Herpetologists' League
Electrophoretic Evidence for the Specific Distinctness of Egernia modesta and E. whitii (Lacertilia: Scincidae) Author(s): D. A. Milton, J. M. Hughes, P. B. Mather Source: Herpetologica, Vol. 39, No. 2 (Jun., 1983), pp. 100-105 Published by: Herpetologists' League Stable URL: http://www.jstor.org/stable/3892547 . Accessed: 11/04/2011 22:43 Your use of the JSTOR archive indicates your acceptance of JSTOR's Terms and Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp. JSTOR's Terms and Conditions of Use provides, in part, that unless you have obtained prior permission, you may not download an entire issue of a journal or multiple copies of articles, and you may use content in the JSTOR archive only for your personal, non-commercial use. Please contact the publisher regarding any further use of this work. Publisher contact information may be obtained at . http://www.jstor.org/action/showPublisher?publisherCode=herpetologists. . Each copy of any part of a JSTOR transmission must contain the same copyright notice that appears on the screen or printed page of such transmission. JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact
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Herpetologica, 39(2), 1983, 100-105 ? 1983 by The Herpetologists' League, Inc.
ELECTROPHORETIC EVIDENCE FOR THE SPECIFIC DISTINCTNESS OF EGERNIA MODESTA AND E. WHITII (LACERTILIA: SCINCIDAE) D. A. MILTON, J. M. HUGHES, AND P. B. MATHER ABSTRACT: Ten enzyme loci (superoxidase dismutase, glutamatedehydrogenase, alcohol dehydrogenase, isocitrate dehydrogenase, phosphoglucomutase, hydroxybutyratedehydrogenase, lactate dehydrogenase, mannose phosphate isomerase, malate dehydrogenase and malic enzyme) were examined in sympatricpopulations of Egernia modesta and both plain-backed and patternbacked Egernia whitii. Five fixed allele differences (at hydroxybutyratedehydrogenase, lactate dehydrogenase, mannose phosphate isomerase, malate dehydrogenase and malic enzyme loci) indicated reproductive isolation between E. modesta and E. whitii. Apart from color pattern variation within E. whitii, no morphological differentiation was evident.
Key words: Egernia modesta; Egernia whitii; Enzyme loci; Reproductive isolation
THIS study involves two closely related forms of skink within the genus Egernia. The first (referred to here as E. whitii Lacepede) is polymorphic for dorsal pattern, with both patterned and plainbacked individuals occurring together. It occurs in southeastern Australia, as far north as southern Queensland. The second (E. modesta Storr) has a plain dorsum and is found from northern New South Wales north to the latitude of Fraser Island (Fig. 1), overlapping with E. whitii for a considerable part of its range. The two forms can be distinguished by the pattern of white spots on the lateral surface, which is well developed in E. whitii, extending from the ears to the hindlimbs. In E. modesta, the pattern is less distinct and is present only on the neck and shoulders. Storr (1968) recognized three subspecies: Egernia whitii whitii (the patternbacked form of E. whitii mentioned above), E. w. tenebrosa (the plain-backed form of E. whitii), and E. w. modesta, because he found no morphometric differences between them. Later, Horton (1972)
lowed by Cogger (1979). No evidence was provided, however, to suggest that the different color patterns were not produced simply by polymorphisms at individual loci, or closely linked loci. If one type was dominant to the other, intermediates would not be expected. The aims of the present study were twofold: (1) to re-examine morphological differences between the three forms, usstatistical ing the more sophisticated technique of canonical variates analysis (Blackith and Reyment, 1966); (2) to use gel electrophoresis of proteins to determine whether interbreeding occurred between the forms in the areas of range overlap. METHODS
Lizards were collected from a site 3 km west of Amiens in southeastern Queensland, where all three forms are sympatric (Fig. 1). Individuals were caught by moving the granite slabs under which they were resting. All animals caught from the area were included in the sample.
stated that E. w. whitii and E. w. modesta
Morphometric Study
should be regarded as distinct species because of the overlap in distribution without intermediate forms. Storr's E. w. tenebrosa was regarded as a morph of E. whitii. Horton's suggestions were fol-
Eight morphometric and two meristic characters were measured for each individual (Table 1). The technique of canonical variates analysis (Blackith and Reyment, 1966) 100
June 1983]
HERPETOLOGICA
101
was used to determine whether the three forms could be separated on the basis of the measurements of 11 characters. Canonical variates analysis produces maximum separation between groups by deriving components that best separate group means in the measurement space. The analysis was carried out using a FORTRAN 10 computer program developed by Dr. T. Whiffin, La Trobe University, on a DIGITAL DEC10 computer. Electrophoretic Techniques Specimens were killed by freezing and the livers were removed and homogenized with 10 gl of buffer solution (0.1 M Tris EDTA, 2 mg NADP, adjusted to pH 6.2 with concentrated HCI). They were then spun at 10,000 rpm for 30 min below 4 C in a Sorvall RC5B centrifuge. The supernatant was decanted into vials and stored at -20 C until used. Horizontal starch slabs were used for electrophoresis, with one of the following buffer systems: 0.01 M Tris-borateEDTA pH 8.5 (Redfield and Salini, 1980), 0.01 M Tris-maleate-EDTA pH 7.4 (Selander et al., 1971) and the lithium hydroxide buffer system of Eanes and Koehn (1978). For each individual, the following proteins were examined, using staining techniques of Selander et al. (1971): (1) On Tris-borate-EDTA pH 8.5: glutamate dehydrogenase (Gdh), lactate dehydrogenase (Ldh), isocitrate dehydrogenase (Idh), superoxidase dismutase (Sod); (2) On Tris-maleate-EDTA pH 7.4: hydroxybutyrate dehydrogenase (Hbdh), phosphoglucomutase (Pgm), alcohol dehydrogenase (Adh); (3) On Tris-citrate/LiOH: malic dehydrogenase (Mdh), malic enzyme (Me) and mannose phosphate isomerase (Mpi). Interpretation of banding patterns on gels and identification of homozygous and heterozygous individuals followed methods and assumptions discussed by Manwell and Baker (1970). Values of Nei's genetic distance (Nei, 1971) were calculated to examine genetic differentiation between forms.
N
Brisbane
AMIENS
Sydney
tP ?
Soo
R
E. modesta
m:: E. whitil
Scale in Kilometers
FIG. 1.-Map of Eastern Australia showing the distributions of E. modesta and E. whitii.
RESULTS
The means and standard deviations for all morphological characters are shown in Table 2. Using the Student's t-tests, no significant differences were found be-
tween E. whitii and E. modesta (P > 0.05) nor between the two color forms of E. whitii (P > 0.05). Fig. 2 shows the results of the canonical variates analysis, showing the first canonical variate plotted against the second. No separation occurred between the groups, although there was more differentiation between E. whitii and E. modesta than between the two forms of E. whitii. Banding patterns for each of Sod, Gdh, Adh, Idh and Pgm were identical in the three morphs (Table 3), indicating that these loci were monomorphic for the same electromorph in all morphs. Fixed allele
TABLE
1.-Descriptions
of the eight morphometric and two meristic characters measured in this study.
Character
Snout-vent length Right forelimb length Right hindlimb length Head length Head width Snout-ear length Snout-forelimb
length
Axilla-groin length Mid-body scales Lamellae
Description
Distance from anterior margin of the rostrum to the anterior margin of the vent Distance from the posterior margin of the forelimb at its insertion to the extremity of the longest digit Distance from the extremity of the fourth toe to the posterior margin of the hindlimb at its insertion Distance from the anterior margin of the rostrum to the posterior margin of the parietal scales Maximum transverse distance, anterior to the auricular opening Distance from the anterior margin of the rostrum to the anterior margin of the auricular opening Distance from the anterior margin of the rostrum to the anterior margin of the right forelimb at its insertion Distance from the posterior margin of the right forelimb at insertion to the anterior margin of the right hindlimb at insertion Number of scales around the center of the body Number of keeled lamellae under the fourth digit of the right hindlimb
differences between morphs were observed at Hbdh, Ldh, Mpi, Me and Mdh loci. Me was monomorphic in E. modesta, and polymorphic for two different alleles in E. whitii. Ldh patterns were multibanded in both E. whitii and E. modesta, always with three closely spaced slow-moving bands and a single fast band. E. modesta zymograms were clearly distinguishable from those of E. whitii, with corresponding bands running slightly faster in E. modesta. As we were unsure of genetic interpretation of these pat-
TABLE
[Vol. 39, No. 2
HERPETOLOGICA
102
terns, we have included only one locus for Ldh in -Table 3. However, possibly two are involved, as is the case with Ldh in many other vertebrates (Harris and Hopkinson, 1976). The genetic distance calculated between E. modesta and both forms of E. whitii was 0.668 + 0.326 and between the two forms of E. whitii was 0.002 + 0.002. These values should be treated with some caution due to the relatively small number of loci sampled (see Nei and Roychoudhury, 1974).
2.-Means and standard deviations (SD) for each of the characters included in the multivariate analysis. E. whitii (plain-backed) (n =18)
E. modesta (n =40) Character
Snout-vent length (mm) Right forelimb length (mm) Right hindlimb length (mm) Head length (mm) Head width (mm) Snout-ear length (mm) Snout-forelimb length (mm) Axilla-groin length (mm) Mid-body scales Lamellae
E. whitii (pattern-backed) (n=30)
x
SD
I
SD
X
SD
77.9 21.4 30.39 16.01 11.74 17.54 29.88 40.14 34.22 25.65
21.56 4.63 7.22 3.33 3.02 3.94 7.29 11.89 2.37 2.02
83.09 24.09 35.49 16.91 12.85 18.73 32.38 41.57 37.39 27.28
18.07 4.52 6.85 2.79 2.70 3.62 6.22 9.48 2.59 1.71
77.59 22.67 33.65 16.29 12.26 17.93 30.41 39.48 37.13 27.70
19.49 5.92 7.03 3.13 3.01 3.56 6.85 10.56 2.30 1.90
103
HERPETOLOGICA
June 1983]
0
*
0 *
0
2
~~~~~~~~~~~~~~0
U
*
0
0
*
0 0
~~~~~4~~ ~
*
:
-I
0~~~~~~~~~~~~
3r
* * 0
*
0
~~~~~00
0.
0
0
0
2
0
0 .
0
4 Variate I ~~~~~~~~~~~~~~~~~~~~~~ 0
0 0
0
0
00 0
0c
0
0
~~~~~~~~~~~~0
0
* *
E.' modesta E. whitii (p.tter-backed) E. whitii (plain - becked)
-3
FIG. 2.-Results of canonical variates analysis, showing the first canonical variate plotted against the second. Larger symbols represent means for each group.
TABLE
3.-Allele frequencies at the 10 loci analyzed, for Egernia whitii and Egernia modesta. Alleles at each locus are lettered in order from the fastest moving to the slowest moving. E. modesta
E. whitii
(plain-backed)
E. whitii
(pattern-backed)
Allele
(n = 40)
(n = 18)
Superoxidase dismutase Glutamate dehydrogenase Alcohol dehydrogenase Isocitrate dehydrogenase Phosphoglucomutase Hydroxybutyratedehydrogenase
a a a a a a
1.00 1.00 1.00 1.00 1.00 1.00
1.00 1.00 1.00 1.00 1.00
1.00 1.00 1.00 1.00 1.00
b a b
1.00
1.00
Lactate dehydrogenase
1.00
Mannose phosphate isomerase
a
Locus
Malate dehydrogenase Malic enzyme
b a b c a -b
-
1.00
1.00
1.00
1.00
1.00 1.00 -
(n = 30)
-
0.41 0.59
0.58 0.42
1.00
1.00
1.00
HERPETOLOGICA
104 DISCUSSION
The multivariate technique of canonical variates analysis did not separate the two forms into non-overlapping groups, as might be expected if they were distinct species (Mayr, 1971). Also there was no separation between plain-backed and within E. pattern-backed individuals whitii (Fig. 2). When two populations occur in sympatry in at least part of their geographic ranges, then the technique of gel electrophoresis may provide conclusive evidence of reproductive isolation and, therefore, species distinctness. If fixed allele differences are found between the two populations, and if no heterozygotes occur, then it can be concluded that no interbreeding is occurring between them. Electrophoretic data in this study provide convincing evidence of reproductive isolation between E. whitii and E. modesta, with fixed allele differences at five of the 10 loci sampled. Neither plainbacked nor pattern-backed forms of E. whitii appear to interbreed with the plainbacked E. modesta. There is no evidence of reduced interbreeding between the two back pattern morphs of E. whitii. Other comparisons between closely-related species report varying numbers of fixed allele differences. Kim et al. (1978) reported that seven out of 34 loci exhibited fixed differences between species of the genus Typhlosaurus. In contrast, Gartside et al. (1977) found no fixed differences between two species of ribbon snakes (Thamnophis) that occurred sympatrically without interbreeding. Halliday (1981) recorded one fixed allele difference (15 loci sampled) between sympatric species of meat ants, while Redfield and Salini (unpublished data) recorded five fixed differences out of 26 loci sampled in sympatric prawn species. In comparison, the results reported here do not imply unusually low levels of genetic differentiation between the two species, and yet morphological differentiation since speciation appears to have been very limited.
[Vol. 39, No. 2
The lack of morphological divergence between the two species may reflect geographic isolation under similar selection pressures. The present direct contact may be a result of recent alteration of the habitat by humans. E. modesta is normally found in open areas with little or no shrub cover whereas E. whitii usually prefers areas with more vegetation. Clearing in areas originally inhabited by E. whitii may have allowed E. modesta to colonize areas that were originally unfavorable.We suggest that these two species may be in the final phase of the speciation process as hypothesized by Mayr (1971), with reproductive isolation complete and natural selection favoring both morphological and ecological divergence between the two forms. No real morphological divergence has yet taken place. Evidence for ecological divergence is discussed in another paper (Milton and Hughes, unpublished). In order to make more concrete suggestions as to the evolutionary history of the two species, populations from other parts of the species' range would need to be sampled. thank Carla Catterall for Acknowledgments.-We comments on the original draft and Kaye Ingold for typing the manuscript.
LITERATURE CITED K. E., AND R. E. REYMENT. 1966. Multivariate Morphometrics. Academic Press, London. COGGER, H. G. 1979. Reptiles and Amphibians in Australia.Reed, Sydney. EANES, W. F., AND R. K. KOEHN. An analysis of genetic structure in the monarch butterfly Danaus plexippus. Evolution 32:784-797. GARTSIDE, D. F., J. S. ROGERS, AND H. C. DESSAUER. 1977. Speciation with little genic and morphological differentiation in the ribbon snakes Thamnophis proximus and T. sauritus (Colubridae). Copeia 1977:697-705. HALLIDAY, R. B. 1981. Heterozygosity and genetic distance in sibling species of meat ants (Iridomyrmex purpureus group). Evolution 35:234242. HARRIS, H., AND D. A. HOPKINSON. 1976. Handbook of Enzyme Electrophoresis in Human Genetics. North Holland Press, Amsterdam. HORTON, D. R. 1972. Evolution in the genus Egernia (Lacertilia:Scincidae).J. Herpetol. 6:101BLACKITH,
109.
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KiM, Y. J., G. C. GORMAN, AND R. B. HUEY.
1978. prawn enzymes (Penaeus spp. and Metapenaeus Genetic variation and differentiation in two spp.). CSIRO Div. Fish Oceanogr. Rep. 116, Melspecies of the fossorial African skink Typhlosaubourne. rus (Sauria:Scincidae). Herpetologica34:192-194. SELANDER, R. K., M. H. SMITH, S. Y. YANG, W. E. MANWELL, C., AND C. M. A. BAKER. 1970. MoJOHNSON, AND J. B. GENTRY. 1971. Biochemilecular Biology and the Origin of Species: Hetcal polymorphism and systematics in the genus erosis, Protein Polymorphism and Animal BreedPeromyscus. I. Variation in the old field mouse ing. Sidgwick and Jackson, London. (Peromyscus polionotus). Studies in genetics VI. Univ. Texas Publ. 7103:41-90. MAYR, E. 1971. Populations, Species and Evolution. Belknap, Massachusetts. STORR, G. M. 1968. Revision of the Egernia MILTON, D. A., AND J. M. HUGHES. 1982. Possible whitii species group(Lacertilia:Scincidae).J. Roy. evidence for reproductive isolation of three forms Soc. W.A. 51:51-62. of Egernia. J. Herpetol.: In review. Accepted: 14 December 1982 NEI, M. 1971. Genetic distance between populaAssociate Editor: Stephen Tilley tions. Am. Nat. 106:283-292. NEI, M., AND A. K. ROYCHOUDHURY. 1974. SamSchool of Australian Environmental pling variances of heterozygosity and genetic disStudies, Griffith University, Nathan, tance. Genetics 76:379-390. REDFIELD, J. A., AND J. P. SALINI. 1980. Tech- Queensland, 4111, Australia [Note: send niques of starch gel electrophoresis on penaeid reprint requests to JMH] Herpetologica, 39(2), 1983, 105-110 ? 1983 by The Herpetologists' League, Inc.
GASTROTHECAAUREOMACULATA:SYSTEMATIC RESOLUTION OF A CASE OF INTRAPOPULATIONAL POLYCHROMATISM IN A HYLID FROG WILLIAM
E. DUELLMAN
ABSTRACT: Gastrotheca aureomaculata occupies a restricted area on the eastern slopes of the Cordillera Central in Colombia. The dorsum is dark green to brown with many pale spots or uniform green; both patterns and intermediates have been found at the same site. Individuals that were uniformly green formed the basis of the type series of G. mertensi, a name herein placed in the synonymy of G. aureomaculata. The spotted pattem on a dark ground color presumably develops ontogenetically.
Key words: Anura; Hylidae; Gastrotheca aureomaculata; Colombia; Polychromatism
Two apparently distinct species of marsupial frogs, Gastrotheca, from the eastern slopes of the Cordillera Central in southern Colombia were named and described by Cochran and Goin (1970). Gastrotheca aureomaculata was diagnosed as being inky blue with greenish gold flecks, whereas G. mertensi was stated to be uniformly green dorsally. Both species were reported from Moscapan on the basis of specimens sent to the late Doris M. Cochran by Marte Latham, a professional collector. In their gazeteer to collecting localities in Colombia, Cochran and Goin (1970:529) simply list-
ed "Moscapan (region east of Popay'an) Cauca-Huila boundary." The exact location of this locality has been a problem for numerous biologists who have studied material collected by Latham. During field work in Colombia in 1974, 1975 and 1979, I attempted to find Moscapan. Kjell von Schneidern, biologist and long-time resident of Popayan, indicated to me that Moscapainwas a region on the eastern slopes of the Cordillera Central, east of the PaTramo de Purace. Field work in that area in 1974 and 1975 did not result in the location of Moscap'annor the collection of Gastrotheca. In March 1979,
