kidney, and

glomeruli are absent in a small number of marine teleosts as they have minimal functional significance11. Snakes and scincid lizards have lost their limbs12,13, most cetaceans present

missing hind limbs14, and the aquatic frog _Barbourula kalimantanensis_ lacks a lung15. In the platypus, the stomach is completely aglandular and has been reduced to a simple dilatation of

the lower esophagus16. In echidna, the small stomach contains a high gastric fluid pH but lacks a gastric gland16. The secondary stomach loss in Actinopterygii, as well as the loss of

gastric gland in monotremes, is an interesting example to elucidate the cause of the secondary loss of an organ; nevertheless, the physiological benefits and developmental mechanisms

involved in this secondary loss have not yet been clarified. Genome sequences of many ray-finned fishes have been recently published and the number of species available allows some

comprehensive analysis on the genomic difference between gastric and agastric fishes. In particular, agastric fishes from the following four orders of teleosts have been sequenced: zebrafish

(_Danio rerio_; Cypriniformes)17, Japanese medaka (_Oryzias latipes_; Beloniformes)18, pufferfish (_Takifugu rubripes_ and _Tetraodon nigroviridis_; Tetraodontiformes)19,20, and wrasse

(_Labrus bergylta_)21. Genome sequences of gastric fishes such as three-spined stickleback (_Gasterosteus aculeatus_)22, Atlantic cod (_Gadus morhua_)23, and Nile tilapia (_Oreochromis

niloticus_)24 have also been published. Based on these analyses, the H+/K+-ATPase (_atp4a_ and _atp4b_) and pepsinogens (_pga_, _pgc_) genes are co-deleted in the genomes of agastric species

but are present in the genomes of gastric species4,21. In monotremes, convergent gene losses for _atp4a_, _atp4b_, _pgc_, and _pga_ occurred in platypus, and those for _pgc_ and _pga_

occurred in echidna, suggesting that the loss of _pgc_ and _pga_ occurred before the platypus-echidna split at more than 21 mya16,25. During our studies of anion transporters of solute

carrier family 26 (Slc26) in pufferfish and eels26,27, we found that the gene or cDNA for Slc26a9 was absent in the expressed sequencing tag (EST) and genome databases of pufferfish,

zebrafish, and Japanese medaka, but present in those of three-spined stickleback, rainbow trout, Atlantic cod, and Nile tilapia. In mice, Slc26a9 is highly expressed in the stomach and

lung28, and its deletion causes tubulovesicle loss in parietal cells, acid29 and prostaglandin-stimulated HCO3− secretion impairment in the stomach30, and airway mucus obstruction through

airway inflammation31. These results indicate that the absence of _slc26a9_ in fish species is correlated with stomach loss, and that more genes that are important for gastric function could

be lost among agastric fishes in a convergent manner. To confirm this hypothesis, we compared gene losses between agastric and gastric fishes and identified additional genes that are

co-deleted in agastric fishes to demonstrate a novel genotypic convergence in relation to stomach loss. RESULTS SCREENING OF GENES CO-DELETED IN THE GENOMES OF AGASTRIC FISHES Genes which

are commonly absent stomachless fish genomes were screened by database mining. First, a list of all annotated genes in the three-spined stickleback genome database22 was obtained using the

Ensembl BioMart tool32 and compared to those of agastric fishes (zebrafish;17, Japanese medaka;18, spotted green pufferfish;20, and Japanese pufferfish;19); approximately 80 three-spined

stickleback genes were identified that were absent in the gene annotations of the agastric fishes. Second, the presence or absence of the identified genes was confirmed by a homology search

in the genome databases for agastric fishes (zebrafish, Japanese medaka, spotted green pufferfish, and Japanese pufferfish). Blast analyses showed that many of those genes were present in

agastric fishes but not correctly annotated or annotated with a different name. Ten genes, _atp4a_, _atp4b_, _pgc, slc26a9_, _kcne2_, _vsig1_, _pqlc2l_, _pradc1_, _atp6v0d2_, and _ankub1_,

were confirmed to be absent in the genome of these agastric fishes but present in three-spined stickleback. A similar analysis was performed on 23 Actinopterygii species. Phylogenetic

relationships among the 23 species are shown in Fig. 1a. Finally, six genes, _atp4a_, _atp4b_, _pgc, slc26a9_, _kcne2_, and _vsig1_ were confirmed to be absent in the genome of the majority

of the agastric fishes but present in gastric species. Three of these six genes (_atp4a_, _atp4b_, and _pgc_) were also reported absent in agastric fishes by Castro et al.4, corroborating

the validity of this strategy. However, _pga2_ was not included in the list, indicating the incompleteness of this method. We next individually analyzed the presence of genes whose function

or expression in the stomach of mammals was recognized using blast analyses. As previously reported4, _pga2_ was confirmed to be absent in the genomes of agastric fishes. In addition, a

teleost fish-specific ohnolog of the claudin 18 gene, _cldn18a_, was found to be co-deleted in the genome databases of these fishes. Another ohnolog, _cldn18b_ was shown to be present in

gastric fishes and some agastric fishes (zebrafish and Japanese pufferfish). In total, four genes (_slc26a9_, _kcne2_, _cldn18a_, and _vsig1_) were found to be co-deleted in the genome

databases of the most agastric fish species of Actinopterygii (Fig. 1b). IDENTIFICATION OF GENES CO-DELETED IN THE GENOMES OF AGASTRIC FISHES Synteny and dot plot analyses were performed to

evaluate gene loss and pseudogenization, respectively. A synteny analysis of the four identified genes (_slc26a9_, _kcne2_, _cldn18a_, and _vsig1_) and the related _cldn18b_ ohnolog was

performed on 23 Actinopterygii species and shown in Fig. 2 and Supplementary Tables 1–5. The results of dot plot analyses are shown in Supplementary Figs. 1–4. A summary of the presence or

deletion of each exon-coding region is shown in Fig. 3. The results showed that _kcne2_, _vsig1_, and _cldn18a_ were absent or pseudogenized in all 11 species in four agastric lineages

(Cypriniformes, Beloniformes and Cyprinodontiformes, Tetraodontiformes, and Labriformes) but present in all other species in 12 gastric lineages (Figs. 2b, c, e, and 3b–d). _slc26a9_ was

absent or pseudogenized in nine agastric species in three lineages (Cypriniformes, Beloniformes, Cyprinodontiformes, and Tetraodontiformes) but present in the other species including two

species of Labriformes (wrasses) (Figs. 2a and 3a). The _cldn18b_ ohnolog was deleted in seven species in two lineages (Beloniformes, Cyprinodontiformes, and Tetraodontiformes) but existed

in the other species including four species in agastric lineages (Cypriniformes and Tetraodontiformes) (Fig. 2d). Synteny and dot plot analyses of _atp4a_, _atp4b_, _pgc_, and _pga2_ was

similarly performed (Figs. 3e–h and 4, Supplementary Figs. 5–8, Supplementary Tables 6–10). The results revealed that _atp4a_, _atp4b_, _pga2_, and _pgc_ were absent or pseudogenized in all

11 species in four agastric lineages but present in all the other species in 12 gastric lineages. _pga_ orthologs are distributed in three loci in the teleost genome and are named _pga1_,

_pga2_, and _pga3_4,33 (Fig. 4d, e). Phylogenetic analysis of _pga_ orthologs and the details of the evolutionary relationships are shown in Fig. 5 and described in the next chapter. In

contrast to _pga2_, which was deleted in all 11 agastric species, _pga1_ was deleted in eight species of three agastric lineages (Cypriniformes, Beloniformes, and Labriformes) but existed in

other species including Tetraodontiformes (Fig. 4d). The _pga3_ gene was deleted in several agastric and gastric fishes4 (Fig. 4e). EVOLUTION OF _PGA_ IN BONY VERTEBRATES Although teleost

fishes have three _pga_ paralogs, _pga1_, _pga2_, and _pga3_4,33, no clear evolutionary relationship among the paralogs has been uncovered. Therefore, a comprehensive analysis of _pga_ was

conducted on the genomic data of cartilaginous fish, tetrapods, lobe-finned fish, and ray-finned fish. The _pga_ paralog nomenclature in representative species is shown (Fig. 5a), and a

molecular phylogenetic tree was constructed (Fig. 5b). These results suggest that cartilaginous fish, tetrapods, lobe-finned fish, and ray-finned fish each have their own _pga_ paralogs. In

cartilaginous fishes, _pga_ paralogs consist of four major branches, indicating that the divergence of these four branches occurred before the speciation of cartilaginous fishes, and that

they acquired species-specific paralogs after speciation. The tetrapod _cym_ is positioned as a tetrapod-specific paralog. The _pga_ paralogs of the coelacanth, a lobe-finned fish, formed a

single branch, suggesting that _pga_ paralogs evolved independently in lobe-finned fish (Fig. 5b, clear highlight). The _pga_ paralogs of ray-finned fish formed five major branches, each of

which contained _pga_ from diverse species, suggesting that these five branches arose from the common ancestor of ray-finned fish. In gray bichir and reedfish, all _pga_ paralogs were

located in tandem (Fig. 5a), suggesting that these ancestral paralogs arose by tandem duplication. In this study, these ancestral _pga_ paralogs were provisionally named _pga.r1_, _pga.r2_,

_pga.r3_, _pga.r4_ and _pga.r5_, with r1-r5 representing paralogs arising from ray-finned fish-specific tandem duplications. Synteny of extant _pga_ derived from _pga.r1_-_pga.r5_ is shown

in Fig. 5a. Gray bichir, for example, has one ortholog derived from _pga.r1_, _pga.r2_, _pga.r4_, and _pga.r5_, and four from _pga.r3_. The spotted gar had one ortholog derived from _pga.r1_

and three from _pga.r3_. All previously named _pga1_, _pga2_, and _pga3_ in teleost fish are orthologs derived from _pga.r3_. Many teleosts only have orthologs derived from _pga.r3_,

whereas the European eel has orthologs derived from _pga.r1_ and _pga.r3_ and the Indo-Pacific tarpon has orthologs derived from _pga.r1_, _pga.r3_, and _pga.r4_. These results can be

considered an example of a birth-and-death model in gene family evolution34. In the phylogenetic tree, we included the amino acid sequence derived from the _pga2_ pseudogene (_pga2-ps_) of

ocean sunfish. Ocean sunfish _pga2-ps_ was positioned in the teleost _pga2_ group with a long branch. EXPRESSION OF STICKLEBACK GENES WHOSE ORTHOLOGS ARE DELETED IN AGASTRIC FISHES Various

three-spined stickleback tissues were analyzed by semi-quantitative RT-PCR to determine the distributions of mRNAs for the eight genes (Fig. 6a), as well as for _actb_ as a positive control

showing cDNA integrity. The results showed that _atp4a_, _atp4b_, _kcne2_, _slc26a9_, _vsig1_, _cldn18a_, _pgc_, and _pga2_ were highly expressed in the stomach. Several of these genes were

also expressed in stickleback organs other than the stomach: _kcne2_ was observed in the ovary and testis, _pgc_ in the gut and liver, _pga_ in various organs, including the gut, liver, and

kidney, _vsig1_ in the gut and liver, and _cldn18a_ in the gut. To identify the cells expressing the genes at the tissue level, in situ hybridization and histology were performed on the

three-spined stickleback gut (Fig. 7), which is composed of a mucosa, submucosa, muscularis, and serosa (Fig. 7a, b). The mucosa consists of a gastric pit and gastric (oxyntic) gland in the

anterior cardiac or fundic region of the stomach, and a gastric pit only in the posterior pyloric region. All genes tested were expressed in the mucosa of the three-spined stickleback

stomach, with none expressed in the other layers. All eight genes, _atp4a_, _atp4b_, _pgc_, _pga2_, _slc26a9_, _kcne2_, _cldn18a_, and _vsig1_, were expressed in gastric gland cells (Fig. 

7c, d), and three _pga2_, _cldn18a_, and _vsig1_, were expressed in the columnar mucous cells of the gastric pit (Fig. 7c, e) which had characteristic Periodic acid-Schiff (PAS)-positive

mucous granules in the apical region (Fig. 7b). Hybridization using sense probes did not resulted in any labeling (Supplementary Fig. 9). In general, the gastric gland of fishes consists of

only one secretory cell type (oxynticopeptic cells), whereas that of mammals is composed of chief cells for digestive-enzyme secretion and parietal cells for acid secretion5. In the gastric

gland of the three-spined stickleback, most epithelial cells presented positive expressions for genes involved in acid secretion (_atp4a_, _atp4b_, _slc26a9_, _kcne2_) and digestive enzymes

(_pgc_, _pga2_) (Fig. 7c, d), indicating that these eight genes are coexpressed in three-spined stickleback oxynticopeptic cells. EXPRESSION OF WRASSE _SLC26A9_ Intact _slc26a9_ was present

in wrasses but not in the other agastric species (Figs. 2a and 3a). To confirm whether _slc26a9_ is transcribed in organs other than the stomach, total RNA was extracted from various organs

of a humphead wrasse and semi-quantitative RT-PCR was performed. In the humphead wrasse, _slc26a9_ was expressed in the eyes, gills, fins, and skin (Fig. 6b). RAPID EVOLUTION OF _CLDN18B_ IN

AGASTRIC FISHES Gastric teleosts have two orthologs for claudin 18, _cldn18a_ and _cldn18b_, whereas agastric teleosts have a single or deleted claudin 18 gene. The paralogs are

specifically present in Teleostei but not in tetrapods. For both _cldn18a_ and _cldn18b_ loci, the synteny of the neighboring genes, _hs2st1_/_hs2st1a_ and _sox14_, are conserved (Fig. 2c,

d). These results indicate that _cldn18a_ and _cldn18b_ are ohnologs that are generated by teleost-specific genome duplication (TGD)35. The presence of _cldn18a_ is highly associated with

the existence of a stomach, whereas the presence of _cldn18b_ is only partially associated with the possession of this organ. To compare the evolution of _cldn18_ between animals with and

without a stomach, mean rates for non-synonymous and synonymous substitutions, dn and ds, respectively, were calculated for four groups: (i) _cldn18_ of tetrapods/coelacanths, (ii) _cldn18a_

of gastric fish, (iii) _cldn18b_ of gastric fish, and (iv) _cldn18b_ of agastric fish (zebrafish and Japanese pufferfish). Non-synonymous substitutions occurred ~4 times more frequently in

the _cldn18b_ of agastric fishes than in the other groups (_P_ < 0.0001; Fisher’s exact probability test) (Fig. 6c–e). These results suggest that the loss of the stomach allows higher

amino acid substitution rates on _cldn18b_, which is likely due to the relaxation of functional constraints. PSEUDOGENIZATION OF _VSIG1_ IN PLATYPUS AND LOSS OF _KCNE2_ IN ECHIDNA As

reported previously16,25, we confirmed convergent gene losses of _atp4a_, _pgc_, and _pga_ in the platypus and echidna (Fig. 8a–d). In both organisms, _atp4b_ was annotated in the genome

database (XM_039915013.1, and XM_038761646.1, respectively); however, the predicted amino acid sequences lacked the amino-terminal cytoplasmic and the transmembrane domains (Supplementary

Fig. 10), which are encoded by the exons 1 and 2 of _atp4b_ in other species. TBLASTN analysis of the whole-genome databases of platypus and echidna did not reveal regions encoding the

cytoplasmic and transmembrane domains of Atp4b. Because Atp4b is a membrane protein with one transmembrane domain36, _atp4b_ is considered to have lost its original function in the platypus

and echidna and may be pseudogenized in these species (Fig. 8b). The presence or absence of the four genes (_slc26a9_, _kcne2_, _cldn18_, and _vsig1_) was searched using the genome databases

of the coelacanth37, _Xenopus_38, anole lizard39, platypus40, echidna25, and human41 by blast searches of their genome sequences. All genes were present in the genomes of the gastric

species, coelacanth, _Xenopus_, anole lizard, and human. _cldn18_ and _slc26a9_ were retained in the genomes of both platypus and echidna (Fig. 8i). Convergent gene loss for _kcne2_ was

observed in the echidna, but not in the platypus (Fig. 8e). _vsig1_ was pseudogenized in the platypus but not in the echidna (Fig. 8f), and dot plot analysis showed a pattern of deletion of

_vsig1_ in the platypus, with exons 2–7 deleted at the homologous locus of _vsig1_ (Fig. 8g–h). DISCUSSION Genome projects of vertebrate species have allowed the clarification of the

presence of lineage-specific gene losses during evolution42,43,44,45,46,47. In the present comparative genomic analysis, the deletion of four genes was shown to be associated with secondary

stomach losses in Actinopterygii species. The four genes contain the Cl− channel-transporter (_slc26a9_) and a regulatory subunit of the K+ channel (_kcne2_). These molecules are

co-expressed with H+/K+-ATPase in gastric gland cells of the stomach and are involved in gastric acid (HCl) secretion. The four genes also contain cell-cell adhesion molecules that are

involved in the paracellular barrier function against H+ (_cldn18_)48 and control the stomach development (_vsig1_)49. These results, along with those of other studies on the deletion of

genes for H+/K+-ATPase (_atp4a_ and _atp4b_) and pepsinogens (_pga_, _pgc_)4, we summarized the convergent losses of important functional genes in four major independent groups of agastric

fishes, Cypriniformes (golden-line barbel, zebrafish, and fathead minnow), Beloniformes and Cyprinodontiformes (Japanese medaka, turquoise killifish, and platyfish), Tetraodontiformes (ocean

sunfish, Japanese pufferfish and spotted green pufferfish), and Labriformes (humphead wrasse and ballan wrasse). _slc26a9_ was present in wrasses and was expressed in organs other than the

stomach, such as the gills and skin (Fig. 6b). This result suggests that an unidentified non-gastric function of _slc26a9_ prevents its loss from wrasses. Ocean sunfish (_Mola mola_) belongs

to Tetraodontiformes and is closely related to pufferfishes. There is no histological analysis that clarify the presence or absence of gastric glands in the gut of ocean sunfish. In the

digestive tract of ocean sunfish, a stomach-like organ is present50. However, the present analysis indicates that the genome of ocean sunfish has a similar pattern of gastric gene deletions

as pufferfishes and other agastric fishes. This result suggests that the ocean sunfish may be an agastric fish. A stomach-like organ is also present in pufferfishes and is known as the

abdominal pouch51. The abdominal pouch of pufferfishes is often called stomach and can temporarily store food, but the abdominal pouch does not have gastric glands nor the ability to digest

food. In the case for ocean sunfish, further analysis is required to clarify the presence or absence of gastric glands in the stomach-like organ. Gastric H+ secretion is mediated by apical

(luminal) H+/K+-ATPase coupled with the K+ channel/transporter for K+ recycling and is also accompanied by Cl− secretion mediated by the apical Cl− channel/transporter. In mammals, the Cftr,

Clc-2, and Slc26a9 Cl− channels are proposed to mediate Cl− secretion52,53. K+ is recycled by a K+ channel composed of Kcnq1 α and Kcne2 β subunits. In addition, the apical K+-Cl−

cotransporter (Kcc4) secretes K+ and Cl− together. Among the apical components for gastric acid secretion, four genes, _atp4a_, _atp4b_, _slc26a9_, and _kcne2_ are deleted in agastric

fishes, suggesting that the function of those genes is closely associated with gastric acid (H+) secretion. The remaining genes were retained in agastric fishes, suggesting that they have

important functions in non-gastric tissues of the agastric fishes. In non-gastric tissues, Cftr excretes Cl− in the gills of marine teleosts and secretes intestinal Cl− 54,55,56. Kcc4 is

involved in H+ secretion in the renal α-intercalated cells in mammals57, which may explain why these genes are retained. The lost genes code for some of the apical components but not the

basolateral components such as Na+/K+-ATPase, anion exchanger 2 (Ae2), and Na+/H+ exchanger 4 (Nhe4) for gastric acid secretion (Fig. 9). In general, the basolateral membrane of epithelial

cells faces the extracellular fluid with a stable ionic composition, whereas the apical membrane faces the luminal fluid with a variable composition. Therefore, functional proteins on the

apical membrane tend to be tissue-specific, while those on basolateral membrane are shared among epithelia of various tissues. Our results suggest that the basolateral components for gastric

acid secretion are common with those of other epithelial systems, thereby preventing the deletion of these genes, whereas some apical components are specific to the stomach, which are more

prone to gene losses. Our analysis revealed that the platypus genome contains _kcne2_, _slc26a9_, and _cldn18_, whereas the echidna genome contains _vsig1_, _slc26a9_, and _cldn18_. In

mammals, _kcne2_58,59, _slc26a9_ 28,29,60,61, and _cldn18_62,63 are expressed in the lung at high levels as well as in the stomach and their functions are related to both gastric and

pulmonary systems. Slc26a9 is critical for respiratory function in terrestrial vertebrates as loss of _slc26a9_ can create a cystic fibrosis-like phenotype64,65,66. In contrast in the

three-spined stickleback, these genes are expressed in the stomach but not in the swim bladder or gill, which are related to respiratory function. These results suggest that _kcne2_,

_slc26a9_, and _cldn18_ are required mainly for gastric function in Actinopterygii, with the exception of wrasse slc26a9, which has non-gastric functions, whereas those are required for the

gastric and pulmonary functions both in terrestrial vertebrates. Therefore, in platypus, the respiratory function of _slc26a9_, _kcne2_, and _cldn18_ in the lung may prevent the loss of

these genes. In echidnas, the respiratory function of _slc26a9_ and _cldn18_ in the lung may also prevent the loss of these genes. However, _kcne2_ was lost in the echidna, suggesting that

the respiratory function of _kcne2_ was compensated for by another gene in this organism. RT-PCR analysis of sticklebacks showed that _kcne2_, _pga_, _pgc_, _pga_, _vsig1_, and _cldn18a_

were expressed not only in the stomach but also in other organs. This result suggests that these genes function in organs other than the stomach of fish. However, in most agastric fish,

these genes were deleted, probably because these functions were compensated for by another gene. The loss of _vsig1_ was observed in agastric fishes and platypus, but not in echidna. Vsig1

is a cell surface protein characterized by two extracellular immunoglobulin-like domains whose physiological function is still largely unknown. Vsig1 is also known as glycoprotein A34

(Gpa34) of tumor cells67, is expressed in low- or non-metastatic cancer cells68, and inhibits Yap/Taz signaling. Yap and Taz are transcriptional regulators and essential for cancer

initiation or growth of most solid tumors69. As the Yap/Taz signaling is important for organogenesis70, the role of Vsig1 for normal stomach development could be via the TAP/TAZ signaling49.

In human and mice, the _vsig1_ gene is expressed in the stomach and testes49,67, while it was expressed in the stomach, intestine, and liver, but not in testes or other organs in the

three-spined stickleback (Fig. 6a). Our result also indicated that Vsig1 is localized at the gastric gland and pit cells, which is identical to the case in mice49. The loss of _vsig1_ could

impair the development of stomach in platypus. Although the _vsig1_ is an intact gene in echidna, their stomach is glandless. In this case, Vsig1 could be involved in the development of the

stomach but some other factors control the development of the gastric gland. Retention of _cldn18b_, a duplicated _cldn18_ in teleosts, by some agastric fishes is a good example of how a

gene evolves when the functional constraint is reduced. In the gastric epithelium, paracellular H+ leakage is prevented by the tight junctions and associated junctional complexes, e.g.,

claudins. Only one component, claudin-18, has been identified as the paracellular H+ barrier48. Complete deletion of both _cldn18a_ and _cldn18b_ in the genomes of Japanese medaka, turquoise

killifish, platyfish, wrasses, ocean sunfish, and spotted green pufferfish indicates that the secondary stomach loss reduced the functional constraint of the _cldn18_ genes. The _cldn18b_

that is retained in some other agastric species (golden-line barbel, zebrafish, fathead minnow, and Japanese pufferfish) exhibited rapid non-synonymous substitution rates, which were higher

than those of gastric species. Although _cldn18b_ is retained in Japanese pufferfish, no tissues expressed the gene71. However, in zebrafish, _cldn18b_ is also expressed in the kidney72. In

mouse kidney, _cldn18_ is expressed in the thick ascending limb of Henle’s loop (TAL), which additionally expresses _cldn10_, _cldn16_, and _cldn19_. The mouse TAL functions as a site for

the reabsorption of Ca2+ and Mg2+ via the paracellular pathway. In the mouse TAL, claudin-10 (claudin-10a: anion permeability; claudin-10b: cation (Na+ > K+) permeability) and -18 may

contribute to the maintenance of barrier function, and claudin-16 and -19 contribute to Ca2+ and Mg2+ ion selectivity73,74,75. Because zebrafish kidneys also expresses claudin-10b72,

zebrafish claudin-18, together with claudin-10b and others, may contribute to the maintenance of tubular barrier function. Many vertebrates have multiple _pga_ gene paralogs. It is difficult

to evaluate the evolutionary relationships of paralogs using the names of genes, as they are a mixture of those arising from old and new gene duplications. Castro et al. named _pga1_,

_pga2_, and _pga3_ as _pga_ paralogs in three loci of the teleost genome4. Molecular phylogenetic analyses involving _pga_ genes in cartilaginous fish, tetrapods, lobe-finned fish, and

ray-finned fish have shown that teleost _pga1_, _pga2_, and _pga3_ differ from _pga_ paralogs in ancient ray-finned fishes, such as Polypterus, sturgeon, and gar. This confirmed that _pga1_,

_pga2_, and _pga3_ are teleost-specific paralogs, as reported by Castro et al.4. Interestingly, of the four _pga_ paralogs in spotted gar (provisionally named _Locpga1_, _Locpga2_,

_Locpga3_, and _Locpga4_), _Locpga1_ belonged to the same branch as the _pga_ paralogs of polypterus and sturgeons; _Locpga2_, _Locpga3_, and _Locpga4_ belonged to the same branch as

teleosts _pga1_, _pga2_, and _pga3_, which are paralogs that arose after the divergence of gar and teleosts. Synteny analysis suggested that _pga2_ and _pga3_ are present in loci generated

by teleost-specific genome duplication (TGD); however, it remains unclear whether _pga2_ and _pga3_ are ohnologs or paralogs derived from pre-TGD tandem duplication. Species- and

lineage-specific tandem duplications of _pga2_ have been observed in various species (e.g., channel catfish, Mexican tetra, northern pike, and Atlantic cod). In the present analysis, _pga2_

was the _pga_ family member whose absence was most frequently associated with secondary loss of the stomach, whereas _pga1_ and _pga3_ were also observed in various gastric fishes. _pga1_

synteny was conserved in many teleost species, although no synteny was observed with teleost _pga2_, _pga3_, or tetrapod _pga_. Given this, and the fact that _pga1_ is a teleost-specific

paralog, it is possible that _pga1_ arose in the common ancestor of teleost fish via duplication through translocation. Among teleost fishes, _pga1_ was present in most gastric fishes and

some agastric fishes and was absent in some gastric fishes and many agastric fishes. In the agastric Japanese pufferfish, _pga1_ is expressed in non-gastric tissues such as the skin76. The

physiological advantages of secondary stomach loss are still largely unknown5. In the treatment of human gastric cancer, gastrectomy alters physiological properties such as oxygen

availability, pH, food transit time, intestinal motility, and hormonal conditions, and alters the overall microbiome community structure77. Gastrectomy-associated alterations in microbial

functions, such as nutrient transport and biosynthesis of organic compounds, may be related to changes in post-gastrectomy metabolism. In gastric teleost species, the stomach has a variety

of physiological functions, such as food digestion, temporal food storage, pathogen invasion defense, and hormonal secretion5. The differences in the physiological properties between gastric

and agastric fish remain unclear. As the stomach kills microorganisms using gastric acid and provides increased uniformity in the population of gut microbes78, it is presumed that loss of

the secondary stomach has some effect on the gut microbiome, and that the gut microbiome of agastric fish is more susceptible to environmental influences. Studies on the fish digestive tract

microbiome indicate that fish harbor specialized gastrointestinal microbial communities like other vertebrates such as mammals79,80,81, and the gut microbiomes of wood-eating catfishes,

zebrafish, guppies, and others are related to their diets79,82,83,84,85. Further studies are required to better understand the physiological advantages of losing the secondary stomach. Our

results raise the question of whether the gene deletions observed in this study caused the stomach loss, or whether the deletions occurred after the stomach loss. Despite stomach loss, our

study did not show deletion of the genes for transcriptional or growth factors that regulate stomach development in agastric fishes86,87,88. Thus, it is conceivable that the lack of a

stomach is associated with the malfunction of the cis-regulatory elements for stomach development, which cannot be identified using the current strategy. It is also possible that a deletion

of one of the eight genes caused a depletion of stomach function in fishes for which this depletion was neutral or advantageous, and additional gene deletion followed, causing the stomach to

be completely regressed in the gut of fishes. In conclusion, we identified novel genes that were lost in agastric fishes among four major teleost lineages, which suggests a convergent

evolution scenario in relation to stomach loss. These genes encode apical ion channels for gastric acid secretion, and the cell-cell adhesion molecule that forms the paracellular H+ barrier

in the gastric epithelium (Fig. 9). These results indicate that a common cassette of gene losses occurred independently during or after stomach loss in the several agastric fish groups.

Further studies are required to identify the causative genotype that triggered this stomach loss. METHODS SCREENING OF GENES CO-DELETED IN THE GENOMES OF AGASTRIC FISHES Lists of all

annotated genes in the genome databases for zebrafish (_Danio rerio_)17, Atlantic cod (_Gadus morhua_)23, Nile tilapia (_Oreochromis niloticus_)24, Japanese medaka (_Oryzias latipes_)18,

three-spined stickleback (_Gasterosteus aculeatus_)22, Japanese pufferfish (_Takifugu rubripes_)19, and spotted green pufferfish (_Tetraodon nigroviridis_)20 were downloaded from Ensembl

(http://www.ensembl.org/index.html)89 using Ensembl BioMart tool32. After removing characters that indicated gene duplications, the presence or absence of all annotated three-spined

stickleback genes in agastric fishes (zebrafish, Japanese medaka, spotted green pufferfish, and Japanese pufferfish) were determined through a text search using Excel software (Microsoft,

Redmond, WA, USA). From this data, a list of three-spined stickleback genes that were commonly lacking in the gene lists of zebrafish, Japanese medaka, spotted green pufferfish, and Japanese

pufferfish was prepared. To avoid the presence of annotated genes with different gene names or unannotated genes in the agastric genome data, the absence of the genes was confirmed using a

BLAST search (TBLASTN)90 of zebrafish, Japanese medaka, spotted green pufferfish, and Japanese pufferfish with Ensembl, and gene names with one or more orthologs were removed from the list.

The presence of the orthologs of the listed genes for jawed vertebrate species listed Table 1 were analyzed by text search or TBLASTN analyses using Ensembl and NCBI. The synteny of each

gene in the list was compared among the above species using Ensembl and NCBI. DOT PLOT ANALYSIS To analyze the pseudogenization or whole gene deletion of the eight genes _slc26a9_, _kcne2_,

_vsig1_, _cldn18a_, _atp4a_, _atp4b_, _pga2_, and _pgc_, in the 11 agastric fish species, the coding region of each gene and its flanking regions of the gastric species, three-spined

stickleback (_Gasterosteus aculeatus_), and channel catfish (_Ictalurus punctatus_) were compared with the corresponding genomic regions of the 11 agastric fish species listed in Fig. 1. Dot

plot comparisons were performed using the EMBOSS dotmatcher program with a window size of 20 and threshold score of 70 (https://www.ebi.ac.uk/Tools/emboss/). To analyze the pseudogenization

of platypus _vsig1_, a dot plot analysis was performed between echidna _vsig1_ and its flanking regions and the corresponding genome regions of the platypus containing the _vsig1_

pseudogene using the EMBOSS dotmatcher program with a window size of 20 and a threshold score of 70. PHYLOGENETIC AND SYNTENY ANALYSES OF _PGA_ _pga_ orthologs were identified in the genome

data of ray-finned fish, lobe-finned fish, tetrapods, and cartilaginous fish, as listed in Table 1. The deduced amino acid sequences were aligned using ClustalW software, and a phylogenetic

tree was constructed using MEGA1191 using the maximum likelihood method. The synteny of _pga_ was compared among the above species using the Ensembl and NCBI databases. SEMI-QUANTITATIVE

REVERSE TRANSCRIPTION (RT)-PCR Three-spined sticklebacks (_Gasterosteus aculeatus_) and humphead wrasses (_Cheilinus undulatus_) captured in Japan in 2012 and 2023, respectively, were

obtained from local dealers. The animal protocols were in accordance with a manual approved by the Institutional Animal Experiment Committee of the Tokyo Institute of Technology. We have

complied with all relevant ethical regulations for animal use. The fishes were anesthetized by immersion in 0.1% ethyl m-aminobenzoate methanesulfonate (MS222; Sigma, St. Louis, MO, USA),

which was neutralized to pH 7.4 with sodium bicarbonate prior to use, and then decapitated. The tissues for RNA preparation were removed with ophthalmic scissors and frozen in liquid

nitrogen. Tissues other than ovary and testis were once pooled without distinguishing between males and females. Ovary and testis were obtained from females and males, respectively, and

pooled. Total RNA was isolated from the three-spined stickleback and humphead wrasse tissues by acid guanidinium thiocyanate-phenol-chloroform extraction using Isogen reagent (Nippon Gene,

Tokyo, Japan) according to the manufacturer’s manual. Owing to the small size of the three-spined sticklebacks, organs from three or more individuals were pooled for RNA extraction. Because

only one 230-gram individual of humphead wrasse was available, RNA was extracted from organs derived from one individual. The RNA was dissolved in diethyl pyrocarbonate (DEPC)-treated water

and its concentration was estimated by measuring the absorbance at 260 nm. mRNA preparations were reverse-transcribed into cDNA using the oligo(dT) primer and the SuperScript III

First-Strand Synthesis System (Invitrogen). The cDNA (0.25 μL of the Super Script III reaction) was used as the template for PCRs, along with the specific primers shown in Supplementary

Table 17. The PCR reactions were performed as follows92. Each reaction mixture (final volume, 12.5 μL) consisted of 0.25 μL cDNA (template), primers (individual final concentration, 0.25 

μM), and 6.25 μL GoTaq Green Master Mix (2×; Promega, Madison, WI, USA). The PCR conditions were as follows: initial denaturation at 94 °C for 2 min, 28 or 33 cycles of 94 °C for 15 s

(denaturation), 55 °C for 30 s (annealing), 72 °C for 1 min (extension), and a final extension at 72 °C for 7 min. PCR products from the three-spined sticklebacks were separated on agarose

gels and visualized with ethidium bromide. The fluorescence images were analyzed with a Kodak Image Station 2000R system (Eastman Kodak, Rochester, NY, USA). The PCR products from the

humphead wrasse were diluted and loaded onto a Microchip Electrophoresis system for DNA/RNA analysis (MCE-202 MultiNA; Shimadzu, Kyoto, Japan) using a DNA-12000 reagent kit (Shimadzu)

following to the manufacturer’s instructions. Electrophoresis results were analyzed using the MultiNA Viewer software (Shimadzu). Images of the gels are shown in Supplementary Fig. 11. IN

SITU HYBRIDIZATION HISTOCHEMISTRY In situ hybridization was performed as previously described in ref. 93. For tissue fixation, three-spined sticklebacks were anesthetized by immersion in

0.1% MS222, neutralized to pH 7.4, treated with sodium bicarbonate before use, and then decapitated. The stomach of three-spined sticklebacks was fixed in 4% paraformaldehyde in 0.1 M

phosphate buffer at pH 7.4 for 1 d at 4 °C. Tissues were dehydrated, embedded in paraplast (Leica Microsystems, Wetzlar, Germany), and cut in 5 μm slices. For in situ hybridization, sections

were deparaffinized in xylene, rehydrated by serial alcohol solutions, treated with proteinase K (5 μg/mL) for 10 min, and postfixed in 4% paraformaldehyde in 0.1 M phosphate buffer at pH

7.4. The sections were equilibrated in hybridization buffer (5× SSC and 50% formamide) at 58 °C for 2 h. A partial sequence of each target gene was cloned into the pGEM-T Easy vector

(Promega) using the primers listed in Supplementary Table 17. Sense and antisense probes were prepared using a digoxigenin (DIG) RNA labeling kit (Roche Applied Science, Indianapolis, IN,

USA), diluted in hybridization buffer containing calf thymus DNA (40 μg/mL), and denatured at 85 °C for 10 min. Denatured RNA probes were spread on the sections and incubated at 58 °C for

>40 h depending on the expression level in a moist chamber saturated with hybridization buffer. Specific signals were developed using a DIG nucleic acid detection kit (Roche Applied

Science), according to the manufacturer’s protocol. Some sections were stained with hematoxylin and eosin (H&E) or periodic acid–Schiff to determine the basic structure of epithelial

cells. Images were obtained using a TOCO automatic virtual slide system (Path Imaging, Tokyo, Japan) and a microscope equipped with a digital CCD camera (AxioCam HRc; Carl Zeiss, Oberkochen,

Germany), and processed using AxioVision 4.1 software (Carl Zeiss). CALCULATION OF NUCLEOTIDE SUBSTITUTION RATES Nucleotide sequences for claudin 18 were obtained from GenBank or Ensembl.

We used three nucleotide sequences for tetrapod/coelacanth _cldn18_ from human, tropical clawed frog, and coelacanth, three for each of gastric fish _cldn18a_ and _cldn18b_ from Atlantic

cod, Nile tilapia, and three-spined stickleback, and two for agastric fish _cldn18b_ from zebrafish and Japanese pufferfish. We used transcriptional sequences predicted from genome data when

mRNA data was not available from the databases. The coding regions were aligned using ClustalW software94 and sites containing gaps were deleted manually without shifting the reading frame

(Supplementary Fig. 12). Distance values for the non-synonymous substitutions per site (dn) and synonymous substitutions per site (ds) were calculated based on the Nei-Gojobori (NG) method95

using the alignment composed of 11 sequences and 522 positions and the MEGA6 software96. Standard errors were computed using the bootstrap method with 500 replicates. The number of

non-synonymous differences (n), synonymous differences (s), non-synonymous sites (N), and synonymous sites (S) was calculated based on the Nei-Gojobori (NG) method using the MEGA6 software.

Fisher’s exact test was used for the statistical analyses97. SYNTENY ANALYSIS OF MONOTREMES AND RELATED SPECIES The presence or absence of _atp4a_, _atp4b_, _pga_, _pgc_, and _vsig1_ was

confirmed by BLAST search (TBLASTN) and synteny analysis using the genome databases of coelacanth37, _Xenopus_38, anole lizard39, platypus40, echidna25, and human41. Synteny analysis was

performed manually using the Ensembl genome browser (https://www.ensembl.org)98 or the NCBI genome data viewer (https://www.ncbi.nlm.nih.gov/genome/gdv/)99. STATISTICS AND REPRODUCIBILITY

All experiments using the three-spined stickleback and humphead wrasse were repeated at least twice, and reproducibility was confirmed using the same sample. For the statistical analyses of

the of nucleotide substitution rates, we used three nucleotide sequences for tetrapod/coelacanth _cldn18_, three for each of gastric fish _cldn18a_ and _cldn18b_, and two for agastric fish

_cldn18b_. The numbers of sites for the statistical analyses are shown in Fig. 6d. Average numbers of non-synonymous differences (n) and unchanged non-synonymous sites (N-n) of gastric fish

_cldn18b_ were compared with those of agastric fishes, zebrafish, and Japanese pufferfish using by two-tailed Fisher’s exact test using GraphPad Prism (GraphPad, San Diego, CA, USA)

(https://www.graphpad.com/quickcalcs/contingency1/). Average numbers of synonymous differences (s) and unchanged synonymous sites (S-s) were also analyzed similarly by two-tailed Fisher’s

exact test. REPORTING SUMMARY Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. DATA AVAILABILITY All resources are

available from the authors upon reasonable request. REFERENCES * Near, T. J. et al. Resolution of ray-finned fish phylogeny and timing of diversification. _Proc. Natl. Acad. Sci. USA._ 109,

13698–13703 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Nelson, J. S. _Fishes of the world_. 4th edn, (John Wiley and Sons, 2006). * Eschmeyer, W. N. & Fricke, R.

_Catalog of Fishes_. Electronic Version edn, (California Academy of Sciences, 2012). * Castro, L. F. et al. Recurrent gene loss correlates with the evolution of stomach phenotypes in

gnathostome history. _Proc. Biol. Sci._ 281, 20132669 (2014). PubMed  PubMed Central  Google Scholar  * Wilson, J. M. & Castro, L. F. Morphological diversity of the gastrointestinal

tract in fishes. in _Fish Physiology_ Vol. 30, 1–55 (Academic Press, 2010). * Protas, M., Conrad, M., Gross, J. B., Tabin, C. & Borowsky, R. Regressive evolution in the Mexican cave

tetra, _Astyanax mexicanus_. _Curr. Biol._ 17, 452–454 (2007). Article  CAS  PubMed  PubMed Central  Google Scholar  * Romero, A. _Cave biology: life in darkness_. (Cambridge University

Press, 2009). * Moran, D., Softley, R. & Warrant, E. J. Eyeless Mexican cavefish save energy by eliminating the circadian rhythm in metabolism. _PLoS One_ 9, e107877 (2014). Article 

PubMed  PubMed Central  Google Scholar  * Alexander, R. M. Buoyancy. in _The Physiology of Fishes_ (ed D. H. Evans) 75–97 (CRC Press, 1993). * Hawkes, J. W. The structure of fish skin. I.

General organization. _Cell Tissue Res._ 149, 147–158 (1974). Article  CAS  PubMed  Google Scholar  * Hickman, C. P. & Trump, B. F. The kidney. in _Fish Physiology. I_. (eds W. S. Hoar

& D. J. Randall) 91–239 (Academic Press, 1969). * Cohn, M. J. & Tickle, C. Developmental basis of limblessness and axial patterning in snakes. _Nature_ 399, 474–479 (1999). Article 

CAS  PubMed  Google Scholar  * Skinner, A., Lee, M. S. & Hutchinson, M. N. Rapid and repeated limb loss in a clade of scincid lizards. _BMC Evol. Biol._ 8, 310 (2008). Article  PubMed 

PubMed Central  Google Scholar  * Thewissen, J. G. et al. Developmental basis for hind-limb loss in dolphins and origin of the cetacean bodyplan. _Proc. Natl. Acad. Sci. USA._ 103, 8414–8418

(2006). Article  CAS  PubMed  PubMed Central  Google Scholar  * Bickford, D., Iskandar, D. & Barlian, A. A lungless frog discovered on Borneo. _Curr. Biol._ 18, R374–375 (2008). Article

  CAS  PubMed  Google Scholar  * Ordonez, G. R. et al. Loss of genes implicated in gastric function during platypus evolution. _Genome Biol_ 9, R81 (2008). Article  PubMed  PubMed Central 

Google Scholar  * Howe, K. et al. The zebrafish reference genome sequence and its relationship to the human genome. _Nature_ 496, 498–503 (2013). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Kasahara, M. et al. The medaka draft genome and insights into vertebrate genome evolution. _Nature_ 447, 714–719 (2007). Article  CAS  PubMed  Google Scholar  * Aparicio, S. et

al. Whole-genome shotgun assembly and analysis of the genome of _Fugu rubripes_. _Science_ 297, 1301–1310 (2002). Article  CAS  PubMed  Google Scholar  * Jaillon, O. et al. Genome

duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. _Nature_ 431, 946–957 (2004). Article  PubMed  Google Scholar  * Lie, K. K. et al. Loss

of stomach, loss of appetite? Sequencing of the ballan wrasse (_Labrus bergylta_) genome and intestinal transcriptomic profiling illuminate the evolution of loss of stomach function in fish.

_BMC Genomics_ 19, 186 (2018). Article  PubMed  PubMed Central  Google Scholar  * Jones, F. C. et al. The genomic basis of adaptive evolution in threespine sticklebacks. _Nature_ 484, 55–61

(2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Star, B. et al. The genome sequence of Atlantic cod reveals a unique immune system. _Nature_ 477, 207–210 (2011). Article 

CAS  PubMed  PubMed Central  Google Scholar  * Brawand, D. et al. The genomic substrate for adaptive radiation in African cichlid fish. _Nature_ 513, 375–381 (2014). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Zhou, Y. et al. Platypus and echidna genomes reveal mammalian biology and evolution. _Nature_ 592, 756–762 (2021). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Kato, A. et al. Identification of renal transporters involved in sulfate excretion in marine teleost fish. _Am. J. Physiol. Regul. Integr. Comp. Physiol._ 297, R1647–1659

(2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Watanabe, T. & Takei, Y. Molecular physiology and functional morphology of SO42− excretion by the kidney of

seawater-adapted eels. _J. Exp. Biol._ 214, 1783–1790 (2011). Article  CAS  PubMed  Google Scholar  * Chang, M. H. et al. Slc26a9 - anion exchanger, channel and Na+ transporter. _J. Membr.

Biol._ 228, 125–140 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Xu, J. et al. Deletion of the chloride transporter Slc26a9 causes loss of tubulovesicles in parietal cells

and impairs acid secretion in the stomach. _Proc. Natl. Acad. Sci. USA._ 105, 17955–17960 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Demitrack, E. S., Soleimani, M.

& Montrose, M. H. Damage to the gastric epithelium activates cellular bicarbonate secretion via SLC26A9 Cl-/HCO3-. _Am. J. Physiol. Gastrointest. Liver Physiol._ 299, G255–264 (2010).

Article  CAS  PubMed  PubMed Central  Google Scholar  * Anagnostopoulou, P. et al. SLC26A9-mediated chloride secretion prevents mucus obstruction in airway inflammation. _J. Clin. Invest._

122, 3629–3634 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kinsella, R. J. et al. Ensembl BioMarts: a hub for data retrieval across taxonomic space. _Database (Oxford)_

2011, bar030 (2011). Article  PubMed  Google Scholar  * Miura, Y., Suzuki-Matsubara, M., Kageyama, T. & Moriyama, A. Structure, molecular evolution, and hydrolytic specificities of

largemouth bass pepsins. _Comp. Biochem. Physiol. B. Biochem. Mol. Biol._ 192, 49–59 (2016). Article  CAS  PubMed  Google Scholar  * Nei, M. & Rooney, A. P. Concerted and birth-and-death

evolution of multigene families. _Annu. Rev. Genet._ 39, 121–152 (2005). Article  CAS  PubMed  PubMed Central  Google Scholar  * Meyer, A. & Van de Peer, Y. From 2R to 3R: evidence for

a fish-specific genome duplication (FSGD). _Bioessays_ 27, 937–945 (2005). Article  CAS  PubMed  Google Scholar  * Abe, K., Tani, K., Friedrich, T. & Fujiyoshi, Y. Cryo-EM structure of

gastric H+,K+-ATPase with a single occupied cation-binding site. _Proc. Natl. Acad. Sci. USA._ 109, 18401–18406 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Amemiya, C. T.

et al. The African coelacanth genome provides insights into tetrapod evolution. _Nature_ 496, 311–316 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Hellsten, U. et al. The

genome of the Western clawed frog _Xenopus tropicalis_. _Science_ 328, 633–636 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Alfoldi, J. et al. The genome of the green

anole lizard and a comparative analysis with birds and mammals. _Nature_ 477, 587–591 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Pask, A. J. et al. Analysis of the

platypus genome suggests a transposon origin for mammalian imprinting. _Genome Biol._ 10, R1 (2009). Article  PubMed  PubMed Central  Google Scholar  * Lander, E. S. et al. Initial

sequencing and analysis of the human genome. _Nature_ 409, 860–921 (2001). Article  CAS  PubMed  Google Scholar  * Albalat, R. & Canestro, C. Evolution by gene loss. _Nat. Rev. Genet._

17, 379–391 (2016). Article  CAS  PubMed  Google Scholar  * Blomme, T. et al. The gain and loss of genes during 600 million years of vertebrate evolution. _Genome Biol._ 7, R43 (2006).

Article  PubMed  PubMed Central  Google Scholar  * Brunet, F. G. et al. Gene loss and evolutionary rates following whole-genome duplication in teleost fishes. _Mol. Biol. Evol._ 23,

1808–1816 (2006). Article  CAS  PubMed  Google Scholar  * Canestro, C., Catchen, J. M., Rodriguez-Mari, A., Yokoi, H. & Postlethwait, J. H. Consequences of lineage-specific gene loss on

functional evolution of surviving paralogs: ALDH1A and retinoic acid signaling in vertebrate genomes. _PLoS Genet._ 5, e1000496 (2009). Article  PubMed  PubMed Central  Google Scholar  *

Semon, M. & Wolfe, K. H. Reciprocal gene loss between Tetraodon and zebrafish after whole genome duplication in their ancestor. _Trends Genet._ 23, 108–112 (2007). Article  CAS  PubMed 

Google Scholar  * Wang, X., Grus, W. E. & Zhang, J. Gene losses during human origins. _PLoS Biol._ 4, e52 (2006). Article  PubMed  PubMed Central  Google Scholar  * Hayashi, D. et al.

Deficiency of claudin-18 causes paracellular H+ leakage, up-regulation of interleukin-1beta, and atrophic gastritis in mice. _Gastroenterology_ 142, 292–304 (2012). Article  CAS  PubMed 

Google Scholar  * Oidovsambuu, O. et al. Adhesion protein VSIG1 is required for the proper differentiation of glandular gastric epithelia. _PLoS One_ 6, e25908 (2011). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Chanet, B. et al. Visceral anatomy of ocean sunfish (_Mola mola_ (L., 1758), Molidae, Tetraodontiformes) and angler (_Lophius piscatorius_ (L., 1758),

Lophiidae, Lophiiformes) investigated by non-invasive imaging techniques. _C. R. Biol._ 335, 744–752 (2012). Article  PubMed  Google Scholar  * Fagundes, K. R., Rotundo, M. M. & Mari, R.

B. Morphological and histochemical characterization of the digestive tract of the puffer fish _Sphoeroides testudineus_ (Linnaeus 1758) (Tetraodontiformes: Tetraodontidae). _An. Acad. Bras.

Cienc._ 88, 1615–1624 (2016). Article  PubMed  Google Scholar  * Kopic, S., Murek, M. & Geibel, J. P. Revisiting the parietal cell. _Am. J. Physiol. Cell Physiol._ 298, C1–C10 (2010).

Article  CAS  PubMed  Google Scholar  * Schubert, M. L. Gastric secretion. _Curr. Opin. Gastroenterol._ 26, 598–603 (2010). Article  CAS  PubMed  Google Scholar  * Evans, D. H., Piermarini,

P. M. & Choe, K. P. The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. _Physiol. Rev._ 85, 97–177

(2005). Article  CAS  PubMed  Google Scholar  * Hiroi, J. & McCormick, S. D. New insights into gill ionocyte and ion transporter function in euryhaline and diadromous fish. _Respir.

Physiol. Neurobiol._ 184, 257–268 (2012). Article  CAS  PubMed  Google Scholar  * Wong, M. K., Pipil, S., Kato, A. & Takei, Y. Duplicated CFTR isoforms in eels diverged in regulatory

structures and osmoregulatory functions. _Comp. Biochem. Physiol. A. Mol. Integr. Physiol._ 199, 130–141 (2016). Article  CAS  PubMed  Google Scholar  * Boettger, T. et al. Deafness and

renal tubular acidosis in mice lacking the K-Cl co-transporter Kcc4. _Nature_ 416, 874–878 (2002). Article  CAS  PubMed  Google Scholar  * Cowley, E. A. & Linsdell, P. Characterization

of basolateral K+ channels underlying anion secretion in the human airway cell line Calu-3. _J. Physiol._ 538, 747–757 (2002). Article  CAS  PubMed  PubMed Central  Google Scholar  * Soler

Artigas, M. et al. Genome-wide association and large-scale follow up identifies 16 new loci influencing lung function. _Nat. Genet._ 43, 1082–1090 (2011). Article  PubMed  Google Scholar  *

Bertrand, C. A., Zhang, R., Pilewski, J. M. & Frizzell, R. A. SLC26A9 is a constitutively active, CFTR-regulated anion conductance in human bronchial epithelia. _J. Gen. Physiol._ 133,

421–438 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Chang, M. H. et al. Slc26a9 is inhibited by the R-region of the cystic fibrosis transmembrane conductance regulator

via the STAS domain. _J. Biol. Chem._ 284, 28306–28318 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * LaFemina, M. J. et al. Claudin-18 deficiency results in alveolar

barrier dysfunction and impaired alveologenesis in mice. _Am. J. Respir. Cell Mol. Biol._ 51, 550–558 (2014). Article  PubMed  PubMed Central  Google Scholar  * Niimi, T. et al. claudin-18,

a novel downstream target gene for the T/EBP/NKX2.1 homeodomain transcription factor, encodes lung- and stomach-specific isoforms through alternative splicing. _Mol. Cell. Biol._ 21,

7380–7390 (2001). Article  CAS  PubMed  PubMed Central  Google Scholar  * Strug, L. J. et al. Cystic fibrosis gene modifier SLC26A9 modulates airway response to CFTR-directed therapeutics.

_Hum. Mol. Genet._ 25, 4590–4600 (2016). CAS  PubMed  PubMed Central  Google Scholar  * Gong, J. et al. Genetic evidence supports the development of SLC26A9 targeting therapies for the

treatment of lung disease. _NPJ Genom. Med._ 7, 28 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Sun, L. et al. Multiple apical plasma membrane constituents are associated

with susceptibility to meconium ileus in individuals with cystic fibrosis. _Nat. Genet._ 44, 562–569 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Scanlan, M. J. et al.

Glycoprotein A34, a novel target for antibody-based cancer immunotherapy. _Cancer Immun._ 6, 2 (2006). PubMed  Google Scholar  * Bernal, C. et al. Functional Pro-metastatic Heterogeneity

Revealed by Spiked-scRNAseq Is Shaped by Cancer Cell Interactions and Restricted by VSIG1. _Cell Rep._ 33, 108372 (2020). Article  CAS  PubMed  Google Scholar  * Zanconato, F., Cordenonsi,

M. & Piccolo, S. YAP/TAZ at the Roots of Cancer. _Cancer Cell_ 29, 783–803 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhao, B., Li, L., Lei, Q. & Guan, K. L. The

Hippo-YAP pathway in organ size control and tumorigenesis: an updated version. _Genes Dev._ 24, 862–874 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Loh, Y. H.,

Christoffels, A., Brenner, S., Hunziker, W. & Venkatesh, B. Extensive expansion of the claudin gene family in the teleost fish, Fugu rubripes. _Genome Res._ 14, 1248–1257 (2004). Article

  CAS  PubMed  PubMed Central  Google Scholar  * Baltzegar, D. A., Reading, B. J., Brune, E. S. & Borski, R. J. Phylogenetic revision of the claudin gene family. _Mar. Genomics_ 11,

17–26 (2013). Article  PubMed  Google Scholar  * Hou, J. et al. Claudin-16 and claudin-19 interaction is required for their assembly into tight junctions and for renal reabsorption of

magnesium. _Proc. Natl. Acad. Sci. USA._ 106, 15350–15355 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Meoli, L. & Gunzel, D. The role of claudins in homeostasis.

_Nat. Rev. Nephrol._ 19, 587–603 (2023). Article  CAS  PubMed  Google Scholar  * Yu, A. S. Claudins and the kidney. _J. Am. Soc. Nephrol._ 26, 11–19 (2015). Article  CAS  PubMed  Google

Scholar  * Kurokawa, T., Uji, S. & Suzuki, T. Identification of pepsinogen gene in the genome of stomachless fish, _Takifugu rubripes_. _Comp. Biochem. Physiol. B. Biochem. Mol. Biol._

140, 133–140 (2005). Article  PubMed  Google Scholar  * Erawijantari, P. P. et al. Influence of gastrectomy for gastric cancer treatment on faecal microbiome and metabolome profiles. _Gut_

69, 1404–1415 (2020). Article  CAS  PubMed  Google Scholar  * Cahill, M. M. Bacterial flora of fishes: A review. _Microb. Ecol._ 19, 21–41 (1990). Article  CAS  PubMed  Google Scholar  *

Tarnecki, A. M., Burgos, F. A., Ray, C. L. & Arias, C. R. Fish intestinal microbiome: diversity and symbiosis unravelled by metagenomics. _J. Appl. Microbiol._ 123, 2–17 (2017). Article

  CAS  PubMed  Google Scholar  * Clements, K. D., Angert, E. R., Montgomery, W. L. & Choat, J. H. Intestinal microbiota in fishes: what’s known and what’s not. _Mol. Ecol._ 23, 1891–1898

(2014). Article  PubMed  Google Scholar  * Egerton, S., Culloty, S., Whooley, J., Stanton, C. & Ross, R. P. The Gut Microbiota of Marine Fish. _Front. Microbiol._ 9, 873 (2018). Article

  PubMed  PubMed Central  Google Scholar  * German, D. P. Inside the guts of wood-eating catfishes: can they digest wood? _J. Comp. Physiol. B_ 179, 1011–1023 (2009). Article  PubMed  PubMed

Central  Google Scholar  * Roeselers, G. et al. Evidence for a core gut microbiota in the zebrafish. _ISME J_ 5, 1595–1608 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Sullam, K. E. et al. Environmental and ecological factors that shape the gut bacterial communities of fish: a meta-analysis. _Mol. Ecol._ 21, 3363–3378 (2012). Article  PubMed  Google

Scholar  * Leigh, S. C., Catabay, C. & German, D. P. Sustained changes in digestive physiology and microbiome across sequential generations of zebrafish fed different diets. _Comp.

Biochem. Physiol. A. Mol. Integr. Physiol._ 273, 111285 (2022). Article  CAS  PubMed  Google Scholar  * Sakamoto, N. et al. Role for cGATA-5 in transcriptional regulation of the embryonic

chicken pepsinogen gene by epithelial-mesenchymal interactions in the developing chicken stomach. _Dev. Biol._ 223, 103–113 (2000). Article  CAS  PubMed  Google Scholar  * Shin, M., Noji,

S., Neubuser, A. & Yasugi, S. FGF10 is required for cell proliferation and gland formation in the stomach epithelium of the chicken embryo. _Dev. Biol._ 294, 11–23 (2006). Article  CAS 

PubMed  Google Scholar  * Spencer-Dene, B. et al. Stomach development is dependent on fibroblast growth factor 10/fibroblast growth factor receptor 2b-mediated signaling. _Gastroenterology_

130, 1233–1244 (2006). Article  CAS  PubMed  Google Scholar  * Cunningham, F. et al. Ensembl 2015. _Nucleic Acids Res._ 43, D662–669 (2015). Article  CAS  PubMed  Google Scholar  * Altschul,

S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. _J. Mol. Biol._ 215, 403–410 (1990). Article  CAS  PubMed  Google Scholar  * Tamura, K.,

Stecher, G. & Kumar, S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. _Mol. Biol. Evol._ 38, 3022–3027 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Tran, Y. H. et al. Spliced isoforms of LIM-domain-binding protein (CLIM/NLI/Ldb) lacking the LIM-interaction domain. _J. Biochem._ 140, 105–119 (2006). Article  CAS  PubMed  Google Scholar 

* Takei, Y. et al. Molecular mechanisms underlying active desalination and low water permeability in the esophagus of eels acclimated to seawater. _Am. J. Physiol. Regul. Integr. Comp.

Physiol._ 312, R231–R244 (2017). Article  PubMed  Google Scholar  * Thompson, J. D., Higgins, D. G. & Gibson, T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence

alignment through sequence weighting, position-specific gap penalties and weight matrix choice. _Nucleic Acids Res._ 22, 4673–4680 (1994). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Nei, M. & Gojobori, T. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. _Mol. Biol. Evol._ 3, 418–426 (1986). CAS  PubMed 

Google Scholar  * Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. _Mol. Biol. Evol._ 30, 2725–2729 (2013).

Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhang, J., Rosenberg, H. F. & Nei, M. Positive Darwinian selection after gene duplication in primate ribonuclease genes. _Proc.

Natl. Acad. Sci. USA._ 95, 3708–3713 (1998). Article  CAS  PubMed  PubMed Central  Google Scholar  * Martin, F. J. et al. Ensembl 2023. _Nucleic Acids Res._ 51, D933–D941 (2023). Article 

CAS  PubMed  Google Scholar  * Rangwala, S. H. et al. Accessing NCBI data using the NCBI Sequence Viewer and Genome Data Viewer (GDV). _Genome Res._ 31, 159–169 (2021). Article  PubMed 

PubMed Central  Google Scholar  * Braasch, I. et al. The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons. _Nat. Genet._ 48, 427–437 (2016).

Article  CAS  PubMed  PubMed Central  Google Scholar  * Bian, C. et al. The Asian arowana (_Scleropages formosus_) genome provides new insights into the evolution of an early lineage of

teleosts. _Sci. Rep._ 6, 24501 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Yang, J. et al. The Sinocyclocheilus cavefish genome provides insights into cave adaptation.

_BMC Biol._ 14, 1 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Burns, F. R. et al. Sequencing and de novo draft assemblies of a fathead minnow (_Pimephales promelas_)

reference genome. _Environ. Toxicol. Chem._ 35, 212–217 (2016). Article  PubMed  Google Scholar  * Chen, X. et al. High-quality genome assembly of channel catfish, _Ictalurus punctatus_.

_Gigascience_ 5, 39 (2016). Article  PubMed  PubMed Central  Google Scholar  * Warren, W. C. et al. A chromosome-level genome of _Astyanax mexicanus_ surface fish for comparing

population-specific genetic differences contributing to trait evolution. _Nat. Commun._ 12, 1447 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Palti, Y. et al. A second

generation integrated map of the rainbow trout (_Oncorhynchus mykiss_) genome: analysis of conserved synteny with model fish genomes. _Mar Biotechnol (NY)_ 14, 343–357 (2012). Article  CAS 

PubMed  Google Scholar  * Rondeau, E. B. et al. The genome and linkage map of the northern pike (_Esox lucius_): conserved synteny revealed between the salmonid sister group and the

Neoteleostei. _PLoS One_ 9, e102089 (2014). Article  PubMed  PubMed Central  Google Scholar  * Araki, K. et al. Whole Genome Sequencing of Greater Amberjack (_Seriola dumerili_) for SNP

Identification on Aligned Scaffolds and Genome Structural Variation Analysis Using Parallel Resequencing. _Int. J. Genomics_ 2018, 7984292 (2018). Article  PubMed  PubMed Central  Google

Scholar  * Reichwald, K. et al. High tandem repeat content in the genome of the short-lived annual fish _Nothobranchius furzeri_: a new vertebrate model for aging research. _Genome Biol_ 10,

R16 (2009). Article  PubMed  PubMed Central  Google Scholar  * Schartl, M. et al. The genome of the platyfish, _Xiphophorus maculatus_, provides insights into evolutionary adaptation and

several complex traits. _Nat. Genet._ 45, 567–572 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Tan, M. H. et al. Finding Nemo: hybrid assembly with Oxford Nanopore and

Illumina reads greatly improves the clownfish (_Amphiprion ocellaris_) genome assembly. _Gigascience_ 7, 1–6 (2018). Article  PubMed  Google Scholar  * Liu, D. et al. Chromosome-level genome

assembly of the endangered humphead wrasse _Cheilinus undulatus_: Insight into the expansion of opsin genes in fishes. _Mol. Ecol. Resour._ 21, 2388–2406 (2021). Article  CAS  PubMed 

Google Scholar  * Pauletto, M. et al. Genomic analysis of _Sparus aurata_ reveals the evolutionary dynamics of sex-biased genes in a sequential hermaphrodite fish. _Commun. Biol._ 1, 119

(2018). Article  PubMed  PubMed Central  Google Scholar  * Pan, H. et al. The genome of the largest bony fish, ocean sunfish (_Mola mola_), provides insights into its fast growth rate.

_Gigascience_ 5, s13742-13016-10144-13743, https://doi.org/10.1186/s13742-016-0144-3 (2016). * Bi, X. et al. Tracing the genetic footprints of vertebrate landing in non-teleost ray-finned

fishes. _Cell_ 184, 1377–1391e1314 (2021). Article  CAS  PubMed  Google Scholar  * Cheng, P. et al. Draft Genome and Complete Hox-Cluster Characterization of the Sterlet (_Acipenser

ruthenus_). _Front. Genet._ 10, 776 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Cheng, P. et al. The American Paddlefish Genome Provides Novel Insights into Chromosomal

Evolution and Bone Mineralization in Early Vertebrates. _Mol. Biol. Evol._ 38, 1595–1607 (2021). Article  CAS  PubMed  Google Scholar  * Peterson, R., Sullivan, J. & Pirro, S. The

Complete Genome Sequences of 38 Species of Elephantfishes (Mormyridae, Osteoglossiformes). _Biodivers. Genomes_ 2022, https://doi.org/10.56179/001c.56077 (2022). * Gallant, J. R., Losilla,

M., Tomlinson, C. & Warren, W. C. The Genome and Adult Somatic Transcriptome of the Mormyrid Electric Fish Paramormyrops kingsleyae. _Genome Biol. Evol._ 9, 3525–3530 (2017). Article 

CAS  PubMed  PubMed Central  Google Scholar  * Coppe, A. et al. Sequencing, de novo annotation and analysis of the first _Anguilla anguilla_ transcriptome: EeelBase opens new perspectives

for the study of the critically endangered European eel. _BMC Genomics_ 11, 635 (2010). Article  PubMed  PubMed Central  Google Scholar  * Pettersson, M. E. et al. A Long-Standing Hybrid

Population Between Pacific and Atlantic Herring in a Subarctic Fjord of Norway. _Genome Biol. Evol_. 15, https://doi.org/10.1093/gbe/evad069 (2023). * Zimin, A. V. et al. A whole-genome

assembly of the domestic cow, _Bos taurus_. _Genome Biol._ 10, R42 (2009). Article  PubMed  PubMed Central  Google Scholar  * International Chicken Genome Sequencing, C. Sequence and

comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. _Nature_ 432, 695–716 (2004). Article  Google Scholar  * Liu, Y. et al. _Gekko japonicus_

genome reveals evolution of adhesive toe pads and tail regeneration. _Nat. Commun._ 6, 10033 (2015). Article  CAS  PubMed  Google Scholar  * Liu, J. et al. Chromosome-level genome assembly

of the Chinese three-keeled pond turtle (_Mauremys reevesii_) provides insights into freshwater adaptation. _Mol. Ecol. Resour._ 22, 1596–1605 (2022). Article  CAS  PubMed  Google Scholar  *

Marra, N. J. et al. White shark genome reveals ancient elasmobranch adaptations associated with wound healing and the maintenance of genome stability. _Proc. Natl. Acad. Sci. USA._ 116,

4446–4455 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Yamaguchi, K. et al. Elasmobranch genome sequencing reveals evolutionary trends of vertebrate karyotype

organization. _Genome Res._ 33, 1527–1540 (2023). Article  PubMed  PubMed Central  Google Scholar  * Zhao, R. et al. Genomic Comparison and Genetic Marker Identification of the White-Spotted

Bamboo Shark _Chiloscyllium plagiosum_. _Front. Mar. Sci._ 9, 936681 (2022). Article  Google Scholar  * Marletaz, F. et al. The little skate genome and the evolutionary emergence of

wing-like fins. _Nature_ 616, 495–503 (2023). Article  CAS  PubMed  PubMed Central  Google Scholar  * Near, T. J. et al. Phylogeny and tempo of diversification in the superradiation of

spiny-rayed fishes. _Proc. Natl. Acad. Sci. USA._ 110, 12738–12743 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kumar, S., Stecher, G., Suleski, M. & Hedges, S. B.

TimeTree: A Resource for Timelines, Timetrees, and Divergence Times. _Mol. Biol. Evol._ 34, 1812–1819 (2017). Article  CAS  PubMed  Google Scholar  * Grahammer, F. et al. The cardiac K+

channel KCNQ1 is essential for gastric acid secretion. _Gastroenterology_ 120, 1363–1371 (2001). Article  CAS  PubMed  Google Scholar  * Kumar, S. & Hedges, S. B. A molecular timescale

for vertebrate evolution. _Nature_ 392, 917–920 (1998). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Yoko Yamamoto, Nana Shinohara, and the

Biomaterials Analysis Division at the Tokyo Institute of Technology for technical assistance; Megumi Ohmaki and Yuko Akiyoshi for their secretarial assistance; and the anonymous reviewers

for their useful comments. This work was supported by Japan Society for the Promotion of Science KAKENHI (Grants 24651211, 26292113, and 21H02281). The Romero laboratory work was supported

by NIH R01-EY017732, R21-DK129897, and R01-DK128844. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan Akira

Kato, Chihiro Ota, Ayumi Nagashima & Masayuki Komada * Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan Akira Kato, Zinia Islam & Naoko Hayashi *

Center for Biological Resources and Informatics, Tokyo Institute of Technology, Yokohama, Japan Akira Kato * Department of Physiology & Biomedical Engineering, Mayo Clinic College of

Medicine & Science, Rochester, MN, USA Akira Kato, An-Ping Chen & Michael F. Romero * Department of Marine Bioscience, Atmosphere and Ocean Research Institute, The University of

Tokyo, Kashiwa, Japan Supriya Pipil, Makoto Kusakabe, Taro Watanabe, Marty Kwok-Shing Wong & Yoshio Takei * Department of Biological Sciences, Faculty of Science, Shizuoka University,

Shizuoka, Japan Makoto Kusakabe * Department of Biomolecular Science, Toho University, Funabashi, Japan Marty Kwok-Shing Wong * Cell Biology Center, Institute of Innovative Research, Tokyo

Institute of Technology, Yokohama, Japan Masayuki Komada * Department of Nephrology & Hypertension, Mayo Clinic College of Medicine & Science, Rochester, MN, USA Michael F. Romero

Authors * Akira Kato View author publications You can also search for this author inPubMed Google Scholar * Supriya Pipil View author publications You can also search for this author

inPubMed Google Scholar * Chihiro Ota View author publications You can also search for this author inPubMed Google Scholar * Makoto Kusakabe View author publications You can also search for

this author inPubMed Google Scholar * Taro Watanabe View author publications You can also search for this author inPubMed Google Scholar * Ayumi Nagashima View author publications You can

also search for this author inPubMed Google Scholar * An-Ping Chen View author publications You can also search for this author inPubMed Google Scholar * Zinia Islam View author publications

You can also search for this author inPubMed Google Scholar * Naoko Hayashi View author publications You can also search for this author inPubMed Google Scholar * Marty Kwok-Shing Wong View

author publications You can also search for this author inPubMed Google Scholar * Masayuki Komada View author publications You can also search for this author inPubMed Google Scholar *

Michael F. Romero View author publications You can also search for this author inPubMed Google Scholar * Yoshio Takei View author publications You can also search for this author inPubMed 

Google Scholar CONTRIBUTIONS A.K., T.W., M.F.R. and Y.T. conceived the study; A.K., S.P., M.Ku, T.W., A.P.C., Z.I., N.H., M.W., M.Ko, M.F.R. and Y.T. designed and conducted the expression

analyses; A.K., C.O., T.W. and A.N. designed and conducted the bioinformatics analyses; A.K. and Y.T. wrote the paper; and A.K., C.O. and A.N. prepared the revised manuscript. All authors

read and approved the final manuscript. CORRESPONDING AUTHOR Correspondence to Akira Kato. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW

PEER REVIEW INFORMATION _Communications Biology_ thanks Donovan German and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor:

Luke R. Grinham. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. SUPPLEMENTARY

INFORMATION SUPPLEMENTARY TABLE AND FIGURES REPORTING SUMMARY RIGHTS AND PERMISSIONS OPEN ACCESS This article is licensed under a Creative Commons Attribution 4.0 International License,

which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link

to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless

indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or

exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Reprints

and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Kato, A., Pipil, S., Ota, C. _et al._ Convergent gene losses and pseudogenizations in multiple lineages of stomachless fishes. _Commun

Biol_ 7, 408 (2024). https://doi.org/10.1038/s42003-024-06103-x Download citation * Received: 03 April 2023 * Accepted: 25 March 2024 * Published: 03 April 2024 * DOI:

https://doi.org/10.1038/s42003-024-06103-x SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not

currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative