Evolution of Gene Families and Relationship with Organismal Evolution: Rapid Divergence of Tissue-Specific Genes in the Early Evolution of Chordates

Naoyuki Iwabe, Kei-ichi Kuma, and Takashi Miyata Department of Biophysics, Faculty of Science, Kyoto University, Japan

To determine a possible relationship between organismal and molecular evolution, the divergence patterns of gene families were examined by taking special notice of functional difference, tissue distribution, and intracellular lo- calization of the members. A phylogenetic analysis of 25 different gene families revealed interesting patterns of divergence of these families: Most gene duplications giving rise to different functions antedate the vertebrates- arthropods separation. On the other hand, in a group of members carrying virtually identical function to one another but differing in tissue distribution (tissue-specific isoform), most gene duplications have occurred independently in each of vertebrates and arthropods after the separation of the two animal groups. In family members encoding molecules localizing in cell compartments (compartmentalized isoforms), the gene duplications antedate the ani- mals-fungi separation. In the cases of the Ca2+ pump and rab subfamilies, the compartmentalized isoforms were shown to have diverged during the early evolution of eukaryotes. A phylogenetic analysis of the tissue-specific isoforms from 26 different subfamilies revealed extensive gene duplications and rapid rates of amino acid substi- tutions in the early evolution of chordates before the separation of fishes and tetrapods. On the contrary, the genetic variations are relatively low in the later period. This pattern of evolution observed at the molecular level is correlated well with that of tissue evolution based on fossil evidence and morphological data, and thus evolution at the two levels may be related.

Introduction

It is generally thought that evolutionary changes at the phenotypic and molecular levels are caused by dis- tinct mechanisms, Darwinian selection of advantageous mutants on one hand and random fixation of selectively neutral mutants on the other hand (Kimura 1983). To obtain a clue for understanding evolution at the two lev- els in a unified way, several lines of evidence suggesting evolutionary links between the two levels should be ac- cumulated. Kimura (1991) suggested the presence of the evolutionary link from the viewpoint of the neutral the- ory and emphasized the importance of relaxed con- straints at the phenotypic level for rapid genetic diver- sities. Molecular phylogenetic analyses of members of gene families would provide a unique opportunity for finding the relationship between evolution at the two levels. Sidow (1992) showed an extensive diversifica- tion of the Wnt gene family on the ancestral lineage of vertebrates in a relatively short time period before the emergence of jawed vertebrates and suggested the re- laxation of functional constraints as a cause of the rapid evolution. Pendleton et al. (1993) presented evidence showing a relationship between the number of Hox gene clusters and developmental complexity of chordates. Re- cently Kuma, Iwabe, and Miyata (1995) showed that the

Key words: gene family, gene duplication, tissue distribution, cell compartment, phylogenetic tree, phenotypic evolution.

Address for correspondence and reprints: Takashi Miyata, De- partment of Biophysics, Faculty of Science, Kyoto University, Kyoto 606-01 Japan. E-mail: miyata@biophys.kyoto-u.ac.jp.

Mol. Biol. Evol. 13(3):483493. 1996 0 1996 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038

evolutionary rates of molecules depend strongly on tis- sues where they are expressed specifically, and suggest- ed the presence of functional constraints against molec- ular variations from the tissue level. This is consistent with evidence that a partial lack of tissue functions re- sults in the elevated rate of molecular evolution, as dem- onstrated by the lens crystallin of a blind mole rat (Hen- driks et al. 1987).

Because family members often express tissue spe- cifically and the evolutionary rates differ for different tissues, the analysis of divergence patterns of gene fam- ilies would be useful in finding a relationship between tissue and molecular evolution. In this paper the diver- gence patterns of gene families during evolution are ex- amined based on phylogenetic analyses, by taking spe- cial notice of tissue distribution and intracellular local- ization (preliminary reports have already been published [Miyata et al. 1993, 19941). We present evidence show- ing extensive gene duplications and rapid rates of amino acid substitutions during the early evolution of chor- dates. In contrast the genetic variations are observed only in a reduced amount during the later period of evo- lution. Since various tissues and organs evolved exten- sively in the early period (Romer 1959; Romer and Par- sons 1977; Fingerman 1981), these results suggest a link between tissue and molecular evolution.

Materials and Methods Sequence Data Sources

All the sequence data used in the present analyses were taken from GenBank release 86.0 and NBRF da-

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484 Iwabe et al.

tabase release 43.0, except for Drosophila RH4 opsin (Monte11 et al. 1987); accession numbers of sequences used in figures 1, 2, and 4 are as follows (for abbrevi- ations, see legends of the figures): figure l-human Na+-K+ a-l (X04297, DOOO99), Hydra Na+-K+ (Y (M75140), human H+-K+ (M63962), human PMCAl (JO4027), rat SERCAla (M99223), Drosophila DRSER- CA (M62892), Trypanosoma pump (M73769), rat Ca2+ pump (M93017, S43533), S. cerevisiae PMRl (M25488), S. cerevisiae PMR2 (M25489), S. pombe cta3 (505634) S. cerevisiae PMAl (X03534); insert a- human 1 (X04297, DOOO99), chicken 1 (JO3230), Xen- opus 1 (UlOlOS), Catostomus 1 (X58629), human 2 (JO5096), chicken 2 (M59959), human 3 (M37457), chicken 3 (M59960), Drosophila Da-47 (X14476), Ar- temia franciscana (S634 10, X56650), Artemia salina (Y075 13), Hydra (M75 140); insert b-rat 1 a (M99223), chicken 1 (M26064), Rana 1 (X63009), human 2 (M23114), chicken 2 (M66385, S51963), rat 3 (M30581), Drosophila (M62892), Artemia (X5 1674); figure 2a-human rhodopsin (K0228 l), chicken rhodop- sin (D00702), lamprey rhodopsin (M63632), chicken green opsin (M92038, M88178), Gecko opsin P467 (M92035), goldfish green- 1 opsin (L11865), goldfish green-2 opsin (L11866), chicken blue opsin (M92037), goldfish blue opsin (L11864), human blue opsin (M13299), chicken violet opsin (M92039), human green opsin (K03494), human red opsin (M13305), chicken iodopsin (X57490), goldfish red opsin (L11867), cave fish opsin R007 (M38630), cave fish opsin GlOl (M38624), cave fish opsin G103 (M60945), Gecko opsin P521 (M92036), octopus rhodopsin (X07797), LoZigo rhodopsin (S 14332), Drosophila opsin RHl (K023 15), Drosophila opsin RH2 (M12896), crayfish opsin (S53494), Limulus lateral eye opsin (L03791, LO3781), Drosophila opsin RH3 (Ml77 18, YOOO43), human ml mAChR (X52068, M35128); figure 2b-human 01 (YO0762, X02508), chicken cx (X07338), Xenopus (Y (X07067), Xenopus cxlA (X17244), Torpedo cx (JOO963), human (~5 (M83712), chicken o5 (JO5642), rat B3 (JO4636), goldfish no2 (X14786), goldfish ncx3 (M29529), human a3 (M37981), chicken a3 (M37336), goldfish a3 (X54051), rat a6 (LO8227), rat o2 (M20297), chicken a2 (X07344) rat o4 (M15682), chicken 014 (X07399), human B2 (X53179), chicken B2 (X53092, X07357), rat B4 (M33953), chicken no3 (A39218 [NBRF]), goldfish B2 (X54052), human B (X14830), Torpedo p (JOO964), mouse 6 (K02582), chicken 6 (K02903), Torpedo 6 (JOO965), human y (Ml 181 l), chicken y (KO2904), Torpedo y (500966, VO1394), mouse E (504698, X557 18), Drosophila Do12 (X52274, X53583), Drosophila ALS (X07194), Dro-’ sophila SBD (X55676), Drosophila ARD (X04016), rat (x7 (M85273), chicken a7 (X68586), chicken BgtBP a2

(X52296), mouse 5HT-3 receptor (M74425); figure 4- rat rab7 (X12535), Dictyostelium rab7 (UO2928), dog rab5 (M35520), Nicotiana Nt-rab5 (X63875), human rab6 (G34323 [NBRF]), Schizosaccharomyces ryhl (X52475), rat rab4 (X06890), human rab2 (X12953), Volvox yptV4 (LO8130), human rab3A (C34323 [NBRF]), mouse rabl (YOOO94), Volvox yptV1 (M93438), Leishmania YPT (L12031), human TC4 (M3 1469), Schizosaccharomyces spil (A40039 [NBRF]), Giardia ran (U02589).

Sequence Alignment and Phylogenetic Tree Inference

Optimal alignments of sequences were obtained by the methods of Needleman and Wunsch (1970) and Ber- ger and Munson (1991), together with manual inspec- tions. The aligned sequences were applied to phyloge- netic inferences for regions where unambiguous align- ment is possible. For each family or subfamily the dis- tance matrix was calculated by correcting multiple substitutions (Jukes and Cantor 1969) and a phyloge- netic tree was inferred by the neighbor-joining method (Saitou and Nei 1987). The bootstrap analyses were car- ried out by the method of Felsenstein (1985).

Results and Discussion Patterns of Functional Divergence of Family Members

Figure 1 shows a phylogenetic tree of the members of the eukaryotic P type ATPase (ion pump) family con- sisting of Na+-K+ pump, H+-K+ pump, and Ca2+ pump. The tree was inferred based on the aligned sequences of 308 amino acid positions where unambiguous alignment is possible. Inserts a and b are phylogenetic trees of family members belonging to the plasma membrane Na+-K+ pumps from vertebrates and invertebrates and those belonging to the Ca2+ pumps from vertebrates and invertebrates, respectively, which were inferred based on the alignments of 983 and 812 amino acids in length, respectively.

As figure 1 shows, the divergence of family mem- bers with different ion specificities antedates the diver- gence of vertebrates and invertebrates. In the Ca2+ pump subfamily, there exist at least four distinct groups of members which differ from one another in the localiza- tion of intracellular compartment (Fagan and Saier 1994; Rudolph et al. 1989; Ghislain et al. 1990) (hereafter we will refer to this type of genes as compartmentalized isoform genes). Judging from the phylogenetic position of the branching point between human and Trypanoso- ma sarcoendoplasmic reticulum (SER) Ca2+ pumps, all the gene duplications giving rise to the four compart- mentalized isoforms are likely to have occurred during the early evolution of eukaryotes, because Trypanosoma diverged from a deep branch among protozoa, as shown by the 18SrRNA tree (e.g., Cavalier-Smith 1993). The

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Evolution of Gene Families 485

Plasma Membrane

Sarco-endoplasmic 1 &2+

W 90 ratla

100 chickan 1

100 Ranal

95 100 human 2 -4z chicken2

rat3

100 DIVSOphih9

ArhMdi3

FIG. l.-The phylogenetic tree of the eukaryotic ion pump family. Details of the branching patterns of the two clusters corresponding to the members of the Na+-K+ pump and those of SERCA, a Ca*+ pump group localized in the sarcoendoplasmic reticulum, are shown in inserts a and b, respectively. The intracellular localization of the ion pumps is also shown. The tree was inferred by the neighbor-joining method (Saitou and Nei 1987) based on the alignment of conserved regions of 308 amino acid positions, where unambiguous alignments are possible; for inserts a and b, the alignments of 983 and 812 amino acids in length were used, respectively. The number at each node indicates the probability that two lineages are joined together at the node to form a single cluster, which has been estimated by a standard bootstrap procedure with 1,000 resamplings (Felsenstein 1985). The deepest root was determined by assuming prokaryotic ion pump sequences as outgroups. Double circle represents divergence of eumetazoa and protozoa; open circle represents divergence of vertebrates and other eumetazoa; filled circle represents divergence of fishes and tetrapods; filled circle in insert b represents the inferred branch point of fishes and tetrapods, which was calculated by assuming the constant evolutionary rate of the SERCA and 350 million years for the time of divergence of amphibian and bird- mammal clade; shaded double rhombus represents gene duplication prior to the eumetazoa-protozoa separation; open double rhombus represents gene duplication prior to the vertebrates+ther eumetazoa separation; shaded rhombus represents gene duplication on invertebrate lineages; open box represents gene duplication whose divergence time is uncertain.

Na+-K+ pump subfamily of vertebrates contains at least three members which are identical to one another in ion specificity, but differ in tissue distribution (Herrera et al. 1987) (hereafter we will refer to this group of genes as tissue-specific isoform genes). In sharp contrast to the divergence pattern of compartmentalized isoforms, gene duplications giving rise to the three tissue-specific iso- forms postdate the divergence of vertebrates and arthro- pods (figure 1, insert a). A similar divergence pattern is also observed in the SER Ca2+ pump subfamily (figure 1, insert b).

To confirm the patterns of divergence by gene du- plications found in the ion pump family, similar phy- logenetic analyses were carried out for 25 different fam- ilies. The results are summarized in table 1 (details will be published elsewhere). In these analyses, the function- al difference between members in each family was dis- tinguished by ligands, ion specificities, substrates, struc- tural difference of functional domains, and difference of directly interacting molecules. The divergence patterns of members in these families are almost identical to

those found in the ion pump family: Most gene dupli- cations that give rise to different functions antedate the vertebrates-arthropods separation. Since no protein ty- rosine kinase is thought to exist in organisms other than animals, it is highly likely that all the gene duplications giving rise to different functions occurred on the animal lineage between the animals-fungi separation and the vertebrates-arthropods separation. The phylogenetic trees of the G protein (x subunit family showed that the major functional diversification of animal genes post- dates the animals-fungi separation. Also in the other families listed in table la, the major functional diver- sification is likely to have occurred within the same time interval, although no ample sequence data from fungi and plants are available at present. Because most of the members of these gene families are involved in the sig- nal transduction or cell-to-cell communications, it re- mains possible that the functional diversification is re- lated to the evolution of multicellularity.

On the other hand, the tissue-specific isoform genes that carry virtually identical functions to each other

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486 Iwabe et al.

Table 1 Classification of Gene Families Based on the Time of Di- vergence of Different Functions

a. Divergence separations

of different functions prior to vertebrates-arthropods

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

G-protein-coupled receptor (rhodopsin) family Functional difference: ligand specificity Subfamilies: opsin, adrenarine receptor (R), serotonine (5HT) R, dopamine R, acetylcholine R (mAChR), histamine R, aden- osine R, cannabinoid R, thyrotoropin-releasing hormone R, ta- chykinin peptide R, neuropeptide Y R, cholecystokinin R, pros- taglandin R, odorant R, pituitary glycoprotein hormone R G protein OL subunit (Go) family Functional difference: interaction with effecters Subfamilies: Gas, Goi, Gao, Gaz, Got, Gaq Adenylyl cyclase (AC) family Functional difference: regulation by GBy and Ca*+-calmodulin Subfamilies: AC-I, AC-II, AC-V Guanylyl cyclase (GC) family Functional difference: functional domain structure Subfamilies: receptor GC, cytoplasmic GC Phospholipase C (PLC) family Functional difference: functional domain structure Subfamilies: PLCB, PLCy, PLCS Phosphodiesterase (PDE) family Functional difference: substrate specificity, functional domain structure Subfamilies: CAMP-specific PDE, cGMP-specific PDE, Ca*+- calmodulin-dependent PDE Nicotinic acetylcholine receptor (nAChR) family Functional difference: ligand specificity Subfamilies: nAChR, serotoninR (5HT3R), GABAAR, GlyR Neurotransmitter transporter (NIT) family Functional difference: transmitter specificity Subfamilies: dopamine transporter (T), GABA T, Glycine T, serotonine (5HT) T Voltage-gated channel family Functional difference: ion specificity, domain structure Subfamilies: Na+-channel, Ca*+-channel, K+-channel Ryanodine receptor (RR) family Functional difference: second messenger Subfamilies: RR, inositol 1,4,5_triphosphate receptor (IP3R) Steroid/thyroid hormone receptor family Functional difference: ligand specificity Subfamilies: thyroid receptor (TR), retioic acid R (RAR), re- tinoid X R (RXR), peroxisome-proliferator-activated R (PPAR), chicken ovalbumin upstream promotor transcription factor (COUPTF), vitamin D R (VDR), steroid receptor Protein tyrosine kinase family Functional difference: functional domain structure Subfamilies: src, abl, CSK, syk, eph, fps, HGFR, IR, ltk, I-OS, trk, Dtrk, FGFR, ret, PDGFR, tie, torso, EGFR, JAKl Protein tyrosine phosphatase (PTPase) family Functional difference: functional domain structure Subfamilies: PTPB, PTPy, LAR, PTPlC, PTPlB Ion pump (P-type ATPase) family Functional difference: ion specificity Subfamilies: Na+/K+ pump, K+/I-I+ pump

b. Divergence of different functions prior to animals-fungi separation 1. Protein serine/threonine kinase family

Functional difference: functional domain structure Subfamilies: PKC, cdc2, CK-I, weel, MAPK, MAPKK, GSK- 3, CK-II cx, cdc5 (polo), CaMK, snfl, AK, GK, mos, MLCK, PhrK y, GRK, PKB, S6K, ActR, ru$ ZmPKl

Table 1 Continued

2.

3

4

5.

6.

7.

8.

9.

10.

11.

12.

13

Protein serine/threonine phosphatase (PPase) family Functional difference: regulatory subunit specificity Subfamilies: PPl, PP2A, PP2B (calcineurin), PPY, PPX, PPV Ion pump (P-type ATPase) family Functional difference: ion specificity Subfamilies: Ca*+ pump, H+ pump, Cu*+ pump Calcium ion pump (P-type Ca*+ ATPase) subfamily Functional difference: intracellular localization Subfamilies: PMCA, SERCA, PMRl, cta3 Heat shock protein 70 (hsp70) Functional difference: intracellular localization Subfamilies: hsp70, BiP rab family Functional difference: intracellular localization Subfamilies: rabl, rab2, rab3, rab4, rab5, rab6, rab7 ADP ribosylation factor (ARF) family Functional difference: intracellular localization Subfamilies: ARE SAR Myosin heavy chain (MHC) family Functional difference: functional domain structure Subfamilies: myosin II, dilute, myosin I (brush border), myosin IB, ninaC, 95F Dynein heavy chain family Functional difference: intracellular localization Subfamilies: cytoplasmin dynein, flagellar dynein B, S. cerev- isiae DYN 1 (nuclear segregation) Kinesin heavy chain family Functional difference: functional domain structure, intracellular localization Subfamilies: kinesin, unc-104, KIF3, KAR3, bimC, nod Tubulin family Functional difference: intracellular localization Subfamilies: (Y tubulin, B tubulin, y tubulin Actin family Functional difference: intracellular localization Subfamilies: actin, Arpl, Arp2, Arp3 rho family Functional difference: regulation of actin filaments Subfamilies: rho, rat, cdc42

Nom-In the G-protein-coupled receptor family, for example, the functional differences among the subfamilies listed in the table were tentatively determined by the difference of ligand specificities. On the basis of the phylogenetic tree of the family members inferred from the aligned sequences including all the sub- families from various eukaryotic species, mostly from vertebrates, arthropods, and fungi, the divergence times of these subfamilies with different functions were estimated. In most cases, each subfamily forms a single cluster, undergoing further gene duplications which give rise to the tissue-specific isoform genes in each lineage of vertebrates and arthropods. For tissue-specific isoform genes, see table 2.

show rather recent divergence: They diverged indepen- dently in both vertebrates and arthropods after the sep- aration of the two groups (see table 2). The divergence times of different compartmentalized isoform genes are very old, going back to dates before the animals-fungi separation. Thus the timing of gene diversification dif- fers for different gene types. It may therefore be possible to relate the genetic diversification to the organismal evolution by taking special notice of function, intracel-

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Evolution of Gene Families 487

Table 2 Comparisons of the Number of Gene Duplications and Evolutionary Rates between the First (Period I) and Latter (Period II) Periods in the Chordate Lineage after the Separa- tion from Arthropods

Subfamily n0 4 41 VIh

nAChR .............................

Opsin ............................... mAChR* ............................ FGFR* ............................. EGFR .............................. IR* ................................ CaMKII* ............................ rap ................................ CDK2 ..............................

actRII* ............................. MAPK* ............................. S6K* ............................... MAPKK ............................ ras* ................................ rap1 (Krev) ......................... Gaq”. ..............................

Gai* ............................... Gas* ............................... Na+ channel .........................

K+ channel* ......................... SERCA* ............................ Na+-K+ ATPase ...................... Ryanodine receptor* .................. IP3R*. ..............................

EF-lo* ............................. Aldolase ............................

Total ...............................

2 1

10 (3.3) 3 (0.75) 6 (2.0) 3 (0.75) 2 (0.67) 0 (0.0) 3 (1.0) 1 (0.25) 3 (1.0) 0 (0.0) 2 (0.67) 1 (0.25) 2 (0.67) 0 (0.0) 2 (0.67) 0 (0.0) 2 (0.67) 0 (0.0) 1 (0.33) 2 (0.50) 1 (0.33) 1 (0.25)

1 (0.33) 1 (0.25) 1 (0.33) 0 (0.0) 3 (1.0) 0 (0.0) 1 (0.33) 0 (0.0) 2 (0.67) 1 (0.25) 2 (0.67) 0 (0.0) 1 (0.33) 0 (0.0) 4 (1.3) 2 (0.50) 4 (1.3) 0 (0.0) 2 (0.67) 0 (0.0) 1 (0.33) 0 (0.0) 2 (0.67) 0 (0.0) 2 (0.67) 0 (0.0)

2 (0.67) 1 (0.25) 1 (0.33) 2 (0.50)

63 (21.0) 18 (4.6)

2.4 6.4 2.9 4.5 2.5 2.7

12 3.6 - 3.1 2.9 4.9 3.3 2.3 8.6 8.7 2.8 2.8 3.4 3.2 4.7 1.8 1.4 6.1

11 2.2 0.7

4.3 2 2.8

No=.-The lineage from the common ancestor of chordates and artbropods to extant vertebrates was divided into the first (period I) and latter (period II) periods by tentatively defining the divergence time of fishes and tetrapods as a boundary. IZ,, is the number of isoforms shared between arthropods and vertebrates. n, and n,, are the numbers of gene duplications occurring in periods I and II, respectively; the number of gene duplications per 100 million years is shown in parentheses, which has been calculated by assuming 700 million years for the time of divergence of chordates and arthropods (Dayhoff 1978; Margulis and Schwartz 1988), and 400 million years for the time of divergence of fishes and tetrapods (Dayhoff 1978). The average evolutionary rate vi of amino acid substitutions per 100 million years in period I and that (vu) in period II were calculated from the branch lengths of the inferred phylogenetic tree. An asterisk indicates that, because no fish data were available, the time of divergence of fishes and tetrapods was inferred from that of amphibians and mammals, by assuming 400 million years for the time of divergence of the former and 350 million years (Dayhoff 1978) for that of the latter, and by assuming the constant evolutionary rate of molecules in period II. Abbreviations: nAChR, nicotinic acetyl- choline receptor; mAChR, muscarinic acetylcholine receptor; FGFR, fibroblast growth factor receptor; EGFR, epidermal growth factor receptor; IR, insulin receptor; CaMKII, calmodulin-dependent protein kinase II; actRI1, activin receptor II; MAPK, microtubule-associated protein kinase; S6K, ribosomal protein S6 kinase (~90); MAPKK, microtubule-associated protein kinase kinase; Gaq, G protein a subunit q subtype; Gai, G protein a subunit i subtype; Gas, G protein a subunit s subtype; SERCA, sarcoplasmic/endoplasmic reticulum type Ca 2+ ATPase (calcium ion pump); IP,R, inositol 1,45tri- phosphate receptor; EF-la, elongation factor la subunit. Data on tissue distributions were taken from the following ref- erences: nAChR, Schuetze and Role (1987), Galzi et al. (1991), Sargent (1993); opsin, Nathans, Thomas, and Hogness (1986); mAChR, Peralta et al. (1987), Bonner et al. (1987, 1988); FGFR, Partanen et al. (1991); EGFR, Kraus et al. (1989); IR, Shier and Watt (1992); CaMKII, Tobimatsu, Kameshita, and Fujisawa (1988); raj Storm, Cleveland, and Rapp (1990); CDK2, Meyerson et al. (1992); actRI1, Attisano et al. (1992); MAPK, Gonzalez et al. (1992); S6K, Alcorta et al. (1989); MAPKK, Brott et al. (1993); ras, Leon, Guerrero, and Pellicer (1987); rap1 (Krev), Matsui et al. (1990), Klinz et al. (1992), Quarck et al. (1994); Gaq, Strathmann, and Simon (1990), Wilkie et al. (1991); Gai, Jones and Reed (1987); Gas,

Jones and Reed (1989); Na+ channel, George, Knittle, and Tamkun (1992); K+ channel, Baumann et al. (1988), Roberds and Tamkun (1991), Attali et al. (1992); SERCA, Burk et al. (1989); Na+-K+ ATPase, Herrera et al. (1987); ryanodine receptor, Hakamata et al. (1992); IPsR, Blonde1 et al. (1993); EF- la, DjC et al. (1990), Ann et al. (1991); aldolase, Tsutsumi et al. (1984), Mukai et al. (1986), Kukita et al. (1988).

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488 Iwabe et al.

lular localization, and tissue distribution of family mem- bers. The tissue-specific isoform genes would be useful in determining a possible link between tissue and mo- lecular evolution, and the compartmentalized isoform genes would provide useful information about the early evolution of eukaryotic cells. Also from the analysis of functional diversification in a gene family, we may ob- tain a clue for understanding the early evolution of mul- ticellular organisms in relation to the genetic diversity.

a)

100 111

Rapid Divergence of Tissue-specific Isoform Genes in the Early Evolution of Chordates

Several lines of evidence from fossil records and comparative analyses of morphological characters indi- cate that during chordate evolution a variety of tissues and organs evolved rapidly from very primitive ones within 300 million years or so before the emergence of higher fishes. From then on, the evolutionary rate mark- edly reduced in the basic structure (Romer 1959; Romer and Parsons 1977; Fingerman 1981). To know whether a relationship between evolution of tissues or organs and that of tissue-specific isoform genes exists or not, we have analyzed the degree of divergence of tissue-specific isoform genes in the early and late periods of chordate evolution based on the phylogenetic tree of these mol- ecules.

b)

Figure 2a and b shows the phylogenetic trees of opsin and nicotinic acetylcholine receptor (nAChR) sub- families, respectively. Since members of each subfamily are identical to one another in ligand-binding specificity, but differ in tissue distribution, they are considered to be tissue-specific isoforms. In these trees, the branching point (filled circle in fig. 2a and b) corresponding to the divergence of fishes and tetrapods was determined by comparing sequences from several vertebrate species and by testing the approximate rate constancy of amino acid substitutions among vertebrate lineages, by which paralogous comparisons were excluded. Tentatively as- signing the divergence time of fishes and tetrapods as a boundary, the entire chordate lineage covering from the separation (open circle) of chordates and arthropods to modern vertebrates was divided into two periods, the first period or period I (shaded region) and the latter period or period II. On the basis of the phylogenetic trees, the number of gene duplications and the rate of amino acid substitutions were estimated for each period. Similar analyses were performed for 26 different sub- families including opsin and nAChR subfamilies. The results are summarized in table 2 and figure 3.

chicken BgtBP a2

Table 2 shows interesting branching patterns char- acteristic of the tissue-specific isoforms: In 22 subfam- ilies out of the 26 analyzed here, the number of gene duplications in period I is larger than that in period II; of the 81 eene duolications in total. 63 (78%) have been

FIG. 2.-Phylogenetic trees of (a) opsins and (b) nicotinic ace- tylcholine receptors (nAChRs) from vertebrates, mollusks, and arthro- pods. The trees in a and b were inferred by the neighbor-joining meth- od (Saitou and Nei 1987), based on the alignment of sequences for conserved regions of 250 and 367 amino acid positions, respectively, where unambiguous alignments are possible. The deepest roots in a and b were determined by assuming a muscarinic acetylcholine recep- tor and a serotonin-3 receptor as outgroups, respectively. The number at each node indicates the probability that two lineages are joined to- gether at the node to form a single cluster, which has been estimated by a standard bootstrap procedure with 1,000 resamplings (Felsenstein 1985). Filled circle and open circle represent the divergence (400 mil- lion years ago) of fishes and tetrapods and that (700 million years ago) of chordates and arthropods, respectively. By tentatively assigning the divergence time of fishes and tetrapods as a boundary, the whole chor- date lineage after the separation from arthropods was divided into two periods, period I (shaded area) and period II. Filled rhombus and open rhombus represent gene duplications in period I and period II, respec- tively. Shaded rhombus represents gene duplication on arthropod lin- eages. A double rhombus in b represents gene duplication prior to the

” I I \ I chordates-arthropods divergence.

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Evolution of Gene Families 491

early evolution of metazoa; the divergence of structur- ally and functionally different members by gene dupli- cations mostly predates at least the Radiata-Bilateria separation (Suga et al., manuscript in preparation). Since no protein tyrosine kinase is found in unicellular organ- isms as far as we know and these enzymes have impor- tant implications in cell-to-cell communications (van der Geer, Hunter, and Lindberg 1994), the extensive diver- sification may be related to the evolution of multicel- lularity. If the relationship between evolution at different levels is really the case, it follows that rapid divergence of genes specific to higher vertebrates like genes in- volved in the immune system is expected to have oc- curred very recently.

Acknowledgments

We thank Profs. M. Kimura, T Ohta and J. May- nard-smith for discussion and suggestions. This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan.

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