Evolutionary Transition From Water to Land in Vertebrates Illuminated by Basal Ray-Finned Fish Vomeronasal Type 2 Receptor (OlfC) Genes

20 The vomeronasal type 2 receptor ( V2R , also called OlfC ) multigene family is found in a broad range 21 of jawed vertebrates from cartilaginous fish to tetrapods. V2R s encode receptors for food-related 22 amino acids in teleost fish, whereas for peptide pheromones in mammals. In addition, V2R s of teleost 23 fish are phylogenetically distinct from those of tetrapods, implying a drastic change in the V2R 24 repertoire during terrestrial adaptation. To understand the process of diversification of V2R s in 25 vertebrates from “fish-type” to “tetrapod-type”, we conducted an exhaustive search for V2R s in 26 cartilaginous fish (chimeras, sharks, and skates) and basal ray-finned fish (reedfish, sterlet, and spotted 27 gar), and compared them with those of teleost, coelacanth, and tetrapods. Phylogenetic and synteny 28 analyses on 1897 V2R s revealed that basal ray-finned fish possess unexpectedly higher number of 29 V2R s compared with cartilaginous fish, implying that V2R gene repertoires expanded in the common 30 ancestor of Osteichthyes. Furthermore, reedfish and sterlet possessed various V2R s that belonged to 31 both “fish-type” and “tetrapod-type”, suggesting that the common ancestor of Osteichthyes possess 32 “tetrapod-type” V2R s although they inhabited underwater environments. Thus, the unexpected 33 diversity of V2R s in basal ray-finned fish illuminates the process of how the osteichthyan ancestors 34 adapt from water to land. 35


Introduction 38
Olfaction is a critical chemosensory system for eliciting social behaviors in vertebrates, including 39 reproduction, kin recognition, aggression, and feeding. The vertebrate olfaction system has 40 experienced drastic changes in anatomy and neurophysiology during adaptation from water to land, 41 one of the most important events is vertebrate evolution. Specifically, aquatic vertebrates detect water-42 soluble chemicals using their olfactory epithelium (OE), whereas terrestrial vertebrates detect both 43 volatile and nonvolatile chemicals by differentiating their OE into main olfactory epithelium (MOE) 44 and vomeronasal organ (VNO). Accompanied by the terrestrial adaptation of olfaction, the 45 chemosensory receptors are proposed to have undergone major innovative diversification, although 46 the detailed evolutionary process of diversification remains to be poorly understood at the DNA level. 47 Olfaction of vertebrates is mainly composed of four types of G protein-coupled receptors (GPCRs), 48 namely, the olfactory receptor (OR), vomeronasal receptor type I (V1R), vomeronasal receptor type II 49 (V2R), and trace amine-associated receptor (TAAR), all forming multigene families 1 . In teleost, V2Rs, 50 also referred to as OlfCs (olfactory receptors classified as type C GPCRs) 2,3 , are expressed in OE of 51 the nasal cavity. Several independent studies have shown that teleost V2Rs detect amino acids that 52 elicit certain feeding behaviors. For example, V2Rs are expressed in microvillous sensory neurons of 53 zebrafish and respond to amino acids, but not bile acids or sex pheromones 4 . In addition, the genetic 54 blockage of neural transmission in the V2R-expressing neurons abolishes the attractive response to a 55 mixture of amino acids 5 . However, given that Yang et al. 6 have proposed a possible contribution of 56 some V2Rs to elicit fright reactions, it is premature to rule out the possibility that V2Rs detect some 57 chemicals other than amino acids. In tetrapods, two anatomically distinct organs, that is, MOE and 58 VNO, each mainly detecting odorants and pheromones. V2Rs are specifically expressed in VNO of 59 type" V2Rs 16 , showing that the coelacanth is an important organism as it serves as a missing link to 74 fill an evolutionary gap between vertebrates under water and land 20 . Recent studies on several shark 75 genomes have shown that olfaction in cartilaginous fish is dominated by V2Rs rather than conventional 76 ORs 21,22,23 , which is consistent with the ultrastructural observation that the presence of only 77 microvillous sensory neurons in OE 24 and with the immunohistochemical observations that most 78 neurons in OE are positive for Go antibodies 25 . In addition, a previous study has shown that V2Rs were 79 absent in lamprey genomes 26 . Based on the above findings, V2Rs are considered to have originated in 80 the jawed vertebrate ancestor before the split between the two extant descendant lineages, that is, 81 cartilaginous fish and Osteichthyes (ray-finned and lobed-finned fish including tetrapods). 82 In this study, we have performed a comprehensive exploration and phylogenetic analyses for 83 V2Rs in nine cartilaginous fish species (one elephant shark, one rabbit fish, four sharks, two skates, 84 and sawfish), three basal ray-finned fish (reedfish, sterlet, and spotted gar), two teleost fish (eel, 85 zebrafish), a coelacanth, two amphibians (western clawed frog and caecilian), an anole lizard, and a 86 mouse to elucidate the process of diversification of "fish-type" and "tetrapod-type" V2Rs in vertebrates. 87 As a result, we have characterized 9 to 42 V2Rs in cartilaginous fish, and a large amount (47 to 189) 88 of V2Rs in basal ray-finned fish. Phylogenetic analyses of V2Rs of 19 various vertebrate species have 89 revealed the existence of "tetrapod-type" V2Rs in the genomes of reedfish and sterlet, implying the 90 presence of an evolutionary seed for mammalian peptide pheromone receptors in the basal ray-finned 91 fish. The result of this fine-scale phylogenetic study would provide important insights into 92 understanding the process of olfactory adaptations in the V2Rs of vertebrates from cartilaginous fish 93 to tetrapods. 94

Characterization of V2Rs from the genomes of a broad range of vertebrates 96
We explored V2R gene repertoires from the genomes of nine cartilaginous fish (one elephant 97 shark, one rabbit fish, four sharks, and three rays), three basal ray-finned fish (reedfish, sterlet, and 98 spotted gar), two teleost fish (zebrafish, Japanese eel), a coelacanth, two amphibians (western clawed 99 frog, caecilian), an anole lizard, and a mouse using an original software "fate" 27 . The copy numbers 100 of V2Rs in individual species are summarized and provided in Table 1. The number of V2Rs in teleost 101 fish was mostly comparable to previous studies with some update. For example, zebrafish that 102 possessed 72 V2Rs, which was larger than in previous studies 13,20 , comprising 19 genes from unplaced 103 scaffolds and 53 genes from chromosome 18. The numbers of V2Rs in cartilaginous fish ranged from 104 8 (rays) to 41 (chimera), which were found to be smaller than those of the teleost fish. In contrast, 105 basal ray-finned fish possessed an unexpectedly large number of V2Rs. In reedfish, we identified 188 106 intact V2Rs, including 68 genes from unplaced scaffolds, which was the largest in all ray-finned fish 107 studied until now. The numbers of V2Rs in sterlet and spotted gars were also high (46 and 49, 108 respectively). The copy number of V2Rs in the western clawed frog (691), which far exceeded that 109 previous studies 15 , was the largest among vertebrates studied so far; it is much higher than those of 110 other tetrapods, such as caecilian (275), anole lizard (64), and mouse (154). It is noteworthy that we 111 found no V2Rs in the genomes of agnathans and amphioxus. According to a recent study by Bi et al. 28 , 112 reports on more than 50 V2R-like sequences in hagfish and a few in amphioxus were observed. 113 However, our phylogenetic analyses revealed that they did not form a cluster with known V2Rs. 114 Therefore, we need to be still cautious to designate these sequences as V2Rs, reaching a conclusion 115 that no typical V2Rs exist in agnathans and amphioxus. 116 117 Phylogeny and classification of the identified V2Rs 118 "a1" for convenience in classification. Over half of the V2Rs of the basal ray-finned fish belonged to 131 this subfamily "a1" (Table 1, reedfish 101; sterlet 21; spotted gar 29). Also, we have found a 132 cartilaginous fish-specific subfamily "a2" and "a3" at the stem of subfamily 4-10 and at the stem of 133 subfamily 15, 16 (Table 1, Fig. 1A, Fig. S1). In particular, the large number of V2Rs of the cartilaginous 134 fish belonged to subfamily "a2," in which the genes were expanded in a species-specific manner ( Table  135 1, Supplementary Fig. S1). Taken together, a large fraction of V2Rs in cartilaginous and basal ray-136 finned fish were classified into these new subfamilies "a1"-"a3, which were the ancestral clades for 137 some known 16 subfamilies. In addition to the subfamily "a1", species-specific expansion of V2Rs in 138 basal ray-finned fish was observed in subfamily two of sterlet (11) and in subfamily 16 of reedfish 139 (67) and spotted gar (9). In contrast to ray-finned fish in which V2R subfamilies were highly diverse, 140 cartilaginous fishes were relatively less diverse in that they lacked some subfamilies (Table 1). One 141 exceptional finding was that only one sequence of caecilian V2R was classified as being a member of 142 the "fish-type" subfamily 14 (Table 1, Fig. 1A, marked with an asterisk). 143 The "tetrapod-type" V2Rs were shown to be dominated by closely related V2Rs that have 144 duplicated more recently than those of cartilaginous and ray-finned fish 9,13,15 . Notably, V2Rs of 145 amphibians (western clawed frog and caecilian) are highly diverse in that they are subdivided into 146 more than 10 and 3 species-specific clusters, while all of the 147 V2Rs of mouse belonged to a single 147 cluster. The 63 V2Rs of anole lizard were grouped into 2 clusters, which were close to those of 148 mammals (Fig. 1B, Table 1). The 75 V2Rs of coelacanth were classified into the "tetrapod-type," while 149 13 V2Rs were classified into the "fish-type" (Fig. 1A, Table 1). The existence of both the "fish-type" 150 and "tetrapod-type" V2Rs in coelacanth was consistent with a previous study 16 . One of the most 151 striking results of this study was that some V2Rs of ray-finned fishes (three genes from each of reedfish 152 and sterlet) were classified into "tetrapod-type". Indeed, in the phylogenetic tree (as shown in Fig. 1B  153 and Supplementary File S1), these V2Rs are nested within the "tetrapod-type" V2Rs. These "tetrapod-154 type" V2Rs of basal ray-finned fish were close to those of coelacanth. 155 156

Newly identified V2R orthologs conserved from cartilaginous fish to amphibians 157
Previous studies have shown that V2Rs were divided into three well-supported clades, namely, 158 the V2R2s, "fish-type" V2Rs, and "tetrapod-type" V2Rs 16 . The orthologous V2R2s were shared in all 159 vertebrates from cartilaginous fish to mammals with a few species-specific duplications in mice 26,29 . 160 In this study, only one V2R2 ortholog of all species investigated was located at the basal position of 161 the V2R tree (Table 1, Fig. 1C, Supplementary Fig. 1). In addition to the three clades, we identified a 162 novel clade, in which V2Rs of cartilaginous fish, basal ray-finned fish, and amphibians were included and "tetrapod-type" V2Rs, which amplify in a species-specific manner. Therefore, we designated them 167 as anc (ancient) V2R. In this analysis, highly conserved ancV2R sequences were found in the genomes 168 of cartilaginous fish to amphibians, but not in teleost fish, coelacanth, and mammals (Fig. 1C). 169 170 Conserved gene clusters of the "fish-type" V2Rs 171 In addition to phylogenetic analysis, the synteny relationships have provided important insight 172 into the classification of V2Rs. Previous studies have shown that the V2Rs in teleost were clustered in 173 one particular chromosomal region, which was flanked by two landmark genes, phospholipase C 174 (PLC) eta1 and neprilysin. In contrast, no V2Rs were found between these two genes in tetrapods, and 175 "tetrapod-type" V2Rs were scattered into several chromosomes 13,18,19 . Therefore, to ascertain if the 176 V2Rs are "fish-type" or "tetrapod-type", we examined the synteny relationships in vertebrates. Figure  177 2 shows the gene arrangements in the genomic region of two landmark genes in various vertebrates 178 from cartilaginous fish to mammals. Notably, V2R2 and ancV2R was found in tandem of the 179 neighboring regions of the PLC eta1, implying the evolutionary conservation of these two genes and 180 evolutionary distinction from known "fish-type" V2Rs. Consistent with the previous study, no V2R 181 was found in tetrapods in this region, except for only one caecilian V2R, which was classified as "fish-182 type" in the phylogenetic tree (Fig. 1A). In basal ray-finned fish (reedfish, sterlet spotted gar), it was 183 obvious that the V2Rs were clustered between PLC eta1 and neprilysin. Importantly, we revealed that 184 all "fish-type" V2Rs were located in this cluster region, while three "tetrapod-type" V2Rs identified in 185 reedfish and sterlet were located on different chromosomes (chr.3 in reedfish, chr.52, 53 and 186 VTUV01000346.1 in sterlet, Supplementary Table S1). In sterlet, we identified two distinct 187 chromosomal regions of the "fish-type" V2R clusters, which were due to polyploidization specific to 188 this group 13 . The synteny of coelacanth also showed a conservation of the "fish-type" V2R cluster. In 189 the elephant shark and bamboo shark, some "fish-type" V2Rs were located outside the cluster, but 190 considering that they were all found in short scaffolds, it is likely that the cluster region was not 191 properly assembled. Overall, the phylogenetic and synteny analyses both supported the conservation 192 of the cluster for "fish-type" V2Rs as well as the existence of the "tetrapod-type" V2Rs in basal ray-193

Expression of V2Rs in the olfactory epithelium of basal ray-finned fish 196
To evaluate the functional role of V2Rs found in the genomes of basal ray-finned fish, we examined 197 the cellular expression patterns for these receptors in the OE. Figure 3 shows the location of the 198 transcripts detected by in situ hybridization on frozen sections of the OE of Polypterus senegalus 199 (bichir), which is a basal ray-finned fish closely related to reedfish. The probes of the four V2Rs -200 member of the "fish-type" V2R showed a sparse pattern in the sensory cells of the OE, typical of 203 canonical V2Rs (Fig. 3A). The expression of a member of "tetrapod-type" V2R, which was newly 204 identified in basal ray-finned fish, also showed similar sparse pattern in the OE of P. senegalus (Fig.  205   3B). The expression of V2R2, of which the ortholog was highly conserved among jawed vertebrates, 206 has showed widespread patterns in their OE (Fig. 3C). The expression pattern of V2R2 in P. senegalus 207 was consistent with the ubiquitous expression in zebrafish 31 and mouse 29 . The expression of ancV2R, 208 of which the orthologs were also highly conserved from cartilaginous fish to amphibians, showed a 209 sparse pattern in their OE (Fig. 3D) distinct from that of V2R2. Overall, the V2Rs belonging to four 210 clades were all expressed in the OE, suggesting their functions as olfactory receptors. However, the 211 patterns of expressions were ubiquitous in V2R2, while they were sparse in V2Rs of other clades. In this study, we have conducted a comprehensive exploration of V2R sequences from the 216 genomes of 19 vertebrate species. Phylogenetic analyses of a large number of V2Rs allowed us to gain 217 a panoramic view of the diversity in terms of copy number and repertoire of V2Rs across vertebrates. 218 Here, we discuss the tempo and mode of evolution of V2Rs and how these factors drive the adaptive 219 evolution of the olfactory system in vertebrates. 220 It is obvious that V2Rs were abundant in ray-finned fish compared to cartilaginous fish, both in 221 terms of copy number and repertoire, which is achieved by a species-specific expansion of "fish-type" 222 V2Rs. The synteny analyses revealed that "fish-type" V2Rs constituted a large gene cluster between 223 PLC eta1 and neprilysin in ray-finned fish (Fig. 2). Although the V2R clusters were ambiguous in the 224 elephant shark, bamboo shark and saw fish, which may be due to the complex chromosomal 225 rearrangements in cartilaginous fish 21 , the "fish-type" V2Rs exist near the two marker genes. Notably, 226 species-specific expansions of V2Rs did not occur uniformly in all subfamilies, but were rather 227 concentrated in certain ones. For example, the expansion of V2Rs was mainly observed in subfamilies 228 4-9, 16, 'a1', and 'a2', while the copy numbers of other subfamilies remained one or two. Therefore, 229 distinction in copy numbers between subfamilies reflects the difference in ligand recognition and 230 biological functions of V2Rs among each subfamily. At present, V2Rs are expected to detect amino 231 acids and their derivatives, eliciting feeding behaviors in teleost fish 4,5,32 . It is reasonable to assume 232 that a limited number of amino acids in diets were received by evolutionarily conserved V2R 233 subfamilies. However, it is plausible to assume that the V2R subfamily with frequent lineage-specific 234 gene duplications is responsible for receiving some species-specific variable chemicals for social 235 communication. For example, Yambe et al. 33 showed that an amino acid derivative, L-kynurenine, 236 secreted in the female urine, acts as the male-attracting pheromone in masu salmon. In addition, a 237 previous study showed a possible correlation between expansions of V2Rs in subfamily 9 and the 238 evolution of fright reactions in teleost fish 6 . Thus, to elucidate the function of V2Rs in ray-finned fish 239 in addition to amino acid reception, it is necessary to further examine the V2Rs from a 240 multidisciplinary framework, including the ligand binding, and behavioral experiments using 241 candidate chemicals. 242 We showed that orthologous sequences of V2R2 and newly identified ancV2R have long been 243 conserved during the evolution of vertebrates. The conservation pattern of orthologs in V2R2 and 244 ancV2R is distinct from the canonical V2Rs, such as those in the "fish-type" and "tetrapod-type" V2Rs, 245 which were diversified via species-specific gene duplications. The existence of V2R2 and ancV2R in 246 close genomic proximity was also conserved in many vertebrates (Fig. 2) in a broad area of the OE and was co-expressed with one of the many canonical V2Rs. Consistent with 3C), while the "fish-type" and "tetrapod-type" V2Rs showed sparse patterns (Figs. 3A, B). In contrast, 251 although ancV2R was similar to V2R2 in terms of evolutionary conservation and genomic proximity, 252 the pattern of expression in the OE was sparse rather than widespread. Thus, it is implicative to note 253 that ancV2R has characteristics between V2R2 and canonical "fish-type" and/or "tetrapod-type" V2Rs. 254 Thus, taking the evolutionary conservation as well as the sparse pattern of expression into account, 255 ancV2R may have retained ancestral nature inherited from a protogene before the split between "fish-256 type" and "tetrapod-type" V2Rs, which are now highly diversified in jawed vertebrate genomes. This 257 study showed that all four clades of V2Rs, including the "tetrapod-type" V2Rs, were expressed in the 258 olfactory organs of basal ray-finned fish, which propose that they have functional roles in olfaction. 259 At the same time, the degree of conservation and expression patterns was distinct among those clades. 260 In particular, it was of interest that the expression patterns of V2R2, ancV2R, and "fish-type" V2Rs 261 were distinct despite their location on the same genomic cluster. A detailed investigation into this 262 genomic region would lead to the elucidation of a cis-regulatory mechanism that controls the 263 expression of canonical V2Rs, as to say "one neuron one receptor" rule 34 . 264 possessed only specific clades of V2Rs, namely, "tetrapod-type" and "fish-type" V2Rs, respectively. 275 In contrast, basal ray-finned fish and amphibians possessed both "tetrapod-type" V2Rs and "fish-type" 276 V2Rs. This finding suggests that all four clades of V2Rs were present at least in the common ancestor coelacanth compared with those in cartilaginous fish (Table 1). In addition, all four clades of V2Rs 287 were shown to be present in basal ray-finned fish and amphibians. The abundance of copy numbers 288 and repertoires in these groups show that V2Rs were highly diversified in the common ancestor of 289 Osteichthyes. Given that some of the olfaction-related genes also emerge in the common ancestor of 290 the Osteichthyes (e.g., ancV1R 36 ; OMP 37 ), it might be possible that an innovative evolution of the 291 olfactory system occurred in this timing. It is worth mentioning here that the polypterids (bichir and 292 reedfish) possesses large paired openings (spiracles) on top of their head, in which they use for 293 breathing air 38 . Similar spiracle-like structures were observed in the fossil records of stem tetrapods 39 . 294 Thus, breathing air using spiracles may have been an important respiratory strategy in the stem 295 Osteichthyes, which inhabit shallow freshwater environments and use lungs in addition to gills for 296 respiration 40 . Specifically, the evolution of air-breathing by spiracles may increase the opportunity to 297 raise their head above water, which led to the acquisition of the primitive capabilities of detecting 298 airborne chemicals before terrestrial adaptation. Thus, such dual functional roles of the olfactory 299 system in stem Osteichthyes were related to the diversification of V2Rs, including "tetrapod-type" 300 V2Rs. Deorphanization of V2Rs of various vertebrates in the near future is necessary to evaluate the 301 above possibility. 302 303

Materials and methods 304
Sequence retrieval 305 To estimate the evolutionary history of V2Rs, we conducted a comprehensive exploration of V2R 306 sequences in the genome assemblies of a broad range of vertebrates, including nine cartilaginous fishes 307 and three basal ray-finned fishes. In addition to two teleost fishes (Japanese eel and zebrafish), 308 coelacanth, two amphibians (caecilian and western clawed frog), anole lizard, and mouse were 309 explored (Table 1). To identify the V2R sequences from the genome assemblies, we performed NM_001081141.2), for constructing an initial gene tree. All genes in the sister clade to CaSR were 345 named homologs of V2R (including V2R2). Using these genes, we constructed the V2R gene tree again 346 (CaSR was used as an outgroup). We also included sequences of all 16 teleost fish subfamilies 347 classified in previous studies 13,14 , as markers to indicate V2R subfamilies. 348 The synteny relationships of "fish-type" V2Rs of vertebrates were then illustrated based on the 349 genomic location of the identified V2Rs (Supplementary Table S1). The numeric data for the genomic 350 position of V2Rs, which were identified as "fish-type" V2Rs, V2R2, and ancV2R in the phylogenetic accordance with the institutional, governmental ARRIVE guidelines. TRIzol (Invitrogen) was then 360 used for total RNA extraction from the olfactory organs of the bichir. Using the total RNA extracted 361 from the olfactory organs of the bichir, cDNA was synthesized by reverse transcription reaction using 362 SuperScript III RTase (Invitrogen). Coding regions of V2R were amplified by PCR using the primer 363 sets, which were designed on the basis of V2R sequences of reedfish, as has been summarized in 364 Supplementary Table S2. The PCR products were cloned using the pGEM-T vector (Promega) and the 365 DH5α strain of E. coli. Digoxigenin-labeled RNA probes were synthesized using the plasmid vector 366 as a template using T7 or SP6 RNA Polymerase (Roche) and DIG RNA labeling mix (Roche) as well. 367 The olfactory organs of the bichir were then fixed with 4 % PFA, replaced with sucrose, and embedded 368 in an O.C.T compound (Sakura Finetek). In situ hybridization was performed according to the method 369 as previously described 36,37 . Briefly, hybridization was performed using DIG-labeled RNA probes. to tetrapods. (A) Phylogenetic tree of "fish-type" V2Rs. Note that "fish-type" V2Rs were subdivided 507 into 16 known and 3 novel subfamilies, as indicated by gray thick bars. Triangles in red, orange, blue, 508 white, and brown indicate the expanded V2R clusters specific to reedfish, sterlet, spotted gar, 509 cartilaginous fish, and teleost fish, respectively. Only one "fish-type" V2R found in the caecilian 510 species was marked using an asterisk. (B) Phylogenetic tree of "tetrapod-type" V2Rs. Triangles in 511 violet, pink, yellow, green, and gray indicate expanded V2R clusters specific to coelacanth, caecilian, 512 western clawed frog, anole lizard, and mouse, respectively. Asterisks were used to mark the "tetrapod-513 type" V2Rs identified in reedfish and sterlet. Note that the "tetrapod-type" V2Rs, in contrast to the 514 "fish-type" V2Rs, are composed of many clusters that are expanded in a species-specific manner. (C) 515 Overview of the phylogenetic tree of all V2Rs showing novel orthologous clade ancV2R. The calcium-516 sensing receptor (CaSR) gene was used for outgrouping all V2Rs. The OTU names consist of the 517 common name and locus as summarized in Supplementary Table S1. The "fish-type" V2Rs, "tetrapod-518 type" V2Rs, and V2R2 clades were compressed into black triangles. The number on the branches 519 indicates the bootstrap support values for particular nodes. Note that the grouping of the orthologous 520 ancV2Rs of cartilaginous fish, basal ray-finned fish, and western clawed frog was suggested by 521 maximum bootstrap support (100 %). The numbers next to triangles indicate the copy number of V2Rs 522 figure. Note that "fish-type" V2Rs were flanked by two landmark genes and that V2R2s and ancV2Rs 533 were located in tandem close to the clusters. No "tetrapod-type" V2Rs were observed in these cluster 534 regions. In the elephant shark, some "fish-type" V2Rs are located outside the cluster because the cluster 535 regions were not properly assembled. 536 537 Figure 3. The expression patterns of V2Rs in the olfactory epithelium of the basal ray-finned fish, and "tetrapod-type" V2R (B), V2R2 (C), and ancV2R (D). Green indicated the expression signals. The 541 blue area indicates the cell nucleus stained with DAPI. V2R2 was globally expressed in the deep layers 542 of olfactory folds (C). In contrast, "fish-type" V2R, "tetrapod-type" V2R, and ancV2R were sparsely four major V2R clades was plotted on the phylogenetic tree of vertebrates from agnathans to 549 mammals. The red circle with "2," yellow with "a," gray with "t," and blue with "f" indicate V2R2, 550 ancV2R, "tetrapod-type" V2R, and "fish-type" V2R, respectively. In contrast to basal ray-finned 551 fish with all four clades of V2Rs, teleost fish, mammals, and lizards were determined to possess 552 only two of them. The reduction of specific V2R clades in these lineages would be due to 553 adaptation to specific oceanic and terrestrial environments. Note that the origin of "tetrapod-type" 554 V2Rs dates back to the era of the common ancestor of extant Osteichthyes, but its antiquity in the 555 jawed vertebrate ancestor remains to be examined with complete genome sequences of more 556 cartilaginous fish (dotted circle with "t" inside).   Figure S1. A phylogenetic tree of subfamily "a2" and the cartilaginous fish-specific 566 V2Rs. Note that this subfamily consists of 132 V2Rs, most of which were expanded in species-specific 567 birth and death processes. Only one orthologous V2R was shared among cartilaginous fish (indicated 568 by asterisk). 569 570 Supplementary File 1. The Newick format phylogenetic tree of all 1897 intact V2Rs identified by a Fig. 1(A)   Fig. 1(B)   Fig. 1(C)   Fig. 2   Fig. 3   Fig. 4