Isozyme evidence for independently derived

Isozyme evidence for independently derived, duplicate G3PDH loci among squamate reptiles JACKW . SITES,JR. Department of Zoology, Brigham Young University, Provo, UT, USA 84602 AND

ROBERTW. MURPHY Department of Ichthyology and Herpetology, Royal Ontario Museum, 100 Queen's Park, Toronto, Ont., Canada M5S 2C6

Can. J. Zool. Downloaded from www.nrcresearchpress.com by Renmin University of China on 05/28/13 For personal use only.

Received September 11, 1990 SITES,J. W., JR.,and MURPHY, R. W. 1991. Isozyme evidence for independently derived, duplicate G3PDH loci among squamate reptiles. Can. J. Zool. 69: 238 1-2396. We report evidence for several independent gene duplications for the locus encoding the enzyme glycerol-3-phosphate dehydrogenase (G3PDH) in squamate reptiles. Evidence for the duplication comes from population genetic studies demonstrating "fixed" heterozygosity in all members of some lizard species, the documentation of independent allelic heterozygosity at each of the two G3PDH loci in these same species, and tissue-specific gene expression surveys in a taxonomically diverse array of groups. The duplicated condition is present at both low and high taxonomic levels (selected populations of the phrynosomatid lizard Sceloporus grammicus, and almost all snakes, respectively), and appears to represent the derived condition in most of these groups. One notable exception is the colubrid snake genus Masticophis, which appears to be characterized by an apomorphic secondary silencing event. Evolutionary implications of the duplication and silencing events within squamates are discussed, and we suggest that the overall phylogenetic utility of this marker is,low in this radiation as a result of extensive homoplasy. SITES,J. W., JR., et MURPHY,R. W. 1991. Isozyme evidence for independently derived, duplicate G3PDH loci among squamate reptiles. Can. J. Zool. 69 : 238 1-2396. Nous avons constat6 l'existence de plusieurs duplications indkpendantes de gknes au locus codant l'enzyme glycCrol-3phosphate dCshydrogCnase (G3PDH) chez des reptiles squamates. L'existence de la duplication a CtC mise en lumikre au cours d'Ctudes gCnCtiques de populations qui ont dCmontrC une hCtCrozygotie > chez tous les individus de certaines espkces de 1Czards; l'existence de la duplication est Cgalement dCmontrCe par I'hCtCrozygotie allClique indkpendante B chacun des deux locus G3PDH chez ces m6mes espkces; des Ctudes de l'expression de gknes dans certains tissus spkcifiques au sein d'un groupe de taxons divers ont Cgalement rCvC1C I'existence de la duplication. La condition de duplication se retrouve B la fois aux Cchelons bas et hauts d'Cchelles taxonomiques (chez des populations choisies du lCzard phrynosomatidC Sceloporus grammicus d'une part, chez presque tous les serpents d'autre part) et semble une caracteristique dCrivCe chez la plupart de ces groupes. Une exception cependant, celle du genre de colubridC Masticophis qui semble caractCrisC par un CvCnement secondaire masquant apomorphe. La signification Cvolutive de la duplication et des CvCnements masquants chez les squamates fait l'objet d'une discussion; il semble que I'utilitC phylogCnCtique gCnCrale de ce marqueur soit faible au sein de cette branche B cause d'une importante homoplasie. [Traduit par la rkdaction]

Introduction Understanding the nature and frequency of gene duplication events is important for several reasons. First, duplication of some loci provides a mechanism for the production of gene products needed in large quantities in certain environments o r during select stages of development. Second, duplications initially produce functionally redundant loci which are thought to serve as the raw material for novel adaptations (Ohta 1987). Many duplicate enzymes, for example, are functionally redundant because they catalyze the same reactions (albeit often with different efficiencies), and thus provide greater regulatory "flexibility" in that one locus is free to accumulate new mutations randomly and perhaps acquire a novel function (Ohno 1970; Whitt 1983, 1987). Gene redundancy also accelerates the spead of "compensatory~' mutations (those correcting the effect of an earlier deleterious mutation), owing to relaxation of selective constraints (Ohta 1989). Third, duplicate enzymecoding loci often produce electrophoretically distinct isozyme patterns, and these may have systematic utility (Buth 1984). The fact that strong evidence for duplication events can be collected electrophoretically (MacIntyre 1976; Li 1982) has allowed for widespread and relatively inexpensive screening of isozyme patterns, especially in plants (Gottlieb 1982) and fishes (Buth 1983; Whitt 1983,1987). Frequently, these enzymes show Pnnted In Canada / Irnpnrnk au Canada

a correlation between an increase in locus number and tissue specificity with phyletic advancement. Similar patterns might be expected for some of the same enzyme systems in various tetrapod groups, but tetrapods have generally not been screened as intensively as fishes (for a very limited survey see Fisher et al. 1980). The monophyletic group Reptilia, as defined by Gauthier et al. (1988), occupies a key phyletic position among tetrapods, and discoveries concerning the frequencies and distribution of gene duplications in this group are of interest because they have implications for patterns of genome evolution in most other living tetrapods. W e have electrophoretically surveyed several different groups of squamate reptiles during the course of ongoing population, genetic, and systematic studies in our laboratories, and have discovered isozyme evidence for independent duplications of an originally single locus coding for the enzyme glycerol-3-phosphate dehydrogenase (G3PDH, EC 1.1.1.8; also known as a-glycerophosphate dehydrogenase, a-GPD). Previously, Fisher et al. (1980) reported only one G3PDH locus in reptiles, on the basis of screening liver and muscle extracts from a single specimen of the lizard Anolis carolinensis, while Hall and Selander (1973) and Densmore and Dessauer (1983) (see also Gartside et al. 1977) reported a twolocus system in some populations of another lizard, Sceloporus


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grammicus, and in Alligator mississippiensis, respectively. In S. grammicus, both loci were expressed in the same tissue extracts, and appeared as three-banded "fixed" heterozygotes (see also Sites and Greenbaum 1983; Sites et al. 1988). A threebanded pattern was also reported by Thompson and Sites (1986) in the sagebrush lizard, Sceloporus graciosus. In Alligator, the two loci are predominantly expressed in different tissues, one in liver and a second in skeletal muscle. Finally, Murphy and Crabtree (1985) reported two G3PDH loci with the same mutually exclusive patterns of expression in the prairie rattlesnake, Crotalus viridis viridis. G3PDH appears to evolve slowly in vertebrates with respect to the rate of amino acid substitution (Ballas et al. 1984), but the emerging pattern from our work and other published reports on reptiles suggests that (i) some lineages show evidence of relatively old duplications, as inferred from restricted patterns of tissue-specific expression (A. mississippiensis and C. viridis); (ii) other lineages appear to have undergone more recent duplications, in which both loci are expressed in the same tissue, and are structurally similar enough to form interlocus heterodimers (S. graciosus and some populations of S. grammicus); and (iii) other lineages either have never fixed duplications at this locus, or these may have occurred once and then were followed by a silencing of one locus, so that only a single locus is now expressed (Anolis carolinensis (?), Fisher et al. 1980). Other reports of single-locus systems include those of Dessauer and Cole (1984) in the teil'd lizard Cnemidophorus tigris, and Murphy and Matson (1986) in the tuatara, Sphenodonpunctatus. In this paper, we summarize patterns of isozyme expression in lizards and snakes, including literature reports and unpublished work from our laboratories, and document that (i) the threebanded G3PDH isozyme phenotypes reported as present in most or all individuals of some samples are most parsimoniously interpreted as the expression of two loci, rather than two electromorphs segregating at a single locus or epigenetic modification of a single locus product; and that (ii) this presumed duplication has probably occurred independently in several groups of squamates.

Materials and methods Our electrophoretic and histochemical staining methods follow Murphy et al. (1990) unless specified otherwise. The isozyme nomenclature follows Murphy and Crabtree (1985) in abbreviating the entire enzyme system with capital letters (G3PDH), identifying a specific locus by a capital letter postscript following the enzyme abbreviation with only the first letter capitalized (G3pdh-A), and identifying specific electromorphs (i.e., presumed alleles, see Allendorf 1977)by lower case letters in parentheses following the locus designation (G3pdh-A(aa)). Ideally, demonstration of two distinct structural loci encoding the same enzyme should be based on breeding studies showing independent inheritance of the separate gene products (Soltis et al. 1987). In a broad survey such as this, it is impossible to identify intraspecific allozyme polymorphisms at each locus and then undertake a breeding program for each species. We therefore indirectly inferred the number of G3PDH loci (see below). The data set consists of previously published surveys of squamates for which the number of G3PDH loci was reported, and unpublished

data from our own laboratories. All of these are included in the Appendix, which summarizes for each species the number of individuals and types of tissues screened and the observed number of G3PDH loci. For some taxa, two loci were inferred by the within-sample presence of a three-banded isozyme phenotype in frequencies much greater than would be expected from a simple diallelic segregation at a single locus. For two species, we have demonstrated apparent independent allelic segregation at each of the two loci, and have illustrated the resulting multibanded isozyme phenotypes. In the majority of taxa, tissuespecific mobility and activity differences were used to infer the presence of two loci, even though these species were usually represented by few individuals and localities. However, we are reasonably confident about the reported number of loci encoding G3PDH in these species because most electrophoretic surveys included liver and muscle samples. G3PDH is either most strongly expressed in liver and skeletal muscle, or entirely restricted to one or both of these tissues in species for which extensive tissue surveys have been carried out, including the alligator (Dessauer and Densmore 1983), several species of the lizard genera Cnemidophorus (Dessauer and Cole 1984) and Sceloporus (this paper), C. v. viridis (Murphy and Crabtree 1985), and the tuatara (Murphy and Matson 1986). To infer patterns of G3PDH evolution within squamates, the distribution of duplications was mapped onto two different cladograms (conceptual issues are discussed by Felsenstein 1985 and Donoghue 1989). The first was derived by Hall (1973, 1980) for the lizard genus Sceloporus on the basis of a chromosome data set in which speciesgroups were defined by one or more synapomorphic autosomal or sexchromosome rearrangements relative to the symplesiomorphic 2n = 34 (XY male) karyotype (see also Paul1 et al. 1976). A second cladogram was modified from those produced for all Squamata by Estes et al. (1988) on the basis of extensive morphological and osteological data sets. Estes et al. produced several alternative cladograms, but since no single hypothesis was heavily supported relative to the alternatives, we adopted their-conservative consensus hypothesis for our comparisons (Estes et al. 1988, Fig. 6, p. 140).

Results Evidence for a G3PDH gene duplication The best-documented examples of the presumed G3PDH duplication are found in two species of the North American - lizard genus Sceloporus. The S. grammicus complex includes a minimum of seven chromosomally differentiated populations (cytotypes) ranging across most of mainland Mexico and into southern Texas. A number of intensive studies have examined the interplay between population structure and the fixation of chromosomal rearrangements in this group (reviewed by Sites et al. 1987; Arevalo et al. 199l), and four electrophoretic studies have been undertaken on various portions of this complex (Hall and Selander 1973; Sites and Greenbaum 1983; Sites et al. 1988; Sites and Davis 1989). Results of thse studies have revealed between-populations variability in the number of G3PDH isozymes expressed in homologous tissues. Figure 1 shows the geographic locations from which these populations have been sampled, and Table 1 summarizes relevant data concerning sample sizes and number of expressed G3PDH loci. Table 1 shows that nine populations of the 2n = 32 cytotype and one of the 2n = 34 race consistently display two loci, while all others show only a single locus. The pattern within the 2n = 32 race is

FIG. 1. Approximate ranges of several chromosome races of Sceloporus grammicus, and the localities of origin of the samples that were electrophoretically screened for G3PDH. Sample sizes and G3PDH character states (one or two loci) are given for each locality in Table 1; "FM" refers to three different "multiple fission" races with diploid numbers ranging from 38 to 46 (see details in Arevalo et al. 1991). The heavy broken line indicates the Texas-Mexico border, and the light broken lines the borders of Mexican states, which are indicated as follows: Coah, Coahuila; Gto, Guanajuato; Hgo, Hidalgo; Mich, Michoacan; Mx, MCxico; NL, Nuevo Leon; Qto, Queretaro; SLP, San Luis Potosi; Tamp, Tamaulipas; Ver, Veracruz; and Zac, Zacatecas. The shaded areas at the bottom identify areas of mountain ranges above 3000 m that surround the Valley of Mexico.


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CAN. 1. ZOOL. VOL. 69, 1991

TABLE1 . Localities (numbers are as indicated in Fig. l ) , cytotype designation, and sample sizes of S. grammicus populations surveyed for G3PDH variation


Locality

Cytotype

N

Number of G3PDH loci

Source Sites and Greenbaum 1983 Sites and Greenbaum 1983 Sites and Greenbaum 1983 Sites and Greenbaum 1983 Sites and Greenbaum 1983 Hall and Selander 1973 Sites et al. 1988 Sites et al. 1988 Sites et al. 1988 Sites et al. 1988 Sites et al. 1988 Sites et al. 1988 Sites et al. 1988 Sites et al. 1988 Sites and Greenbaum 1983 Sites and Greenbaum 1983 Hall and Selander 1973 Hall and Selander 1973 Sites et al. 1988 Sites and Greenbaum 1983 Sites and Greenbaum 1983 Sites and Greenbaum 1983 Sites and Greenbaum 1983 Sites and Greenbaum 1983 Sites and Greenbaum 1983 Sites and Davis 1989 Sites and Davis 1989 Sites and Davis 1989 Sites and Davis 1989 Sites and Davis 1989 Sites and Davis 1989 Sites and Davis 1989 Sites and Davis 1989 Sites and Davis 1989 Sites and Davis 1989

NOTE:L and H refer to low and high-elevation samples of the 2n = 32 cytotype, respectively. *Twenty-nine of 31 individuals had two loci.

especially interesting in that populations from the northern part of the range (localities 1-5 in Fig. 1) possess the one-locus system, whereas southern populations, including both low(localities 10- 14) and high-elevation (localities 6-9) populations possess the two-locus system. In all these samples, all individuals displayed either the "fixed heterozygosity" three-banded isozyme phenotype or, in a very few cases, a five-banded phenotype suggestive of electromorphic heterozygosity at one of the two loci (see below). A second species for which the same "fixed heterozygosity" pattern has been documented on a widespread geographic scale is Sceloporus graciosus, which is indigenous to most of the western United States and a few isolated mountain ranges in the Baja California peninsula (see map in Thompson and Sites 1986). Thompson and Sites (1986) reported the presence of a presumed G3PDH duplication in 278 specimens from 13 localities, but another 61 specimens from 4 other localities displayed identical enzyme phenotypes. The only exceptions to the threebanded fixed heterozygosity pattern were four individuals displaying independent electromorphic heterozygosity at one of the loci. As in S. grarnrnicus, these individuals all had five-

banded G3PDH phenotypes. In both these species, tissue expression studies were carried out to determine the overall patterns of expression of G3PDH, and all populations examined consistently displayed activity in only liver and skeletal muscle; no activity was expressed in heart, kidney, brain, stomach, duodenum, or whole blood, and the patterns expressed in liver and muscle were identical in all cases. If these patterns are generally chracteristic of squamate reptiles, then studies involving the use of these two tissues alone may give an accurate picture of the number of G3PDH loci expressed in a given species. Figure 2 presents examples of G3PDH isozyme patterns resolved in both S. graciosus and S. grarnrnicus in the studies described above, and Fig. 3 is a diagrammatic interpretation of the hypothesized molecular composition of each isozyme in the phenotypes illustrated in Fig. 2. Figure 2A shows a single isozyme pattern typical of many S. grarnrnicus cytotypes scored for a single locus but with the occasional appearance of an electromorphic heterozygote. Figure 3A gives the standard interpretation of these three-banded phenotypes in dimer molecules, in which two electromorphs of different mobilities (designated "a" and "b") should recombine in a heterozygote


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FIG.2. Isozyme patterns in Sceloporus graciosus and S. grammicus, showing four classes of phenotypes consistently resolved in these species. (A) Single G3PDH locus in S. grammicus in which one individual (second from left) is electromorphically heterozygous in an otherwise monomorphic sample. (B) "Fixed heterozygosity" condition in S. graciosus, resulting in three-banded G3PDH phenotypes generated by expression of interlocus heterodimers in all individuals. (C and D) Alternative heterozygotes in the two-locus systems (both in S. graciosus), producing independent five-banded isozyme phenotypes at the slow (C) and fast (D) loci. All panels are oriented so that the cathodal end of the gel is at the bottom.

to form two homodimer isozymes (aa and bb) and a single heterodimer (ab) of intermediate mobility (for a review of gel interpretation of isozyme banding patterns see Murphy et al. 1990). These "aa/ab/bb" isozymes should form in a 1:2: 1 ratio if both electromorph products are synthesized in equal concentrations, recombine at random, and are equally stable, but there are a number of reasons why this may not always be the case (Buth 1984; Murphy and Crabtree 1985). Our results differ slightly from the expected 1:2: 1 ratio of staining intensities, but otherwise are consistent with an interpretation of electromorph heterozygosity. This interpretation is further strengthened by the fact that these phenotypes appeared only as occasional heterozygotes in what were otherwise uniformly monomorphic samples, and never deviated from Hardy-Weinberg genotypic ratios (Sites and Greenbaum 1983; Thompson and Sites 1986; Sites et al. 1988). Figure 2B shows the fixed heterozygosity G3PDH phenotypes for one sample of S. graciosus, and this was identical for all other samples of this species and for all samples of S. grammicus scored for two G3PDH loci in the above-cited allozyme studies (Table 1). Figure 3B provides the molecular interpretation, which in this case involves the formation of an interlocus heterodimer between the original (A') and duplicate (A') loci, which are interpreted to recombine at random to produce A'A'/A1A2/A2A2isozymes in approximately a 1:2:1 ratio. Figures 2C and 2D show examples of five-banded G3PDH isozyme phenotypes, both in S. graciosus, which we interpret to result from single-locus heterozygosity for different-mobility electromorphs superimposed on the duplication pattern. This pattern is shown to occur independently at both the original A' locus (Fig. 2C) and the duplicated A2 product (Fig. 2D). As an example, if all three electromorphs in Fig. 2C, ~ ' ( a )~, ' ( b ) , and A2(a),had different mobilities, six dimeric isozymes are pos-

sible in a single individual, including A1(bb)/A1(ab)/A1(aa)/ A1(b)A2(a)/A1(a)A2(a)/A'(aa). In all our zymograms, however, only five bands are present, and the middle (third) isozyme is always the most intensely expressed. We interpret this isozyme to actually represent co-migration of the products of two genetically distinct isozymes with identical mobilities. In Fig. 2C, the middle band would actually represent both the A1(aa)and the A'(b)A2(a) isozymes "stacked" on top of one another. -This hypothesized molecular basis for both classes of five-banded phenotypes is diagrammatically illustrated in Fig. 3C. Taxonomic distribution of the G3PDH duplication among squamates The Appendix summarizes the known occurrence of the G3PDH duplications in amphisbaenians, saurians, and snakes. The only amphisbaenian surveyed, Bipes, and the vast majority of lizards dislay a single G3PDH locus, but there are several interesting exceptions. The genus Sceloporus has been moderately well surveyed and, with the exceptions of S. graciosus and some populations of S. grammicus, shows no other evidence of duplication at this locus. The only other moderately wellsurveyed radiation is the very large genus (200+ species, Paul1 et al. 1976) Anolis (as defined by Canatella and de Queiroz 1989); all the 43 species surveyed to date express a single G3PDH locus. All other lizard radiations show a minimum of one duplication; in the Gekkota, the eublepharid genus Coleonyx expresses two loci, and similar patterns are evident in North American species of the scincid genus Eumeces of scincomorphs, and the xenosaurid and varanid genera Shinisaurus and Varanus, respectively, of the Anguimorpha. Since many of these surveys are based on small sample sizes with respect to number of individuals and (or) number of species surveyed per genus,


A

~ 3 ~ dA'h (bb) G3pdh-

A' (ab)


G3pdh-A (aa)

- -= m = fi

G3pdh-AZ(aa)

G3pdh- A'(@ A2(a) I 1

2

1

G 3 ~ d h -A (b) A (@+A (aa) G3pdh- A' (ab) G3pdh- A' (bb) -

G3pdh- A2 (bb) G3pdh- A''l (ab) I

G3pdh- A' (b)A1(a)+AL (aa) Gjpdh-

A' (a) A2 (a)

G3pdh- A' (aa)

FIG. 3. Diagrammatic interpretation of recombination patterns among G3PDH subunits (the superscript notation is explained in the text) hypothesized to produce isozyme patterns illustrated in Fig. 2. (A) Intralocus pattern resulting from heterozygosity for different-mobility electromorphs at the original nonduplicated (A') locus (see Fig. 2A). (B) Interlocus three-banded patterns resulting from recombination among products of separate loci (A' and A2) having different mobilities (see Fig. 2B). (C) Intralocus allozyme heterozygosity in duplicate system at the A ~ 2D). , least anodal (G3pdh-A', Fig. 2C) and most anodal loci ( ~ 3 ~ d h - Fig.

and frequently also in terms of numbers of tissues screened, the G3PDH duplications listed in the Appendix conservatively must be considered a minimum. The same kinds of data are also summarized for snakes, and the results are reversed from the pattern of duplications evident in lizards. The vast majority of snakes (16 of 17 genera representing five families, see the Appendix) that have been sampled express two G3PDH loci. Again surveys are limited in terms of species diversity and number of individuals sampled from most taxa, but the overall picture is one in which a single silencing event of one of the two loci has occurred within the Colubridae; six species of Masticophis express only a single G3PDH locus.

Discussion Evolution of the G3PDH duplication within Sceloporus The documentation of two G3PDH loci within the genus Sceloporus is based upon surveys of about one-fourth of the 65+ species in this genus, in which both liver and muscle were screened. Since these tissues are the only or the predominant ones in which this enzyme is expressed in reptiles (Densmore and Dessauer 1983; Dessauer and Cole 1984; Murphy and Crabtree 1985; Murphy and Matson 1986), species expressing only a single isozyme only one of these tissues, or a single electromorph of identical mobility in both, may have only a

single G3PDH locus. However, if a duplication is very recent (in which case sequence homology is absolute), or if one of the duplicate products has been secondarily silenced, electrophoresis will still reveal only a single isozyme. We therefore refer to a "single locus" only in the general sense that one isozyme is expressed, and acknowledge the limitation of our methods. In the two lineages within Sceloporus possessing the twolocus G3PDH isozyme pattern, we have demonstrated that (i) independent electromorphic heterozygosity is present at each locus; and (ii) there is consistently strong expression of an interlocus heterodimer isozyme in both liver and muscle extracts. The first of these observations is the strongest evidence that we can obtain for two loci in the absence of controlled breeding studies. The second of these observations may be used to infer that the duplication of the original G3PDH locus has occurred relatively recently, because duplicate loci at early stages of divergence are thought to be structurally and metabolically similar, and therefore capable of interacting to form heteropolymer isozymes (Whitt 1983). Time-dependent divergence of the two loci from each other is hypothesized to result in eventual restriction of heteropolymer formation as the loci diverge in function. These trends are documented for other enzymes in advanced teleost fishes (Ferris and Whitt 1978; Fisher and Whitt 1978; Toledo and Ribiero 1978; Whitt 1981; Rainboth et al.

SITES AND MURPHY

small-scaled, smalluse 2 ,FORMOSUS (13 sp more m iscrosd=22

,

/YSR'&

(2n=341xy@

more micros MAGISTER

2n=30

large scaled, largebodied radiation Can. J. Zool. Downloaded from www.nrcresearchpress.com by Renmin University of China on 05/28/13 For personal use only.

'HORRIDUS (11 sp?)

vfuse /

Y?'

fuse 2nd micro pr

2n=32 XY @

Em 9 mutation

(4 sp)

\ ZOSTEROMUS

cLARw (2 SP.)** 2n=40 4 3 s macro prs 1, 3-5

\

fix XI X2Y sex chromosome system

'GRAMMICUS (5 sp.)

b

2n=32 X l X2Y@

MEGALEPIDURUS (2 sp.)** TORQUATUS (12 sp?)

FIG. 4. Generalized cladogram of species-groups in the large-scaled, large-bodied radiation of Sceloporus, based on chromosomal data summarized by Hall (1973, 1980). Numbers in parentheses identify approximate numbers of morphologically recognized species in all polytypic groups, with uncertainties in poorly known groups being indicated by "?." Vertical and horizontal hatqh marks denote inferred rearrangements in macro- or micro-chromosomes (Em, "enlarged micro"; fuse, Robertsonian fusion; Gss, Robertsonian fission), or sex chromosomes, used by Hall to hypothesize monophyletic groups. Open squares identify derived synapomorphic karyotypes (2n = 32,30, etc.), and solid circles on the graciosus and grammicus branches identify known duplicate G3PDH systems, albeit restricted to only some races of S. grammicus (*, see Table I). (**, species-groups for which no allozyme data are available.)

1986), although at variable taxonomic levels. Since we have no reason to assume that reptilian systems would not follow this general trend, we infer that the G3PDH duplication in Sceloporus is a relatively recent occurrence. Figure 4 presents an abbreviated phylogenetic hypothesis of relationships among various species-groups within the largescaled, large-bodied radiation of the genus Sceloporus. Sceloporus graciosus, a monotypic group with a 2n = 30 (XY indistinct) karyotype, is part of a lineage independent of that containing the S. grammicus species-group (2n = 32, X,X,Y male). Given this relationship, our data strongly suggest an independent origin for each duplication within this genus. The duplication appears to be fixed in the entire species S. graciosus, while absent in four other species-groups in the same clade, including the magister group (from which the species magister, monserratensis, and rufidorsum have been surveyed), the zosteromus group (monotypic), the horriduslundulatus group (occidentalis, spinosus, undulatus, virgatus, and woodi), and the formosus group (fbrmosus and taeniocnemis, see Appendix).

The duplication is evident in some populations only of S. grammicus but absent in the closely related torquatus speciesgroup (represented by the species dugesii, jarrovi, mucronatus, and torquatus, see the Appendix). We have not surveyed representatives of the asper, clarki, megalepidurus, or orcutti groups, but the duplication is absent in Sceloporus representing other species-groups not included in Fig. 4 (merriami and parvus, see the Appendix) and representatives of four other closely related sceloporine genera (Holbrookia, Petrosaurus, Uma, and Uta, see the Appendix). Within the grammicus group, the species S. heterolepis and S. shannonorum have not been electrophoretically surveyed. Of the other species, S. palaciosi expresses a single locus (Sites et al. 1988), while the distribution of the duplication among chromosome races of S. grammicus can be interpreted in several ways. The presence of the G3PDH duplication in central Mexican populations of the 2n = 32 race, and its absence from northern populations with the same standard karyotype (Fig. 1, Table I), suggest that what is now referred to as the 2n = 32


2388


cytotype may not be a single monophyletic unit. This race may have had independent northern and southern origins in the absence of chromosomal change, or this race may be a monophyletic unit but may have been separated into northern and southern populations early in its history, followed by the origin and fixation of a G3PDH duplication in the southern populations only (localities 6- 14 in Fig. 1). A third possibility is that the duplication event may have been fixed in the ancestral population of a monophyletic 2n = 32 race, and has subsequently been silenced in the northern populations of this cytotype (localities 1-5 in Fig. 1) and in all other derived cytotypes (see the phylogenetic arguments in Sites and Davis (1989) for placement of other cytotypes of S. grammicus). Another alternative is that populations characterized by a single G3PDH isozyme but scored for occasional "allelic" heterozygotes actually have duplicate loci with identical electrophoretic mobilities, but in which one locus had acquired a second differentially migrating allele. Under these conditions electrophoresis would reveal a three-banded isozyme phenotype on a gel, but fixation of this pattern would only occur with fixation of the new allele at one locus. Similar alternatives could be proposed for the 2n, = 34 cytotype, in that one population (locality 16 in Fig. 1) is fixed for the G3PDH duplication, whereas all others are not. However, we cannot ascertain for either case whether duplicate loci in these races arose before or after the origin of the races themselves. Ongoing allozyme surveys will include a wider geographic sampling of all S. grammicus chromosome races from central Mexico, as well as S. heterolepis, and should help define the geographic and taxonomic limits of the fixed duplication. Evolution of the G3PDH duplication within the Squamata The frequencies of the two-locus G3PDH systems summarized in the Appendix can be interpreted in a comparative evolutionary framework if they can be overlaid onto a wellcorroborated phylogenetic hypothesis (Donoghue 1989). In Fig. 5, we present a modified version of the cladogram from Estes et al. (1988); the family names recognized within the Iguania in our cladogram are those recognized by Frost and Etheridge (1989), those within the Gekkota follow Kluge (1989), and we omitted groups present in the cladograms in Estes et al., but for which no data are available for G3PDH. We can infer that the single-locus condition is the ancestral state by using Sphenodon as the sister-group of all squamates, as it expresses a single G3PDH locus in liver and skeletal muscle (determined from a screen of 15 different tissues, see Murphy and Matson 1986). At least seven duplications can therefore be inferred within the Squamata; two within the Iguania, and one each in the Gekkota (Eublepharidae), Scincomorpha (Scincidae), Xenosauridae (Shinisaurus), Varanidae (Varanus), and Serpentes (Fig. 5). An alternative view is that there were fewer duplications, perhaps only one or two occurring deeper in the cladogram. If this were the case, the distribution to two-locus systems in Fig. 5 must be accounted for by silencing events due to the mutational formation of pseudogenes in one of the two G3PDH loci originating from the duplication event, or an absence of mobility divergence between the products. We cannot rule out these possibilities, and in fact we make a very strong case for such an interpretation within one clade (see below). However, we do not favor the silencing explanation for the overall pattern for the following reason. The overall pattern of isozyme expression obtained from other

vertebrate groups is one in which evolutionarily recent duplications are characterized by the expression of both loci in the same tissue and their ability to recombine to form stable interlocus heterodimers. As the two products diverge (possible mechanisms are reviewed by Li 1982), they frequently lose the ability to form a stable heterodimer, and (or) specialize toward different functions and become restricted to expression in different tissue types (Whitt 1981,1983,1987). Figure 5 and the Appendix show that in some lineages, the duplications are widespread (in all members of a genus, for example) and in all but Sceloporus are restricted in their patterns of tissue-specific expression (one locus in liver, one in muscle). These patterns suggest that such duplications are old enough to have resulted in interlocus divergence in sequence and function. Many of the groups in Fig. 5 have these patterns, whereas patterns of expression in Sceloporus imply separate and more recent duplications in that genus. Based on the distribution of G3PDH duplications in Fig. 5, we suggest independent and relatively old duplications for Coleonyx, North American Eumeces, the Xenosauridae, Varanus, and the Serpentes. Within the Serpentes there appears to be a single G3PDH duplication; this condition is shared by almost all snakes that have been thoroughly studied, and presumably represents a synapomorphy for the entire clade. The exact placement of snakes and amphisbaenians within the squamates is uncertain, leading Estes et a1.'(1988, p. 235) to designate these groups as "incertae sedis." In Fig. 5, we illustrate this uncertainty by showing alternative positions for both (as did Estes et al. 1988, Fig. 6), but neither position would alter our interpretation of the polarity of G3PDH for the Serpentes. Therefore, within the Squamata, the Serpentes provide the only well-supported instance of an apomorphic secondary silencing event: we interpret the single-locus condition in the colubrid genus Masticophis as a synapomorphic state for the genus. Utility of gene-duplication data for phylogenetic inference The phylogenetic utility of any character is directly proportional to (i) the rate of change in the character under consideration in the group of interest; and (ii) the number of alternative states that may be produced by evolution in the group. The first of these determines the hierarchical level at which a particular character may be useful for phylogenetic inference (Goodman et al. 1979), while the second determines the frequency of parallel, convergent, or reverse changes in character states (Murphy 1988). Conventional electrophoretic techniques will detect only the presence or absence of a duplication with divergent interlocus mobilities, which imposes additional constraints on the phylogenetic utility of these kinds of data. Exceptions are known, especially in plant groups in which duplications of isozyme loci by processes other than polyploidy are relatively rare (Gottlieb 1982), and are therefore useful in defining monophyletic groups at lower hierarchical levels (Gottlieb and Weeden 1979). However, widespread taxonomic surveys such as this one often reveal a mosaic of both one- and two-locus states distributed across several hierarchical levels (for example in plants see Weeden et al. 1989). This survey revealed the presence of G3PDH duplications at both low (Sceloporus) and high (Serpentes) taxonomic levels and, with few exceptions, was inadequate for resolving some important phylogenetic issues. Specifically, we could not determine either the number of duplication or the number of silencing events, although both could theoretically be useful in defining monophyletic groups. Theoretical and empirical studies

SITES AND MURPHY

\

(-) , Crota(-), Polychridae (-), Phrynosomatidae (+ in

SQUAMATA

GEKKOTA


S C L ROGLOSSA ~

{

Eublepharidae (+ in Coleonvx) Gekkonidae (-)

*AMPHlSBAENIA

0 ,

----

I

*SERPENTES

\ AUTARCHOGLOSSA

Gymnophthalmidae (-) Teiidae (-) Lacertidae (-) Xantusiidae (-)

SCINCOMORPHA

Scincidae (+ in North Am. Eumeces)

r VARANOIDEA

Anguidae (-)

Varanidae (+ in Varanus)

0

AhADHlSBAENlA (- in Bipes)

Boidae (+), Colu bridae in Masticophis) (+), Typhlopidae (+), Viperidae (+)

FIG.5. Cladogram of higher category relationships among the Squamata, modified from Estes et al. (1988, Fig. 6, p. 140). The presence or absence of a two-locus G3PDH system is indicated by "+" in association with highlighted familial names, or "- ", respectively, based on data summarized in the Appendix. An asterisk indicates that placement is uncertain.

of other groups suggest that mutational silencing should be the most common evolutionary fate for one of the two products of any gene duplication, and that the rate of silencing will be a function of the effective population size (N,), the number of speciation events, and divergence times (Ferris et al. 1979; Kimura and King 1979; Takahata 1982). These parameters are extremely difficult to quantify over evolutionary time, but certainly would be expected to vary across the range of squamate

lineages listed in the Appendix. Rather than attempt to routinely use duplication or silencing events as phylogenetic markers, except in cases in which they conspicuously define previously well-corroborated clades (Avise and Aquadro 1987; or the Serpentes or Masticophis in this study), we suggest the use of such data to study patterns and processes of genome evolution. Many of the alternative interpretations presented here can only be tested by nucleic acid techniques that will permit identifica-


2390


tion of (i) the number and distribution of G3PDH pseudogenes within these groups, and (ii) the degree of either amino acid or nucleic acid sequence homology necessary to distinguish between orthologous and paralogous isozymes (for examples in other groups see Poorman et al. 1984 and Park et al. 1985). It is clear, however, that squamates constitute a radiation with the potential to provide much new information about patterns and processes of gene duplication from very low to very high taxonomic levels.

Acknowledgments The work of J.W.S. on Sceloporus for the past 5 years has been supported by the National Science Foundation (BSR-8509092 and 88-2275 I), the National Geographic Society (308885), and the Professional Development Committee, College of Biology and Agriculture, Brigham Young University. For aid in collecting, he thanks E. Arevalo, J. L. Camarillo, A. Gonzales, L. Javier, G. Lara, M. Mancilla, F. Mendoza, C. Porter, and J. and H. Sites, and for laboratory assistance, D. Mindell and P. Thompson. R.W.M. was supported by grants from the Natural Sciences and Engineering Research Council of Canada (A-3 148) and the National Institutes of Health (RR-08156-10 to D. J. Morafka (principal investigator) and R.W.M.). Laboratory assistance was provided by G. Aguirre, C. Costilla, I. Delgado, E. Habacon, J. Hernandez, R. D. MacCulloch, D. J. Morafka, G. D. Scott, and R. Tansiel. s

ADEST, G. A. 1977. Genetic relationships in the genus Uma (Iguanidae). Copeia, 1977: 47-52. ALLENDORF, F. W. 1977. Electromorphs or alleles. Genetics, 87: 82 1-822. AREVALO,E., PORTER, C. A., GONZALES,A., MENDOZA,F., CAMARILLO, J. L., and SITES; J. W., JR. 1991. Population - cytogenetics and evolution of the Sceloporus grammicus complex (Iguanidae) in Central Mexico. Herpetol. Monogr. 5: 79- 1 15. AVISE,J. C., and AQUADRO,C. F. 1987. Malate dehydrogenase isozymes provide a phylogenetic marker for the Piciformes (woodpeckers and allies). Auk, 104: 324-328. BALLAS,R. A., GARAVELLY, J. S., and WHITE,H. B., 111. 1984. Estimation of the rate of glycerol-3-phosphate dehydrogenase evolution in higher vertebrates. Evolution, 38: 658-664. BEZY,R. L., and SITES,J. W., JR. 1987. A preliminary study of allozyme evolution in the lizard family Xantusiidae. Herpetologica, 43: 280-292. BEZY,R. L., GORMAN, G. C., KIM,Y. J., and WRIGHT,J. W. 1977. Chromosomal and genetic divergence in the fossorial lizards of the family Anniellidae. Syst. Zool. 26: 57-7 1. BEZY,R. L., GORMAN, G. C., ADEST,G. A., and KIM,Y. J. 1980. Divergence in the night lizard Xantusia riversiana (Sauria: Xantusiidae). In The California Islands: Proceedings of a Multidisciplinary Symposium. Edited by D. M. Power. Santa Barbara Natural History Museum, Santa Barbara, CA. pp. 565-583. BUSACK,S. D. 1986. Taxonomic implications of biochemical and morphological differentiation in Spanish and Moroccan populations of three-toed skinks, Chalcides chalcides (Lacertilia, Scincidae). Herpetologica, 42: 230-236. BUTH,D. G. 1983. Duplicate isozyme loci in fishes: origins, distribution, phyletic consequences, and locus nomenclature. In Isozymes: current topics in biological and medical research. Vol. 10. Edited by M. C. Rattazzi, J. G. Scandalios, and G. S. Whitt. Alan R. Liss, New York. pp. 38 1-400. 1984. The application of electrophoretic data in systematic studies. Annu. Rev. Ecol. Syst. 15: 501 -522. BUTH,D. G., GORMAN, G. C., and LIEB,C. S. 1980. Genetic divergence between Anolis carolinensis and its Cuban progenitor, Anolis porcatus. J. Herpetol. 14: 279-284.

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Appendix Summary of G3PDH isozyme data for squamate reptiles, on the basis of number of species surveyed per genus within families. Higher taxonomic category arrangements follow Estes et al. (1988), while familial names of iguanians and gekkonoids follow Frost and Etheridge (1989) and Kluge (1987), respectively. Abbreviations for tissues are as follows: H, heart; I, duodenum; K, kidney; L, liver; Lu, lung; M, skeletal muscle; P1, plasma; RBC, red blood cells; S, stomach; and Sp, spleen; ?, data not reported. b

No. of individuals screened Amphisbaenia Amphisbaenidae Bipes biporus B . canaliculatus B . tridactylus Iguania Chamaeleonidae Agama stellio Crotaphytidae Crotaphytus collaris C . dickersonae C . insularis C . reticulatus Gambelia wislizenii Iguanidae Ctenosaura hemilopha Dipsosaurus dorsalis Sauromalus australis S. hispidus S. klauberi S. obesus (including ater) S. slevini Phrynosomatidae Holbrookia propinqua Petrosaurus mearnsi P . thalassinus Sceloporus graciosus S. dugesii S. formosus S. grammicus S. jarrovi S . magister S . merriami

No. of G3PDH loci resolved

Tissues examined

Source

Kim et al. 1976 Kim et al. 1976 Kim et al. 1976

'Nevo 1981 Moiitanucci et al. Montanucci et al. Montanucci et al. Montanucci et al. Montanucci et al. R. W.M., R.W.M., R.W.M., R. W.M., R.W.M.,

1975 1975 1975 1975 1975

unpublished data unpublished data unpublished data unpublished data unpublished data

R.W .M., unpublished data R.W .M., unpublished data J.W. S., unpublished data R.W .M., unpublished data R.W .M., unpublished data Thompson and Sites 1986 J.W .S., unpublished data J. W. S., unpublished data See Table 1 J. W.S., unpublished data R.W .M., unpublished data J. W. S., unpublished data

SITES AND MURPHY

Appendix (continued)


No. of individuals screened

S . "monserratensis" S . mucronatus S . occidentalis S . parvus S . rujidorsum S . spinosus S . taeniocnemis S . torquatus S . undulatus S . virgatus S . woodi S . zosteromus Uma exsul U . inornata U . notata U . paraphygas U . scoparia Uta stansburiana Polychridae Anolis acutus A . aeneus A. angusticeps A. bimaculatus A . blanquillanus A . bonairensis A . carolinensis A. cobanensis A. cooki A. cristatellus A. desechensis A. distichus A. evermanni A . extremus A. ferreus A . gadovi A. gingivinus A . grahami A . griseus A. gundlachi A. krugi A . leachi A. lividus A . luciae A . marmoratus A. milleri A. monensis A. naufragus A . nubilus A. oculatus A . pogus A. polyrhachis A. poncensis A. porcutus A . pulchellus A . richardi A. roquet A . sabanus A . sagrei A . schwartzi A. scriptus A. stratulus


Tissues examined

Source

L,M L,M L,M L,M L,M L,M L,M L,M L,M L,M L,M L,M H,K,L,M* H,K,L,M* H,K,L,M* H,K,L,M* H,K,L,M* Whole body

R. W.M., unpublished data J. W. S., unpublished data J. W .S., unpublished data J. W .S., unpublished data R. W .M., unpublished data J. W.S., unpublished data J. W. S., unpublished data J. W. S., unpublished data J. W .S., unpublished data J. W .S., unpublished data J. W .S., unpublished data R. W .M., unpublished data Adest 1977 Adest 1977 Adest 1977 Adest 1977 Adest 1977 McKinney et al. 1972; Soul6 and Y ang 1973

H,K,L,M* H,K,L,M* Whole body H,K,L,M* H,K,L,M* H,K,L,M* Whole body

Gorman and Kim 1976 Yang et al. 1974 Webster et al. 1972 Gorman and Kim 1976 Yang et al. 1974 Yang et al. 1974 Webster et al. 1972; Buth et al. 1980 Campbell-et al. 1989 Gorman et al. 1980, 1983 Gorman et al. 1980, 1983 Gorman et al. 1980 Webster et al. 1972;Case and Williams 1984 Gorman et al. 1983 Yang et al. 1974 Gorman and Kim 1976 Gorman et al. 1983 Gorman and Kim 1976 Taylor and Gorman 1975;Gormanet al. 1983 Yang et al. 1974 Gorman et al. 1983 Gorman et al. 1983 Gorman and Kim 1976 Gorman and Kim 1976 Gorman and Kim 1975;Yang et al. 1974 Gorman and Kim 1976 Campbell et al. 1989 Gorman et al. 1980 Campbell et al. 1989 Gorman and Kim 1976 Gorman and Kim 1976 Gorman and Kim 1976 Campbell et al. 1989 Gorman et al. 1983 Buth et al. 1980 Gorman et al. 1983 Yang et al. 1974 Yang et al. 1974; Gorman and Kim 1975 Gorman and Kim 1976 Webster et al. 1972 Gorman and Kim 1976 Gorman et al. 1980 Gorman et al. 1983

3

H,K,L,M* H,K,L,M* H,K,L,M* H,L,M* H,K,L,M* H,K,L,M* H,K,L,M* H,K,L,M* . H,K,L,M* H,K,L,M* H,K,L,M* H,K,L,M* H,K,L,M* H,K,L,M* H,K,L,M* H,K,L,M* H,K,L,M* ? H,K,L,M* ?

H,K,L,M* H,K,L,M* H,K,L,M* ? H,K,L,M* H,L,M H,K,L,M* H,K,L,M* H,K,L,M* H,K,L,M* Whole body H,K,L,M* H,K,L,M* H,K,L,M*

CAN. J . ZOOL. VOL. 69, 1991



A. trinitatis A. wattsi Gekkota Eublepharidae Coleonyx switaki C . variegatus Gekkonidae Cyrtodactylus lousidensis Gehyra catenata G. dubia G . minuta G . montium G . nana G . oceanica G . purpurascens G . variegata Heteronotia binoei H. spelea Heteronotia sp. nov . Nactus arnouxii (bisexual) HemidactyEus brooki Lepidodactylus lugubris Phyllodactylus bugastrolepis P . nocticolus P. paucituberculatus P. unctus P. xanti Sphaerodactylus nicholsi S. roosevelti S. townsendi



31 21

1 1

H,K,L,M* H,K,L,M*

Yang et al. 1974 Gorman and Kim 1976

5 15

2 2

H,L,M H,L,M

R. W .M., unpublished data R. W .M., unpublished data

Tissues examined

Moritz 1985 Moritz 1985 Moritz 1985 Moritz 1985 Moritz 1985 Moritz 1985 Moritz 1985 Moritz 1985 Moritz 1985 Moritz et al. 1989, 1990 Moritz et al. 1989, 1990 Moritz et al. 1989, 1990 Moritz 1987 Pasteur et al. 1978 Pasteur et al. 1987 R. W .M., unpublished data R. W .M., unpublished data Murphy and Papenfuss 1980 R. W.M., unpublished data R. W .M., unpublished data Whole body Whole body Whole body

Scincomorpha Gymnophthalmidae Pholidobolus afJinis P. annectens P. macbrydei P. montium P. prefrontalis Proctoporus "A" Pr. hypostictus Pr. unicolor Teiidae Ameiva ameiva A. auberi Cnemidophorus lemniscatus (bisexual) C . murinus C . nigricolor C : tigris C . septemvittatus C . sexlineatus Lacertidae Lacerta melisellensis L. oxycephala L. sicula Podarcis erhurdii P. milensis P. muralis

Source

R. W.M., unpublished data R.W .M., unpublished data R. W .M., unpublished data

Sites et al. 1990 Sites et al. 1990 Sites et al. 1990 Sites et al. 1990 Sites et al. 1990 Parker and Selander 1976; Gorman et al. 1977; Dessauer and Cole 1984 Parker and Selander 1976 Parker and Selander 1976 Whole body Whole body Whole body ? ? ?

Gorman et al. 1975 Gorman et al. 1975 Gorman et al. 1975 Mayer and Tiedemann 1980 Mayer and Tiedemann 1980 Mayer and Tiedemann 1980

2395

SITES AND MURPHY



No. of individuals screened P. sicula P. taurica P. tiliguerta Podarcis sp. Xantusiidae Xantusia henshawi X. riversiana X. vigilis Lepidophyma j!avimaculatum L. gaigeae L. micropholis L. smithii L. sylvaticum L. tuxtlae Scincidae Chulcides chalcides C . mertensi Sphenomorphus jobiensis (4 species) Eumeces copei E. fasciatus E. inexpectatus E. laticeps E. skiltonianus Typhlosaurus gariepensis T . lineatus Anguimorpha Anguidae Anniella geronimensis A. pulchra Xenosauridae Shinisaurus crocodilurus Varanidae Varanus gouldi Serpentes Boidae Lichanura trivirgata Colubridae Chilomeniscus cinctus Eridiphas slevini Hypsiglena torquata Lampropeltis getulus Leptodeira maculata L. septentrionulis Masticophis aurigulus M. bilineatus M. $flagellum M. lateralis M. striolatus M. taeniatus Phyllorhynchus decurtatus Thamnophis couchi T . elegans T . ordinoides Trimorphodon biscutatus Elapidae Emydocephalus annulatus


Tissues examined

Source Mayer Mayer Mayer Mayer

198 1

and Tiedemann 1980 198 1

and Tiedemann 1980

Bezy et al. 1980; Bezy and Sites 1987 Bezy et al. 1980; Bezy and Sites 1987 Bezy et al. 1980; Bezy and Sites 1987 Bezy and Sites Bezy and Sites Bezy and Sites Bezy and Sites Bezy and Sites Bezy and Sites

1987 1987 1987 1987 1987 1987

Busack 1986 Busack 1986 Donnellan and Aplin 1989 R.W.M., unpublished data R. W .M., unpublished data R. W .M., unpublished data R. W. M., unpublished data R. W. M . , unpublished data Kim et al. 1978 Kim et al. 1978

Bezy et al. 1977 Bezy et al. 1977 R. W.M., unpublished data ,

R. W.M., unpublished data

R. W .M., unpublished data R. W .M . , unpublished R. W.M., unpublished R. W.M., unpublished R. W .M., unpublished R. W .M., unpublished R. W. M . , unpublished R. W .M . , unpublished R. W .M., unpublished R. W .M., unpublished R. W.M., unpublished R. W.M., unpublished R. W .M . , unpublished

data data data data data data data data data data data data

Murphy and Ottley 1980 Lawson and Dessauer 1979 Lawson and Dessauer 1979 Lawson and Dessauer 1979 R. W .M . , unpublished data R. W .M., unpublished data

CAN. J . ZOOL. VOL. 69, 1991

Appendix (concluded)



Laticauda colubrina L. laticaudata L. semifasciata Pelamis platurus Typhlopidae Leptotyphlops humilis Viperidae Agkistrodon bilineata A. contortrix A. piscivorous Bothrops godmani B. numifer Crotalus atrox C . catalinensis C . cerastes C . durissus C . horridus C . intermedius C . lepidus C . mitchellii C . molossus C . polystictus C . pricei C . pusillus C . ruber C . scutulatus C . tortugensis C . triseriatus C . viridis C . willardi Lachesis muta Sistrurus catenatus S. miliarius S. ravus Trimeresurus albolabris


Tissues examined

Source

R. W. M., R. W .M., R. W .M., R. W.M.,

unpublished unpublished unpublished unpublished

data data data data

R. W .M., unpublished data R. W .M., unpublished data R. W .M., unpublished data R. W .M., unpublished data R. W.M., unpublished data R. W .M., unpublished data R. W .M., unpublished data R. W .M., unpublished data R. W .M., unpublished data R. W .M., unpublished data R. W.M., unpublished data R. W .M., unpublished data R. W .M., unpublished data R. W .M., unpublished data R. W .M., unpublished data R:W. M., unpublished data R. W .M., unpublished data R. W .M., unpublished data R. W .M., unpublished data R. W .M., unpublished data R. W .M., unpublished data R. W .M., unpublished data R. W .M., unpublished data R. W .M . , unpublished data R. W .M., unpublished data R. W .M., unpublished data R. W .M., unpublished data R. W.M., unpublished data R. W .M . , unpublished data

*All tissues were homogenized together to produce a single extract. **Only a single locus was reported by Murphy et al. (1983), but a second was consistently present in all Eumeces examined, though not sufficiently resolved for allozyme scoring.