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1 Creatine Kinases of Amphibians and Reptiles: Evolutionary and Systematic Aspects of Gene Expression Author(s): Donald G. Buth, Robert W. Murphy, Michael M. Miyamoto, Carl S. Lieb Source: Copeia, Vol. 1985, No. 2 (May 3, 1985), pp Published by: American Society of Ichthyologists and Herpetologists Stable URL: Accessed: 04/02/ :26 Your use of the JSTOR archive indicates your acceptance of JSTOR's Terms and Conditions of Use, available at. JSTOR's Terms and Conditions of Use provides, in part, that unless you have obtained prior permission, you may not download an entire issue of a journal or multiple copies of articles, and you may use content in the JSTOR archive only for your personal, non-commercial use. Please contact the publisher regarding any further use of this work. Publisher contact information may be obtained at. Each copy of any part of a JSTOR transmission must contain the same copyright notice that appears on the screen or printed page of such transmission. JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. American Society of Ichthyologists and Herpetologists is collaborating with JSTOR to digitize, preserve and extend access to Copeia.

2 Copeia, 1985(2), pp Creatine Kinases of Amphibians and Reptiles: Evolutionary and Systematic Aspects of Gene Expression DONALD G. BUTH, ROBERT W. MURPHY, MICHAEL M. MIYAMOTO AND CARL S. LIEB The creatine kinases of amphibians and reptiles are evaluated in terms of their genetic variability beyond that of allelic differentiation. One character, the number of loci controlling creatine kinase, is invariant; all investigated species express two loci (Ck-A and Ck-C). Amphibians and reptiles vary in their tissue specificity of creatine kinase expression and in the ability to form interlocus and intralocus heterodimers. The restriction of expression to fewer tissues and the loss of the ability to form heterodimers are interpreted to be derived states. The inferred evolutionary polarity in these variable characters allows their application in phyletic studies of amphibians and reptiles. THE tissue and developmental expressions of multilocus isozymes have considerable potential for application in phylogenetic studies (Whitt, 1981; Buth, 1984). These characteristics of genome structure and regulation have been largely overlooked with greater emphasis given to allelic differences and inferences about genetic distances based on computed allele frequencies. Nevertheless, gene expression data have been used as characters in a few systematic studies especially using the lactate dehydrogenase system, e.g., tissue specificity (Shaklee et al., 1973; Shaklee and Whitt, 1981) and restriction of heterotetramer assembly (Gorman, 1971; Buth et al., 1980; Murphy, 1984). Miyamoto (1983a) discussed the application of the restriction of purine-nucleoside phosphorylase heterotrimer assembly in systematic studies. General patterns of gene expression and genic evolution of the creatine kinase enzyme system (CK: EC ) have been partially elucidated for fishes (Eppenberger et al., 1967; Ferris and Whitt, 1978a; Fisher and Whitt, 1978, 1979; Fisher et al., 1980; Whitt, 1981). In fishes, creatine kinase varies in the number of structural genes controlling the expression of the enzyme (Scholl and Eppenberger, 1971; Fisher and Whitt, 1978, 1979), the tissue specificity of gene expression (Eppenberger et al., 1971; Perriard et al., 1972; Fisher and Whitt, 1978, 1979), and the ability to form interlocus and intralocus heterodimers (Ferris and Whitt, 1978a; Buth, 1979a; Utter et al., 1979). Furthermore, the evolutionary polarity of the character states has been inferred in these ichthyological studies providing the potential for application in unknown situations. Only recently have creatine kinases been routinely screened in multilocus electrophoresis studies of amphibians and reptiles. Thus, the limited amount of comparative electrophoretic data has restricted herpetological analyses to allelic differentiation, i.e., allozymes and their frequencies, as the sole measure of creatine kinase variability. In the present study, we examine the gene expression attributes of the creatine kinases of amphibians and reptiles and evaluate these characteristics in terms of their utility in phylogenetic studies. MATERIALS AND METHODS Comparative data were obtained through a summary of our studies in the laboratory of G. C. Gorman at UCLA and a literature survey. Deposition of our voucher specimens and exposition of data in greater detail are addressed in treatments of specific taxonomic groups, e.g., Lieb (1981) and Miyamoto (1982). Our investigations of creatine kinases of amphibians and reptiles employed horizontal starch gel electrophoresis with 12.5% Electrostarch gels. The optimal electrophoretic buffer system varies from group to group; the Poulik and Triscitrate II buffers of Selander et al. (1971) and the 0.41 M sodium citrate buffer, ph 8.0, of Brewer (1970) were extensively used. Specific staining procedures were modified from Shaw and Prasad (1970) and have been described elsewhere (Buth and Murphy, 1980). Creatine kinase products were also resolved with the protein staining technique of Massaro and Markert (1968) as modified by Ferris and Whitt (1978a) and Buth (1979a).? 1985 by the American Society of Ichthyologists and Herpetologists

3 280 COPEIA, 1985, NO. 2 [+] /CK-C; a CK-C, 4 fl^^. CK-A'C' , , , ,1 4 rye IrE R JN STO.MACH LIYER HE-RT MU SCLF.,3", c.-a'e -..i \C?'K-AA Fig. 1. Tissue specificity of creatine kinase expression in toads of the genus Bufo: B. cognatus (specimens 1 and 2), B. punctatus (specimen 3), B. boreas x B. microscaphus hybrid (specimen 4) and B. boreas (specimen 5). Both the Ck-A and Ck-C loci are diallelic ("s" and "f" alleles are designated on the basis of the "slow" and "fast" anodal electrophoretic migration of their products). Both interlocus and intralocus heterodimers are formed with the subunit composition of all electromorphs indicated. Adenylate kinase (Ak-A locus) activity is also resolved using the staining procedure of Buth and Murphy (1980). Ak-A activity is limited to muscle tissue in Bufo. RESULTS AND DISCUSSION Creatine kinase loci.-fisher and Whitt (1978) examined nine amphibian and reptilian species and established that, based on immunochemical properties, the two creatine kinase loci expressed in these groups correspond to the Ck-A and Ck-C loci of fishes and mammals. We found that, at most, two CK loci are expressed in diploid amphibians and reptiles. Although Gartside et al. (1977) reported three CK loci in Alligator mississippiensis, Ck-1 and Ck-2 from heart and Ck-3 from skeletal muscle, these data have been reinterpreted; the "Ck-2" product expressed in heart is probably the Ck-AC interlocus heterodimeric product (L. D. Densmore, pers. comm.). A more recent survey of ten tissues for creatine kinase activity in A. mississippiensis revealed the products of two loci; Ck-2 (=Ck-A) predominating in skeletal muscle and Ck-1 (=Ck-C) predominating in kidney and heart (Dessauer and Densmore, 1983). Tissue expression-the pattern of tissue expression of creatine kinase activity shows considerable variation in amphibians and reptiles as is seen in the toad genus Bufo (Fig. 1). In most of the species of Bufo that we have examined, a relatively restricted pattern of tissue expression is observed. Strong Ck-A activity is found in skeletal muscle, whereas Ck-C is not expressed in this tissue. Ck-C activity is substantial in stomach but Ck-A activity is lacking. The Ck-A and K-A; Ck-C loci are both expressed in eye and heart but not to the degree as seen in muscle and stomach, respectively. Creatine kinase activity is negligible, at least as far as being scorable, in brain and liver tissues. These patterns of expression generally correspond to those of Bufo americanus (Fisher and Whitt, 1978) and Bufo marinus (Fisher and Whitt, 1978, 1979). However, minor differences in tissue expression can be noted among some of the species of Bufo in this study and those of Fisher and Whitt (1978, 1979). For example, creatine kinase products are reported from brain in B. americanus and B. marinus, but we failed to resolve activity in this tissue in several other species of Bufo (Fig. 1). We have examined creatine kinase expression in Bufo marinus and have resolved products of the Ck-A locus in brain tissue as reported by Fisher and Whitt (1979) and shown in Fig. 5 of the latter study. Thus, these differences among species are probably due to tissue specific regulation of expression and, in Bufo, may be of systematic value at the subgeneric level. Contrasting this relatively restricted pattern of CK expression in Bufo, is the more widespread pattern of tissue expression observed in other taxa such as the Pacific rattlesnake, Crotalus viridis. In C. viridis, strong Ck-A activity is observed in heart, stomach and skeletal muscle, whereas strong Ck-C activity is observed in brain, kidney, stomach and intestine (Murphy and Crabtree, 1985) and tissue specificity of expression is known to vary between the embryonic stages and adults (Crabtree and Murphy, 1984). Dessauer and Densmore (1983) reported an even greater array of tissue expression in Alligator mississippiensis; products of both Ck-A and Ck-C loci were found in almost all of ten tissues examined. Fisher and Whitt (1978) concluded that am- phibians and reptiles do not express the high degree of tissue specificity usually found in the advanced fishes. We agree with their statement and add that a diversity of tissue expression is observed, with some amphibians and reptiles exhibiting relatively widespread patterns (several tissues) while others are relatively restricted (few tissues). In general, strong Ck-A activity is always observed in skeletal muscle, strong Ck-C activity is always observed in stomach, and the Ck-C locus is rarely expressed in skeletal muscle (e.g., Alligator mississippiensis as reported by Dessauer and Densmore, 1983). Interlocus heterodimer formation.-in fishes, the

4 BUTH ET AL.-HERPETOLOGICAL CREATINE KINASES 281 [+] _M _1-L Origin - [_ KIDNEY HEART -CK-C2 - CK-AC CK-A2 Fig. 2. Zymogram of creatine kinase expression in seasnakes of the genus Laticauda: L. laticauda (specimens 1 and 4), L. colubrina (specimens 2 and 5) and L. semifasciata (specimens 3 and 6). Both the Ck-A and Ck-C loci are monomorphic with interlocus heterodimers formed in heart tissue extracts. loss of ability to form interlocus heteropolymers in multilocus multimeric enzyme systems has been correlated with phyletic advancement in lactate dehydrogenase (Toledo and Ribeiro, 1978) and creatine kinase (Ferris and Whitt, 1978a). The creatine kinases of amphibians and reptiles vary in their ability to form the heterodimer between the products of the Ck-A and Ck-C loci. Seasnakes of the genus Laticauda form the Ck-AC heterodimer in tissues (e.g., heart) that express both Ck-A and Ck-C in this group (Fig. 2). Many toads of the genus Bufo (Fig. 1) can form the Ck-AC heterodimer in tissues that express sufficient amounts of Ck-A and Ck-C gene products. However, only the Ck-A2 and Ck-C2 homodimers are expressed in eye tissue of Bufo marinus (Fisher and Whitt, 1979). The absence of the anticipated interlocus heterodimer in B. marinus may be an artifact of these gene products being produced in separate tissue types, e.g., Ck-C in the retina and Ck-A in extraocular muscles. A more unequivocal situation exists in the prairie rattlesnake, Crotalus v. viridis where both Ck-A and C products are produced in stomach tissue yet no Ck-AC interlocus heterodimer is formed (Murphy and Crabtree, 1985). Failure to form heteropolymers via the random combination of subunits is considered an evolutionary derivation, relative to the primitive condition in which no restriction is placed on the combination of subunits. Unfortunately, few taxa have had multiple tissues screened for CK activity to allow evaluations of Ck-AC formation thus making comparative data scarce. Additional taxa need to be examined in this fashion to ascertain the utility of this character in phyletic evaluations. Intralocus heterodimer formation.-the formation of heteropolymers from different allelic components encoded by a single locus is directly related to heterozygosity at the locus controlling expression of the multimeric enzyme in question. In fishes, Ck-A heterozygosity is relatively uncommon (Ferris and Whitt, 1978a). We found that Ck-A heterozygosity is similarly uncommon in amphibians and reptiles. All known heterozygous taxa, including several interspecific hybrids, are listed in Table 1. Our examination of certain groups in greater depth (Table 2) supports our contention that intraspecific variability (at least at the Ck-A locus) is very low. In all but one species, an intralocus heterodimer is formed in individuals heterozygous at the Ck-A locus. Our examples include an interspecific amphibian hybrid (Fig. 1) and a reptilian species (Fig. 3). Two of five specimens of Petrosaurus mearnsi from central Baja California del Norte expressed a heterozygous condition (Table 1) involving the formation of only two forms (presumptive homodimers) of creatine kinase. The restriction of the Ck-A intralocus heterodimer in P. mearnsi is comparable to such restriction in the teleost fishes (Ferris and Whitt, 1978a; Buth, 1979a). This interpretation is made with some reservation. Presumptive Ck-A products were resolved using a general protein stain (Buth, 1979a) and not a specific histochemical stain. Although we believe these proteins are Ck-A products on the basis of their predominance in skeletal muscle tissue and their relative electrophoretic mobility, fresh samples of P. mearnsi from this particular locality are needed to confirm the identity of these proteins with a specific stain for CK. Statements regarding relative levels of polymorphism at the Ck-C locus are premature because so few amphibians and reptiles have been examined for these gene products. At present, there are no known Ck-C heterozygotes. CONCLUSIONS The evolutionary patterns of creatine kinase expression in amphibians and reptiles are summarized in Table 3. As a group, diploid amphibians and reptiles do not vary in the number of creatine kinase loci expressed (Ck-A and Ck- C). This condition is regarded as primitive, the derived state (more than two CK loci) appearing only in certain groups of bony fishes. In expressing only the plesiomorphic state, this char-

5 282 COPEIA, 1985, NO. 2 TABLE 1. OBSERVED CREATINE KINASE HETEROZYGOSITY AT THE CK-A LOCUS IN AMPHIBIANS AND REPTILES. Number of Total number Taxon heterozygous specimens of specimens examined Reference Class Amphibia Bombina bombina x B. variegata?? Szymura and Farana (1978) Bufo boreas x B. microscaphus 1 1 Present study Eleutherodactylus escoces 1 9 Miyamoto (1982) Eleutherodactylus rugulosus 1 3 Miyamoto (1982) Rana esculentaa Graff et al. (1977) Rana lessonaeb 7 16 Uzzell and Berger (1975) Rana lessonaeb Graf et al. (1977) Rana ridibunda x R. perezi 6 6 Graf et al. (1977) Rana sp.?? Ferris and Whitt (1978a) Class Reptilia Anolis nebuloides 1 4 Lieb (1981) Anolis uniformis 2 3 Lieb (1981) Crotalus scutulatus x C. viridis 1 1 Present study Lacerta armeniacac?? Uzzell and Darevsky (1975) Lacerta unisexualisc?? Uzzell and Darevsky (1975) Petrosaurus mearnsid 2 5 Present study I Hybridogenetic mode of reproduction. b Hybridogenetic source for Rana esculenta. I Unisexual mode of reproduction. d Based on protein stained products rather than specific CK stain. acter is of no phyletic utility in evolutionary studies of amphibians and reptiles. Amphibians and reptiles do vary in the patterns of tissue-specific expression of creatine kinase and in the ability to form interlocus and intralocus heterodimers. The evolutionary polarity shown in Table 3 may be applied in unknown situations to address specific phyletic [+] oz j t~q'. ' e'. y'- >v V (- TABLE 2. GENETIC VARIABILITY AT THE CK-A Locus IN THE AMPHIBIAN GENUS Eleutherodactylus (MIYAMOTO, 1982,1983B) AND THE REPTILIAN GENERA Anolis (GORMAN ET AL., 1980, 1983; LIEB, 1981; LIEB ET AL., 1983), Crotalus (CRABTREE AND MURPHY, 1984, AND UNPUBLISHED DATA), Eumeces (MURPHY ET AL., 1983), AND Sphaerodactylus (MURPHY ET AL., 1984). Number of Number hetero- Number Number of of poly- ozygous of species specimens morphic individ- Genus examined examined species uals Eleutherodactylus Anolis 40 1,017' 2 3 Crotalus b Eumeces Sphaerodactylus *Includes 371 specimens of Anolis cristatellus (Gorman et al., 1980), all of which are monomorphic. b An interspecific hybrid (Table 1). Origin-I [-] Fig. 3. Zymogram of creatine kinase expression as resolved from a tissue mixture (heart-lung-muscle) in iguanid lizards of the genus Anolis. The products of the Ck-A locus form intralocus heterodimers in the heterozygous condition as shown in A. uniformis (specimens 3 and 7). The Ck-C locus is not expressed in these tissues of these species.

6 BUTH ET AL.-HERPETOLOGICAL CREATINE KINASES 283 TABLE 3. EVOLUTIONARY PATTERNS OF CREATINE KINASE GENE EXPRESSION IN AMPHIBIANS AND REP- TILES. Character state Character Ancestral Derived 1. Number of loci [Invariant; only Ck-A and Ck-C expressed] 2. Tissue specificity Widespread Restricted 3. Interlocus hetero- Present Absent dimer formation 4. Intralocus hetero- Present Absenta dimer formation The derived condition has been observed only in Petrosa urus inearnsi. problems. However, these inferences must be applied with caution. In these cases, the derived states (restriction of CK expression to certain tissues, lack of the ability to form lieterodimers) are loss events. Inappropriate synapomorphic status may be assigned in situations where the derived state was achieved through independent loss events. Such parallelism might be minimized if these characters are applied only to treatments of lower taxonomic categories. However, application in higher categories is certainly possible in some situations. Comparable problems exist in the application of gene duplication data in phyletic studies (Ferris and Whitt, 1978b; Buth, 1979b). ACKNOWLEDGMENTS This study was supported by the National Science Foundation (DEB to G. C. Gorman), the UCLA Biomedical Research Support Grant and the UCLA Committee on Research (UR 3674 to D. G. Buth), the Theodore Roosevelt Memorial Fund, the Regents of the University of California Research and Travel Grant and the University of California Patent Fund Research Grant (to R. W. Murphy) and the Allan Hancock Foundation and Sigma Xi (Grant- In-Aid of Research to M. M. Miyamoto). We are indebted to G. S. Whitt whose instruction and assistance made this research possible. We also thank T. L. Vance for laboratory assistance and S. D. Ferris andj. B. Shaklee for comments on an earlier draft of the manuscript. LITERATURE CITED BREWER, G.J An introduction to isozyme techniques. Academic Press, New York. BUTH, D. G. 1979a. Creatine kinase variability in Moxostoma macrolepidotum (Cypriniformes; Catostomidae). Copeia 1979: b. Duplicate gene expression in tetraploid fishes of the tribe Moxostomatini (Cypriniformes, Catostomidae). Comp. Biochem. Physiol. 63B: The application of electrophoretic data in systematic studies. Ann. Rev. Ecol. Syst. 15: ,AND R. W. MURPHY Use of nicotinamide adenine dinucleotide (NAD)-dependent glucose-6-phosphate dehydrogenase in enzyme staining procedures. Stain Technol. 55: , B. M. BURR ANDJ. R. SCHENCK Electrophoretic evidence for relationships and differentiation among members of the percid subgenus Microperca. Biochem. Syst. Ecol. 8: CRABTREE, C. B., AND R. W. MURPHY Analysis of maternal-offspring allozymes in Crotalus viridis. J. Herpetol. 18: DESSAUER, H. C., AND L. D. DENSMORE Biochemical genetics, p In: Alligator metabolism studies on chemical reactions in vivo. R. A. Coulson and T. Hernandez (eds.). Pergamon Press, Oxford. EPPENBERGER, M. E., H. M. EPPENBERGER AND N. O. KAPLAN Evolution of creatine kinase. Nature 214: ,A. SCHOLL AND H. URSPRUNG Tissue specific isoenzyme patterns of creatine kinase ( ) in trout. FEBS Letters 14: FERRIS, S. D., AND G. S. WHITT. 1978a. Genetic and molecular analysis of non-random dimer assembly of the creatine kinase isozymes of fishes. Biochem. Genet. 16: , AND. 1978b. Phylogeny of tetraploid catostomid fishes based on the loss of duplicate gene expression. Syst. Zool. 27: FISHER, S. E., AND G. S. WHITT Evolution of isozyme loci and their differential tissue expression. Creatine kinase as a model system. J. Mol. Evol. 12: , AND Evolution of the creatine kinase isozyme system in the primitive vertebrates. Occ. Pap. Cal. Acad. Sci. 134: ,J. B. SHAKLEE, S. D. FERRIS AND G. S. WHITT Evolution of five multilocus isozyme systems in the chordates. Genetica 52/53: GARTSIDE, D. F., H. C. DESSAUER AND T. JOANEN Genic homozygosity in an ancient reptile (Alligator mississippiensis). Biochem. Genet. 15: GORMAN, G. C Evolutionary genetics of island lizard populations. Year Book Amer. Phil. Soc. p , D. G. BUTH, M. SOULE AND S. Y. YANG The relationships of the Anolis cristatellus species group: Electrophoretic analysis. J. Herpetol. 14: AND The relationships of the Puerto Rican Anolis: Electropho-

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