Biochemical Systematics and Ecology 27 (1999) 201—211 Superoxide dismutase expression in the lizard genus Anolis: systematic significance of a silenced gene Carl S. Lieb!, Donald G. Buth",*, Michael M. Miyamoto# ! Department of Biological Science, University of Texas at El Paso, El Paso, TX 79968-0519, U.S.A. " Department of Biology, University of California (UCLA), Los Angeles, CA 90095-1606, U.S.A. # Department of Zoology, University of Florida, Gainesville, FL 32611-2009, U.S.A. Received 16 May 1996; accepted 2 April 1997 Abstract Data on the expression of a duplicated superoxide dismutase locus are presented for 41 species in the lizard genus Anolis. Expression of a single locus in these forms has resulted from several loss (silencing) events, and is regarded as a derived condition from the primitive duplicated state. There is apparent consistency in the presence or loss of the duplication within several groups known to be related based on morphological or other biochemical evidence. However, it is demonstrated here that the silencing of the gene has occurred independently in several infrageneric lineages. ( 1999 Elsevier Science Ltd. All rights reserved. Keywords: Anolis; Reptiles; Isozymes; Superoxide dismutase; Gene duplication 1. Introduction Superoxide dismutase (SOD; EC 1.15.1.1), formerly known as indophenol oxidase (IPO) or tetrazolium oxidase (TO) (Harris and Hopkinson, 1976; International Union of Biochemistry and Molecular Biology, 1992), catalyzes the reaction O~ 2 #O~ 2 #2H`PH2O2#O2 acting as a defense against superoxide radical toxicity in respiring cells (Fridovich, 1975). This enzyme is known to be under multilocus control in vertebrates (Harris and Hopkinson (1976), and references therein; Healy and Mulcahy (1979)). The low cost * Corresponding author. 0305-1978/99/$ — See front matter ( 1999 Elsevier Science Ltd. All rights reserved. PII: S0 30 5 - 19 7 8( 9 7 )00 0 38 -0 202 C. S. Lieb et al./Biochemical Systematics and Ecology 27 (1999) 201–211 and ease of resolution has made SOD a popular enzyme among arrays commonly examined in electrophoretic studies. In their examination of several multilocus enzyme systems in the chordates, Fisher et al. (1980:74) included SOD as a ‘‘control’’ as this enzyme is ‘‘generally encoded by single loci in diploid species of advanced fishes’’. However, Healy and Mulcahy (1979) reported two forms of superoxide dismutase in the diploid northern pike, Esox lucius. One of these forms was identified as a tetrameric manganoprotein most active in mitochondria (‘‘m-SOD’’); the other form was a dimeric cuprozinic protein that predominates in the cytoplasm (‘‘s-SOD’’). Based on their respective structures and intracellular locations of their products, these loci are probably orthologous to those listed as SOD and SOD , respectively, in humans (Harris and Hopkinson, 1976). B A Herein we designate these loci as mSod-A and sSod-A, respectively; our locus nomenclature modified from Buth (1983). It should be noted that Fisher et al. (1980) were referring to the cytosolic, or supernatant, form of SOD ("sSod-A) which, for most species they examined, expressed only a single zone of activity. However, ‘‘in some cases two or three bands were seen for [SOD]. These could be interpreted as subbands (conformational isozymes) or that the individuals were heterozygous at one locus, or that certain species had acquired additional gene loci. No unequivocal tandem gene duplications were noted for the2 SOD [locus]’’ (Fisher et al., 1980:80). Polyploidy can also yield duplicated genes (Ohno, 1970) and has played an important role in the evolution of many lower vertebrates (Lewis, 1980; Buth, 1983). Duplicated sSod-A loci have been reported in several tetraploid fishes (Ferris and Whitt, 1977; Buth, 1979). These duplicated SOD genes of polyploid origin, given that they are cytosolic in distribution and dimeric in quaternary structure, can interact to form an interlocus heterodimer (Fig. 3 of Buth, 1980). Similarly, all homozygous individuals possessing two homozygous sSod-A loci should show the three-banded pattern of expression comparable to that of a one-locus, heterozygous condition of a single specimen (unless the interlocus heterodimer is lacking due to posttranslational modification or instability). Additional isozyme bands are possible if either or both duplicated sSod-A loci are heterozygous. In contrast to the condition in diploid fishes (Fisher et al., 1980), multiple SOD loci have been commonly reported in amphibians and reptiles (Adest, 1977; Gorman et al., 1977; Kalezic and Hedgecock, 1980; Murphy and Ottley, 1980; Daugherty et al., 1981; Nevo, 1981; Sites et al., 1981; Dessauer and Densmore, 1983; Murphy et al., 1983; Wright and Richards, 1983; Wright et al., 1983; Crabtree and Murphy, 1984; Murphy and Crabtree, 1985). These loci have not often been specifically identified as to mitochondrial or cytosolic origin. Nevertheless, there is reason to believe that the products of some of these SOD loci are present in the same intracellular region; a single interlocus heterodimer of intermediate electrophoretic mobility has usually been observed as in tetraploid fishes (Buth, 1980). The nature of the origin of the SOD duplication in amphibians and reptiles is unknown, although indirect evidence suggests that it may be of tandem origin. Additional duplications in other enzyme systems have not been observed and the retention of several duplications would be expected if polyploidy occurred in a common ancestor (see also Murphy and Crabtree (1985)). C. S. Lieb et al./Biochemical Systematics and Ecology 27 (1999) 201–211 203 In the species-rich lizard genus Anolis (Iguanidae), some species have been reported to express two presumptive sSod-A loci (Yang et al., 1974; Buth et al., 1980; Lieb et al., 1983; Campbell et al., 1989), whereas others have been noted to express a single sSod-A locus (Gorman et al., 1983; Campbell et al., 1989). If the duplicated condition is primitive for reptiles, the loss of expression of one sSod-A locus can be interpreted as a derived condition. This loss event can be a systematically informative character (Ferris and Whitt, 1978; Buth, 1979, 1984) because loss of duplicate sSod-A expression in a common ancestor constitutes a synapomorphy linking daughter species. Obviously, this situation can be confounded by homoplastic expression in the form of: (1) independent loss events, and (2) reversals to the duplicated state, although controversy surrounds the likelihood of the latter phenomenon (Ferris and Whitt, 1978; Buth, 1979, 1982). We examined the distribution of the original sSod-A duplication and its subsequent diploidization in Anolis in the context of current classification of evaluate the systematic potential of this character and the distribution of the derived state in relation to the current taxonomy of the genus. Our data are drawn from the literature and our own studies and are neither random nor taxonomically extensive; certain geographic regions and lineages have been emphasized more than others. Nevertheless, these data are informative in identifying: (1) certain lineages that share a common loss of duplicate sSod-A expression among all members examined (a synapomorphy under a parsimony criterion) or (2) less closely related groups that appear to have lost duplicate sSod-A expression independently. To make these interpretations, we must assume that: (1) duplication of sSOD is the ancestral state, (2) that reduplications following gene silencing are rare, if at all possible, (3) heteropolymer formation is due to interaction within the same subcellular compartment (e.g. cytosol for sSod-A) and possible only between subunits of similar structure (e.g. dimers forming heterodimers), and (4) all of our species of Anolis practice sexual reproduction, i.e. none are parthenogens capable of expressing a ‘‘fixed heterozygote’’ pattern. 2. Materials and methods Specimens were sacrified by freezing. Liver tissue was dissected from each specimen, mixed with an approximately equal volume of 0.1 M Tris-HCl, pH 8.0, mechanically homogenized, and centrifuged at 10 000]g for 15—30 min at 4°C. Supernatant fractions were subjected to horizontal starch gel electrophoresis using 12.5% Electrostarch gels (lot d307; Electrostarch Co., Madison, WI 53701) or 12.5% gels comprised of a mixture of 80% Electrostarch (lot d392) and 20% Connaught starch (Fisher Scientific Company). Multiple buffer systems were used for electrophoretic separation including the Tris-citrate II, Tris-HCl, phosphate-citrate, and ‘‘Poulik’’ systems (Selander et al., 1971). SOD activity was resolved using a staining solution (0.2 M Tris-HCl, pH 9.0) employing phenazine methosulphate and nitroblue tetrazolium as described by Johnson et al. (1970). Voucher specimens were placed in the collections of the Natural History Museum of Los Angeles County (LACM), the Museum of Vertebrate Zoology (MVZ) and 204 C. S. Lieb et al./Biochemical Systematics and Ecology 27 (1999) 201–211 the University of Southern California (USC) holdings now at the University of Miami. The criteria used for scoring of duplicate or single sSod-A gene expression were those of Ferris and Whitt (1978). Presence of a single SOD electromorph was interpreted as the product of a single sSod-A locus although, in theory, products of duplicated loci could have been superimposed with an identical electrophoretic mobility. The methods used to diagnose the latter situation were: (1) departures from an expected 1 : 2 : 1 ratio of staining intensity for presumptive heterozygous individuals, (2) resolution of three allelic products (thus two loci) in a single specimen, and, at times, (3) the use of multiple buffer systems to enhance the possibility of separating different products (Aquadro and Avise, 1983). Ferris and Whitt (1978) noted the reduced likelihood of superimposed products covarying among many species with each species fixed for a different allele. Because sSod-A is often polyallelic among some species of Anolis (e.g. Gorman et al., 1983), the covarying criterion was especially relevant. The interpretation that two loci control sSod-A was primarily based on the observation of multiple SOD electromorphs expressed in every individual of a particular species. These three-banded patterns (two homodimers and one heterodimer) could also be due to a diallelic polymorphism yielding heterozygous conditions at a single locus. This possibility was examined using the sample size criterion of Ferris and Whitt (1978). The probability of heterozygosity in a two-allele model is maximized under Hardy—Weinberg expectations when the frequencies of both alleles equal 0.5. The probability that every individual of a particular sample would display the same two-allele heterozygous condition can be computed as P"0.5N where N is the sample size. A significance level of P(0.05 (P)0.03125) is obtained with a sample of five or more individuals. Determination of single-locus control is not as dependent on sample size and can be based on the observation of a single sSod-A electromorph in a single specimen. 3. Results and discussion Differences in clarity of SOD resolution were related to buffer conditions. Boratebased buffers (‘‘Poulik’’ and Tris-HCl) were optimal for resolving the duplicated sSod-A condition, which yielded a large number of bands. However, the Tris-citrate II buffer yielded greater clarity of resolution for the diploidized, single locus condition. The phosphate-citrate buffer yielded intermediate levels of resolution. Therefore, resolution of the number of sSod-A loci expressed was optimal when both borate and citrate buffers were used. In all instances, the appearance of subbands slightly obscured resolution; each buffer allowed formation of an anodally expressed subband for each zone of SOD activity. When duplicate sSod-A loci were expressed, an interlocus heterodimer was almost always formed; exceptions include A. chlorocyanus, A. equestris, and three members of the schiedii species group. The number of loci controlling sSod-A expression in selected species of Anolis is presented in Table 1. C. S. Lieb et al./Biochemical Systematics and Ecology 27 (1999) 201–211 205 Table 1 Number of loci controlling cytosolic (supernatant) superoxide dismutase sSOD-A expression in selected species of the genus Anolis. Classification modified from Etheridge (1960), Williams (1976a, b, 1989), Gorman et al. (1980, 1983), Lieb (1981, 1997), and Savage and Guyer (1989) Species Alpha section punctatus subsection cybotes series cybotes species group A. cybotes latifrons series frenatus species group A. frenatus punctatus series punctatus species group A. punctatus cristatellus series cristatellus subseries acutus species group A. evermanni A. stratulus A. distichus cristatellus species group gundlachi subgroup A. gundlachi A. krugi A. poncensis A. pulchellus cristatellus subgroup A. cooki A. cristatellus carolinensis subsection carolinensis series chlorocyanus species group A. chlorocyanus carolinensis species group A. carolinensis A. porcatus equestris species group A. equestris Beta section auratus series chrysolepis species group A. nitens crassulus species group A. crasssulus humilis species group A. humilis A. uniformis Sample size Number of SOD Reference! loci expressed Native range 1 2" A Hispaniola 1 1 A South America 1 2" A South America 3 4 8 1 1 1 B B A Puerto Rico Puerto Rico Hispaniola, Bahamas 4 4 2 7 1 1 1 1 B B B B Puerto Puerto Puerto Puerto 5 15 1 1 B A, B Puerto Rico Puerto Rico 6 1# A Hispaniola 17 3 2 2" A, B, C C SE U.S.A. Cuba 6 1# A Cuba 3 2" A South America 1 1 A Mexico, South America 1 3 1 1 A A Central America Mexico, Central America Rico Rico Rico Rico 206 C. S. Lieb et al./Biochemical Systematics and Ecology 27 (1999) 201–211 Table 1—continued Species sericeus species group A. sericeus schiedii species group A. cobanensis A. milleri A. naufragus A. polyrhachis gadovi species group A. dunni A. gadovi A. liogaster A. omiltemanus A. taylori nebulosus species group A. nebuloides A. nebulosus A. guercorum fuscoauratus series fuscoauratus species group A. fuscoauratus A. limifrons lionotus species group A. oxylophus grahami series grahami species group A. grahami petersi series biporcatus species group A. biporcatus A. macrinii sagrei series sagrei species group A. sagrei status incertus A. capito Sample size Number of SOD Reference! loci expressed Native range 1 2" A Central America 8 6 2 2 1 2# 2"# 2"# D D D D Guatamala Mexico Mexico Mexico 2 16 4 6 2 1 1 1 1 1 A A, B A A A Mexico Mexico Mexico Mexico Mexico 8 14 5 2 2 2 A A A Mexico Mexico Mexico 2 3 2" 2" A A South America Central America 1 1 A Costa Rica 4 2" B Jamaica 2 1 2" 1 A A Central America Mexico 27 2 A, E Cuba 2 1 A Central America ! A"Present study; B"Groman et al. (1983); C"Buth et al. (1980); D"Campbell et al. (1989); E"Lieb et al. (1983). " Sample size inadequate for definitive two-locus interpretation. # Problematic expression; see text. Although there are some interpretive difficulties raised by limited samples for some species, the data strongly suggest that the silencing (loss) of one duplicated sSod-A gene may be consistent among closely related taxa. If our single specimens of A. cybotes and A. punctatus are really heterozygotes at a single sSOD locus, the entire punctatus subsection of alpha anoles expressed a derived condition that may have C. S. Lieb et al./Biochemical Systematics and Ecology 27 (1999) 201–211 207 Fig. 1. Superoxide dismutase expression from liver extracts of selected species of Anolis as resolved with tris-HCl buffer. The products of two SOD loci are expressed in A. carolinensis (lane 1), A. porcatus (lanes 2 and 3), and A. biporcatus (lane 5). The product of a single SOD locus is expressed in A. gadovi (lane 4), A. capito (lane 6), and A. uniformis (lane 7). been the result of a single gene silencing event. The classification and phylogeny of the punctatus subsection anoles proposed by Gorman et al. (1980) from karyological and serological (albumin immunological) evidence indicated that the cybotoid anoles form a distinct lineage ancestral to the derived cristatellus series. Although confirmation through additional samples of cybotoid anoles is necessary, interpreting the preliminary data for A. cybotes and A. punctatus as retained sSOD duplications would be consistent with the phylogeny of Gorman et al. (1980); only one additional gene silencing, in A. frenatus, would have to be hypothesized. In the carolinensis subsection, the derived condition was observed in two of three species groups sampled (all carolinensis series). The significance of this distribution is unclear, particularly since the relationships within the carolinensis subsection and series taxa are poorly understood (see Wyles and Gorman, 1980). Furthermore, our scoring of the ‘‘single-locus’’ patterns of expression within the carolinensis subsection may be in error. Our initial interpretation of expression in A. chlorocyanus and A. equestris was that of a single gene product together with a single anodal subband. Hillis (pers. commun.) has provided an SOD zymogram for A. milleri, A. naufragus, and A. polyrhachis that showed a somewhat comparable pattern except that the secondary band is cathodal relative to the zone of greatest activity. The latter pattern was interpreted as that of products of two sSOD loci with a restriction of heterodimer formation (Campbell et al., 1989). Alternatively, whereas duplicate sSOD loci in other anoles are expressed nearly equivalently (Fig. 1), it may be that the ‘‘two-banded’’ pattern is the result of a gross asymmetry of gene expression. The two bands may represent the homodimeric product of one locus and the interlocus heterodimer; the homodimeric product of the second locus may be in insufficient quantity to form a visible third band. Thus, the entire carolinensis subsection may be expressing two sSOD loci, or there may be independent silencing events in several of the species groups. In summary for the alpha anoles, the pattern of sSOD expression could be the result of a single silencing event in the punctatus subsection, or as many as three or more such events involving A. frenatus, the cristatellus subseries, and within the carolinensis series. 208 C. S. Lieb et al./Biochemical Systematics and Ecology 27 (1999) 201–211 Of the Beta section anoles surveyed, the derived condition was not observed in either of the two Caribbean series (sagrei and grahami). Among the three series of mainland beta anoles, however, there are several instances where silencing events have occurred. Our interpretation of sSOD expression among the beta anoles is limited by the many instances in which the sample size was insufficient for a definitive two-locus assignment. In the petersi series, an osteologically heterogeneous and perhaps ancestral assemblage (sensu Etheridge, 1960), A. capito (when included) and A. macrinii evince loss of a second SOD locus, whereas A. biporcatus may retain the ancestral condition. However, the association of A. capito with the petersi series is somewhat questionable (Gorman, 1973), and the placement of A. macrinii within this series is provisional (Lieb, 1981). Heterogeneity in expression in the fuscoauratus series is also compromised by the sample size limitation for species possibly displaying a derived condition. Representatives of several species groups of auratus series anoles were examined. Of these, the humilis and gadovi species groups show the loss of the duplicated SOD locus, whereas the nebulosus species group and possibly many species in the schiedii group retain it. Small sample sizes and uncertainties concerning group relationships make interpretation of these patterns difficult. In summary for the beta anoles, the pattern of sSOD expression could be the result of at least two silencing events: within the auratus series and among the other series to the exclusion of the sagrei series. Alternatively, if enhanced sampling should show that all suspected instances of duplicate expression are true including the problematic expression within the schiedii species group, then a duplicate sSOD locus was silenced perhaps independently in at least six species groups plus A. capito. If the classification used in this study is accepted as reflecting the evolutionary relationships of these species of Anolis and if reversal of a silenced gene is unlikely, the duplicated SOD locus has been silenced at least three times within this assemblage and perhaps as many as ten times or more. As a synapomorphic character, the shared loss of one of the duplicated SOD loci does not extrapolate well to the ‘‘higher’’ infrageneric categories of anoles, although it may well have some practical utility in associating individual species within closely-knit lineages. The present paper reviews SOD expression in less than 15% of the total number of species in the genus; there is thus ample opportunity for further assessment of this character as a tool in anole systematics. Because either sSOD locus could have been silenced, care should be exercised so that dual autapomorphy is not confused with synapomorphy. Of course, the use of any loss character should be applied with caution in systematic studies. However, isozyme characters (Buth, 1984) such as a change in gene number controlling SOD expression can continue to supplement allozyme data in species-rich groups such as the genus Anolis. Acknowledgements This study was supported by the UCLA Department of Biology Fisheries Program, the UCLA Biomedical Research Support Grant, the UCLA Committee on Research C. S. Lieb et al./Biochemical Systematics and Ecology 27 (1999) 201–211 209 Grant (d3674 to D.G. Buth), the University of Miami Department of Biology (to M.M. Miyamoto), Instituto de Ecologia, Mexico (to C.S. Lieb), and the National Science Foundation (NSF DEB 77-03259 to G.C. Gorman). We greatly appreciate the collecting assistance to Jorge A. Moreno and Michele R. Tennant, and the then-unpublished data provided by David M. Hillis. We are indebted to David M. Hillis, Robert W. Murphy, Walter J. Rainboth, Jay M. Savage, and an anonymous reviewed for their valuable comments and critical evaluation of earlier versions of this paper. References Adest, G.A., 1977. Genetic relationships in the genus ºma (Iguanidae). Copeia 1977, 47—52. Aquadro, C.F., Avise, J.C., 1983. Evolutionary genetics of birds. VI. A. reexamination of protein divergence using varied electrophoretic conditions. Evolution 36, 1003—1019. Buth, D.G., 1979. Duplicate gene expression in tetraploid fishes of the tribe Moxostomatini (Cypriniformes, Catostomidae) Comp. Biochem. Physiol. 63B, 7—12. Buth, D.G., 1980. 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