Herpetologica, 39(3), 1983, 20C211 1983 by The Herpetologists' League, Inc.

PHYLOGENETIC RELATIONSHIPS O F T H E NORTH AMERICAN FIVE-LINED SKINKS, GENUS EUMECES

(SAURIA: SCINCIDAE)

ROBERT W. MURPHY, WILLIAM E. COOPER, JR., AND WILLIAM S. RICHARDSON

ABSTRACT: Allozyme data are used to examine the taxonomic validity and phylogenetic re- lationships of the three species of North American five-lined skinks. All three species are found to be electroph~reticall~ distinguishable. A cladistic evaluation ofthe allozymes leads to the conclu- sion that Eumeces laticeps, E . inexpectatus and E. fasciatus form a nionophyletic group with the former two species sharing the most recent common ancestor.

Key words: Reptilia; Sauria; Scincidae; Eumeces inexpectatus; Eumeces laticeps; Euineces fasciatus; Phylogenetics; Biochemical systematics; Genetic variability

EUMECE~ LATICEPS and E. inexpectatus were first recognized as distinct species by Taylor (1932), who subsequently (Taylor, 1935) combined three North American five-lined skinks and nine East Asian species to form the fasciatus group. His hypothetical phylogenetic tree (Tay- lor, 1935:38) depicted E. laticeps, E. fas- ciatus and E. inexpectatus as more closely related to each other than to Asian mem- bers of the fasciatus group. Taylor be- lieved that E. inexpectatus and E. fascia- t u s shared the most recent common ancestor.

Recognition of E. laticeps and E. inex- pectatus as distinct species was generally accepted, but doubts remained until Da- vis (1968) confirmed their taxonomic va- lidity. Using discriminant function anal- ysis to examine morphological characters, Davis was able to assign all but a small fraction of specimens to one of the three

North American species of the fasciatus group. Contrary to Taylor, he believed that E , laticeps and E. fasciatus were more closely related to each other than to E. inexvectatus. Several characters, in- cluding color pattern, the presenck of narrow median subcaudals, the number of caudal scale rows. the degree of head - widening in adult males, and fewer mis- identifications of E. inexpectatus by dis- criminant functions, supported this posi- tion.

Interest in phylogenetic, taxonomic and ecological relationships among the three North American species of the fasciatus group continues in part because of simi- larities in morphology and distribution. Members of the group are broadly sym- patric over most of their ranges (Conant, 1975) and are morphologically very sim- ilar, differing only in minor scalation fea- tures and, to some extent, in maximum

September 19831 HERPETOLOGICA 201

TABLE 1.-Summary of Eumeces utilized in this study.

Populatinu --

E . laticeps 1 2

E . inexpectatus 1 2 3

4 E , fasciatus

1 2 3 4 5 6

E . copei 1

E . skiltonianus 1

Locality Museuni deposition*

Alabama; Montgomery Co. South Carolina; McCormick Co.

Georgia; Marion Co. Virginia; Westmoreland Co. North Carolina; Tyrrell Co.

South Carolina; McCormick Co.

Alabama; Elmore Co. Virginia; Powhatan Co. South Carolina; Abbeville Co. North Carolina; Tyrrell Co. Alabama; Autauga Co. Tennessee; Anderson Co.

Mexico; Morelos

California; Riverside Co.

CAS 155478-155498 CAS 15549S155501

CAS 155502-155503 USNhl 234086, 234340-234345 USNhl 23424S234245,

23427 1-234274, 234303-234304

CAS 155504-155505

CAS 155506155515 CAS 155517 CAS 155516 USNM 234379 CAS 155518-155521 CAS 155522

BYU 37139

CAS 155523

* BYU = Brigham Young University, Provo, Utah; CAS = California Acadenly of Sciences, San Francisco; USNM = Nat~nnal Museuni of Natural History, Smithsonian Institution, Washington, D.C.

body size. Despite sustained interest in these three similar species, there has been little progress since Taylor's (1935) monograph toward substantiating phylo- genetic relationships. Although recent authors (Greer, 1970; Hikida, 1978) have questioned Taylor's (1935) hypothesis of evolution in North America and dispersal to Asia, there have been no attempts to study the ~hylogenetic relationships of North American species nor to show that these members of the fasciatus group form a monophyletic group.

We present allozyme data that corrob- orate the validity of North American species of the fasciatus group and clarify their phylogenetic relationships. Our null hypotheses (following Murphy and Ott- ley, 1980) are the following: E. fasciatus, E . laticeps and E. inexpectatus are not valid biological species because each freely exchanges genes with at least one other member of the North American complex of the fasciatus group. To reject any of these null hypotheses we must demonstrate that the given "species" is not freely exchanging genes with any other "species."

MATERIALS AND METHODS Collection data, sample sizes, and dis-

position of voucher specimens are pre- sented in Table 1.

Lizards were frozen and maintained at -42 C; within 1 mo following freezing, the skinks were partially thawed to facil- itate removal of heart, liver and skeletal muscle (femoral andlor pectoral). Tissues were then minced with scissors, diluted with an approximately equal volume of water, and mechanically homogenized. Tissue homogenates were refrozen for 18 h, then thawed and centrifuged at 20,000 g for 20 min at 2 C. The supernatant frac- tions were used for electrophoretic anal- ysis.

Horizontal starch gel electrophoretic techniques (Selander et al., 1971; Yang et al., 1974), including multiple buffer sys- tems and various potential voltages, were utilized to visualize enzymatic and non- enzymatic presumptive gene products. These products, electrophoretic condi- tions, and stain references are given in Table 2; enzyme nomenclature used fol- lows the recommendations of the No- menclature Committee of the Interna-

TA

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(m

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rial

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P

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P

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Sha

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970)

S

haw

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(197

0)

Har

ris

and

Hop

kins

on (

1976

) H

arri

s an

d H

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nson

(19

76)

Har

ris

and

Hop

kins

on (

1976

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76)

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ris

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1976

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0)

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980)

H

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(19

76)

Sel

ande

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(19

71)

Sel

ande

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(19

71)

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ande

r et

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(19

71)

Sel

ande

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(19

71)

But

h an

d M

urph

y (1

980)

B

rew

er (

1970

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haw

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71)

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(19

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71)

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(19

71)

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(19

71)

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ande

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71)

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71)

Sel

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(197

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(19

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Mur

phy

and

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1980

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(19

80)

Mur

phy

and

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1980

)

September 19831 HERPETOLOGICA 203

tional Union of Biochemistry (1979). Allozyme data were treated as products of codominant alleles at a given locus. Al- lele frequencies (Table 3) were calculat- ed from phenotypic data and used to compute genetic similarity ( I ) and genet- ic distance (D) coefficients (Nei, 1972).

Our basic goals were to biochemically differentiate the North American species of the fc~sciatus group and clarify their phylogenetic relationships. For these purposes, various methods were used to analyze the allozyrne data. Genetic sim- ilarity and distance coefficients within the fasciatus group were compared to those of other sister species of vertebrates and were subjected to cluster analysis by the unweighted pair group method with arithmetic means (Sneath and Sokal, 1973); allozyme data were examined on a locus-by-locus basis considering each locus to be a character and allelic com- position the character states. Data from outside reference species allowed deter- mination of evolutionary polarity of the character states (Watrous and Wheeler, 1981).

The products of 43 presumptive gene loci were scored for five species of Eu- meces from 13 populations. One addi- tional population of E. inexpectatus (pop- ulation 4, Table 1) was scored for 42 of these 43 loci (Acp-1 products were not resolved). We found 14 loci to be mono- allelic in all populations (Table 2). Allelic frequencies of the 29 polymorphic loci scored for 13 populatiorls are given in Ta- ble 3. For population 4 of E. inexpectatus (Table l ) , only the S-Mdh-A locus was found to be polymorphic; the S-Mdh-Aa allele had a frequency of 0.75, and S-Mdh- Ah 0.25. All other gene products were the same as the allele most commoil to the other three populations of E. i t~expecta- tus.

Three of the 43 loci scored could not be specifically identified. The allelic products of one unidentified locus were resolved as a cathodally expressed sys-

204 HERPETOLOGICA IVOZ. 39. N O . 3

0 0 0 0 0 0 0 0 0 0 0 0 0 21 I", I I q l q l q l I z q l I I 112 I q z l 11211 I S 121

m w w o o o o o o o o o o o 3 m 2 2 2 I 1 2 2 I I 1 2 1 2 1 2 19 2 I I I 2 I I 1 2 I 1 2 I 2 I 122

m w t - 0 0 0 0 0 0 0 0 0 0 0 0 0

2 8 2 2 1 2 2 I I 1 2 1 2 12 1 2 I q I I I q I I 1 2 I 1 2 I 2 I 1 2 I

m r - m t-0 0 0 0 0 0 0 0 0 0 0 I z z z 132 I I rq I q I 2 12 I q I I I q l I I q I Is, I s I I ? I

0 o t - m 0 0 0 0 0 0 0 0 0 0 2 1222 1s I I I y q I 2 I 2 I I q I I q I I I q I I", 2 I

d e o d c i o d e o 6 s d e m e d e o d e o-o d s l d e o m e o m d a d e o

September 19831 HERPETOLOGICA 205

0 0 0 0 0 0 0 0 0 0 0 0 0

", I I I " , 12 I I I", I", 112 12 l I 2 I 2 l I l I 2 I IS

206 HERPETOLOGICA [Vol. 39. No. 3

tem appearing in addition to PGM in samples specifically stained for that en- zyme. This "Pgm-like" system (Table 2) was easily resolved and could not be identified with any other system exam- ined in this study. (Frequently this locus is referred to as a second P g ~ n system al- though the enzyme has not been specif- ically identified.) Only a single pheno- type for hemoglobin could b e scored although this protein is a tetramer encod- ed in vertebrates by at least two loci (a and 0; Goodman et al., 1975; Hochachka and Somero, 1973). Thus the single phe- notype was conservatively scored as two monomo~phic loci, Hb-1 and Hb-2.

Estimates of allozyme variability with- in the various populations of the fascia- tus group are given in Table 4 based on the observed number of heterozygotes. Although sample sizes are small, hetero- zygosity values for all three species are fairly low, ranging from only 2.56 to 3.75% in the largest population sampled for each species. Such low values, however, are well within the range reported for other lizards (Gorman et al., 1977).

In general, genetic similarities of I = 0.8 or less (or genetic distances of D = 0.2 or greater) occur among sister species of reptiles and other vertebrates (Adest, 1977; Avise and Ayala, 1976; Murphy, 1983, unpublished data). Genetic simi- larity values (Table 5) between E. inex- pectutus and E. laticeps range from 0.82- 0.87 (f = 0.84), slightly higher than the observed values for other species pairs. Genetic similarity values between E. inexpectatus and E. fasciatus vary from 0.68-0.76 (.r = 0.72). Between E. laticeps and E. fasciatus, genetic similarity val- ues ranged from 0.64-0.71 (.v = 0.67). A phenogram constructed by clustering the genetic similarity values of 11 popula- tions of North American skinks of the fas- ciatus group using the unweighted pair group method with arithmetic means (Sneath and Sokal, 1973) is given in Fig. 1.

The presence andlor absence of unique alleles may be used to differentiate and

September 19831 HERPETOLOGICA 207

TABLE 4.-Summary of genetic variability in sampled populations of three species of North American five-lined skinks. Population numbers refer to Table 1.

Population

Frequency of minor allele 9% loci poly- Alleles % h e t e r o ~ ~ g o s i t y

detection morphic* per locus per individual**

E . laticeps 1 2 1 0.238 14.0 1.16 2.56 2 3 0.167 - - 4.72

E . inexpectatus 1 2 0.250 - - 2.32 2 7 0.071 - - 1.71 3 9 0.056 11.6 1.14 2.96 q*** 2 0.250 - - 1.19

* A locus was cons~dered polymorphic if more than one allele was detected. Percent loci polymorph~c and alleles per locus est~mates are provided only for the largest samples of each species.

** Based on the number of heterozygotes. *** Acp-1 products were not resolved in this population; variability estimates are based upon a total of 42 loci.

identify populations. Table 3 shows E. inexpectatus to have three alleles that are unique to the species: Acp-2% S-Mdh-Ab and Pep-la. Of these only one is fixed (Pep-la); the alternative alleles at Acp-2 and S-Mdh-A are shared with both E. fas- cintus and E. laticeps. These data suggest that perhaps E. inexpectatus is a "good" biological species, but they leave some room for doubt because only a single al- lele is both unique and fixed. We cannot be certain that the locus is not polymor- phic in E. inexpectatus because of rela- tively small sample sizes and limited number of sampled populations. Alleles of' two loci appear to be unique and fixed in E. laticeps, Sod-AIa and Cat-Ab. The Acp-2"allele appears to be unique to E. laticeps. These findings strongly contrast with those of E. fascintus, in which seven loci (Ada-2, Ada-3, Est-1, Est-3, G-6-pdh- A, Mpi-A and Pep-2) exhibit fixed differ- ences, and four additional loci (Gtdh-A, S-Mdh-A, Pep-3 and Pgm-A) have appar- ently unique alternative alleles (Table 3).

Additional elucidation is possible by examining the patterns of shared alleles. In considering the three North American species of the fasciatu,s group, we have seven possible combinations of allelic distributions: (a) alleles unique to a sin-

gle species (three combinations); (b) al- leles common to all three species (one combination); and (c) alleles shared be- tween two species but not by the third (three combinations). We have already examined the first category of possible combinations. The second category can provide only absence, or negative data, and cannot be used to reject our null hy- pothesis. Thus we must rely on the third category for information. The three pos- sible species pairs sharing an allele ab- sent in the third are as follows: (1) E. inexpectatus-E. laticeps; (2) E. inexpec- tatus-E. fasciatus; and (3) E. laticeps-E. fasciatus.

Eumeces inexpectatus and E. laticeps share 12 alleles absent in E. fasciatus (Table 3). Similarly, E. fasciatus and E. inexpectatus share one allele not detect- ed in E. laticeps. And finally, E . laticeps and E. fasciatus share one allele not found in E. inexpectatus. These patterns of sharing, when combined with our pre- vious observations on the distributions of the unique alleles, easily differentiate the three North American species of the fas- ciatus group. We conclude from these electrophoretic data that E. fizsciatus, E. laticeps and E . inexpectatus are indeed distinct species.

208 HERPETOLOGICA TVol. 39, No. 3

September 19831 IIERPETOLOGICA 209

The higher average genetic similarity between E. inexpectatus and E. 1aticep.s (Fig. l) , reflecting the greater number of shared alleles, suggests that these two species may be more closely related to each other than either is to E. fasciatus. Significantly, this implication is in dis- cord with the views of both Taylor (1935) and Davis (1968). However, the data on genetic similarity only imply this conclu- sion-they do not provide proof. To con- clusively show that E. inexpectatzis and E. laticeps shared the most recent com- mon ancestor, s~na~omorph i e s (shared derived character states) must be dem- onstrated.

Stevens (1980) reviewed methods and criteria for determining evolutionary po- larity of character states, noting assump- tions and pitfalls; the best method was determined to be out-group analysis of character state distributions. However, for out-group analysis to be efficient, the nearest sister group need not be used (Watrous and Wheeler, 1981) as Stevens (1980) proposed. Because the phyloge- netic relationships within the genus Eu- meces have not been detailed, we chose two species of North American skinks, E. skiltonianzis from California and E. copei from central Mexico, to form a taxonomic out-group (TOG). The TOG was used to determine the evolutionary polarity of the character states of E . inexpectatzts, E. fas- ciatus and E. laticeps, the taxonomic in- group (TIC).

We based our phylogenetic interpre- tation of the observed character state dis- tributions on Watrous and Wheeler (1981), with the following clarifications. (1) Any allele (character state) at a given locus (character) found to be common to both the TIC and TOG is considered to be a plesiomorphy (primitive character state). (2) When no alleles at a given locus are shared between the TOG and the TIC, no evolutionary polarity is assigned. (3 ) When the plesiomorphic character state is determined, all remaining alleles at a given locus are considered apomorphies (derived character states). (4) A polymor-

E laticeps

7 1 )

Genetic Similar i ty ( / )

FIG. 1.-Phenogram depicting the average value of genetic similarity (I) between 11 populations of five-lined skinks of the North American fasciatus group of Eurneces.

phic condition is considered apomorphic unless both alleles are shared with the TOG--when the derived allele is shared with other populations, the polymorphic condition is considered to be synapomor- phic. (5) Synapomorphies are the direct result of sharing a common ancestor. (6) By using synapomorphies, a cladogram depicting a hypothesis of the true phy- logenetic relationships of organisms can be constructed.

In view of these clarifications, we ex- anlined character state distributions and evolutionary polarities within each char- acter. From Table 3, we determined that only four characters (loci) have synapo- morphic character states: Acp-1, S-Acon- A, Gpi-A and Pep-4. Alleles at the Acp-1 locus confirm our assumption that the TIC constitutes a monophyletic group; Acp-lb and Acp-lc are apomorphic alleles pres- ent in all species of the TIG. (Acp-la is the plesiomorphic allele being shared by at least one species of the TIC and one species of the TOG.)

The presumptive gene loci S-Acon-A, Gpi-A and P e p 4 have character states in- terpreted as synapomorphies linking E. laticeps and E. inexpectatus. The S-Acon- Ab, Gpi-Abnd Pep-4'! alleles are derived. The most parsimonious phylogenetic hy- pothesis based upon these data is shown in Fig. 2. There are no synapomorphies

210 HERPETOLOGICA rvol. 39. NO. 3

FIG. 2.-Cladogram showing the phylogenetic re- lationships of the North American species of the Eumeces fasciatus group. Solid bars represent the synapomorphic character states as follows: 1 = S-Acon-Ab; 2 = Gpi-A\ 3 = Pep-4c.

linking E. inexpectatus and E. fasciatus, or E. fasciatus and E. laticeps; there is no homoplasy. The phylogenetic analysis shows that E. inexpectatus and E. lati- ceps shared the most recent common ancestor; this agrees with the phenetic UPGMA analysis and differs from the phenetic analyses of the morphological characteristics (Davis, 1968; Taylor, 1935).

The electrophoretic data also raise questions concerning relative similarity levels between members of the fasciatus groups within Eumeces. The close elec- trophoretic similarity observed between the TIG and TOG (E. copei and E. skil- tonianus) was surprising. The observed average genetic similarity between E. copei and the TIG is I = 0.57 and for E. skiltonianus is I = 0.62. Indeed, the val- ues of genetic similarity between our TIG and TOG are greater, for example, than those observed for members of the North American lizard genera Phzjllodactzjlus (Murphy and Papenfuss, 1980), Urosau- rus (Murphy, 1983), Cnemidophorus (Murphy, unpublished data), Sceloporus

(Murphy, 1983) and Anolis (Gorman et al., 1983). Although the data are fragmen- tary, they point to the necessity for fur- ther investigation of genetic differentia- tion within Eumece.~.

Ack11ow1edgments.-For assistance in the field andlor donation of specimens, we thank Blair Hedges, George Middendorf, Richard Montanucci, John Ottley, Carl Putnam, Steve Ruth and Stanley Trauth. Donald Buth and Michael Miyamoto pro- vided technical assistance; George Gorman provid- ed necessary laboratory space. Donald Buth, Laurie Vitt and three anonymous reviewers provided valu- able editorial comments. Margaret Kowalczyk pre- pared the figures. This work was partially supported by a grant-in-aid from Auburn University at Mont- gomery and the UCLA Department of Biology Fish- eries Program (D. G. Buth).

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AVISE, J. C., J. 1. SMITH, AND F. J. AYALA. 1975. Adaptive differentiation with little genic change between two native California minnows. Evolu- tion 29:411-426.

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BUTH, D. G., AND R. W. ~ ' ~ U R P H Y . 1980. Use of nicotinamide adenine dinucleotide (NAD).de. pendent glucose-6-phosphate dehydrogenase in enzyme staining procedures. Stain Technol. 55: 173-176.

CONANT, R. 1975. A Field Guide to Reptiles and Amphibians of Eastern and Central North Amer- ica, 2nd ed. Houghton Mifflin Co., Boston.

DAVIS, D. M. 1968. A study of variation in North American lizards of the fasciatus group of the ge- nus Eumeces (Scincidae). Ph.D. Dissertation, Duke University.

GOODMAN, M., G. W. MOORE, AND G. MATSUDA. 1975. Evolution of vertebrate hemoglobin amino acid sequences, pp. 181-206. In C. L. Markert (Ed.), Isozymes IV. Genetics and Evolution. Ac- ademic Press, New York.

CORMAN, G. C., D. G. BUTH, h4. SOULE, AND S. Y. YANG. 1983. The relationships of the Puerto Ri- can Anolis: electrophoretic and karyotypic stud- ies. In A. Rhodin (Ed.), Advances in Herpetology and Evolutionary Biology. Harvard University Press, Cambridge. In press.

GORMAN, G. C., Y. J. KIM, AND C. E. TAYLOR. 1977. Genetic variation in irradiated and control pop- ulations of Cnemidophorus tigris (Sauria, Tei-

September 19831 HERPETOLOGICA 211

idae) from Mercury, Nevada with a discussion of genetic variability in lizards. Theoret. Applied Genet. 49:%14.

GREER, A. E., JR. 1970. A subfamilial classifica- tion of scincid lizards. Bull. Mus. Comp. Zool. 139: 151-184.

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Accepted: 10 March 1983 Associate Editor: Stephen Tilley

RWM: Department of Biology, Univer- sity of California at Los Angeles, Los An- geles, CA 90024, USA, and Research As- sociate, Department of Herpetology, California Academy of Sciences, Golden Gate Park, Sun Francisco, C A 94118, USA; WEC: Department of Biology, Au- burn University at Montgomery, Mont- gomery, A L 36193, USA; WSR: Depart- m e n t of Physical Sciences, A u b u r n University at Montgomery, Montgomery, A L 36193, USA