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Blood Protein Variation in the Bufo americanus Species Group of Toads Author(s): Sheldon I. Guttman Source: Copeia, Vol. 1969, No. 2 (Jun. 3, 1969), pp. 243-249 Published by: American Society of Ichthyologists and Herpetologists (ASIH) Stable URL: http://www.jstor.org/stable/1442072 . Accessed: 28/08/2014 14:56 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp . 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 [email protected]. . American Society of Ichthyologists and Herpetologists (ASIH) is collaborating with JSTOR to digitize, preserve and extend access to Copeia. http://www.jstor.org This content downloaded from 82.29.144.13 on Thu, 28 Aug 2014 14:56:24 PM All use subject to JSTOR Terms and Conditions

Blood Protein Variation in the Bufo americanus Species Group of Toads

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Blood Protein Variation in the Bufo americanus Species Group of ToadsAuthor(s): Sheldon I. GuttmanSource: Copeia, Vol. 1969, No. 2 (Jun. 3, 1969), pp. 243-249Published by: American Society of Ichthyologists and Herpetologists (ASIH)Stable URL: http://www.jstor.org/stable/1442072 .

Accessed: 28/08/2014 14:56

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp

.JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact [email protected].

.

American Society of Ichthyologists and Herpetologists (ASIH) is collaborating with JSTOR to digitize,preserve and extend access to Copeia.

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Blood Protein Variation in the Bufo americanus Species Group of Toads'

SHELDON I. GUTTMAN

The transferrin and hemoglobin components of the six species of toads of the Bufo americanus group were polymorphic. Thirteen molec- ular types of transferrin and ten hemoglobin components appeared in the individuals examined. In several instances interspecific hybridiza- tion may be responsible for the individual variation in these com- ponents.

INTRODUCTION

N investigation of blood proteins of members of the genus Bufo was under-

taken to supplement evidence of evolution- ary relationships derived from other criteria. Great individual variation was found in electrophoretic patterns of plasma proteins of the Bufo americanus species group of toads. This variation was sufficient to negate any possible calculation of affinities between the B. americanus and other Bufo species groups. Analyses of transferrins and hemo- globins, specifically, indicate that these com- ponents are highly variable. This paper presents my findings on the multiple molec- ular forms of plasma transferrin and hemo- globin in the six species of the B. americanus species group. These species are the Amer- ican toad, B. americanus; the Dakota toad, B. hemiophrys; the Houston toad, B. hous- tonensis; the southwestern toad, B. micro- scaphus; the southern toad, B. terrestris; and the Rocky Mountain toad, B. woodhousii.

The B. americanus group has been the subject of an intensive study of genetic com- patibility relationships (W. F. Blair, 1963a). The significance of mating call as an etholog- ical isolating mechanism in the group was discussed by W. F. Blair (1956, 1957a, 1957b, 1958), and ecological differences were found important in maintaining species integrity (A. P. Blair, 1941, 1955). Chromatograms of the parotoid gland secretions of various species in the group were examined by Hunsaker, et al. (1961) and Wittliff (1962). Abramoff, et al. (1964) used serology to test the relationships of B. americanus from islands in upper Lake Michigan. Blood proteins of some species were studied by Fox,

1 Part of a dissertation submitted in partial fulfill- ment of the requirements for the degree of Doctor of Philosophy in the Graduate School of the University of Texas.

et al. (1961), Brown (1964), and Hebard (1964). No transferrin or hemoglobin poly- morphism was noted. Of the nine species of Bufo examined by Dessauer, et al. (1962) the transferrins of only B. terrestris varied.

MATERIALS AND METHODS

Seventy-seven specimens were examined. The sex and collection site of each toad sampled are listed in Table 1.

Blood samples were obtained by severing the left carotid artery and drawing up the flowing blood with a pipette rinsed with a 4% solution of (ethylenedinitrilo) tetraacetic acid (EDTA). Centrifugation for 15 min at approximately 1500 rpm separated the cells from the plasma. The plasma was frozen at -5? C until it was used. Red blood cells were washed three times in 1% saline. They were lysed by the addition of a volume of distilled water five times that of the packed cells, then frozen at -5? C and then thawed.

Upon thawing, the mixture was centrifuged for 15 min. The supernatant was imme- diately subjected to electrophoresis. No mobility differences were noted between fresh and stored (up to three years) trans- ferrin.

The technique employed was vertical starch-gel electrophoresis modified from Smithies (1959), and Kristjansson and Hick- man (1965). Hemoglobin gels were prepared using a tris-borate buffer (0.114 M tris, 0.05 M boric acid), pH 8.5. A sodium-borate buffer (0.06 M NaOH, 0.3 M boric acid), pH 8.0, was used for transferrins. The procedure for transferrin preparation was described by Guttman (1967). Autoradiography with Fe59, using the procedure of Giblett, et al. (1959), confirmed that all components designated as transferrins by their solubility in rivanol were iron-binding proteins. Electrophoresis of transferrins was continued for 20 hr at

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COPEIA, 1969, NO. 2

TABLE 1. SEX AND COLLECTION SITE OF TOADS, Bufo, EXAMINED FOR TRANSFERRIN AND/OR HEMOGLOBIN PHENOTYPE. (Letters refer to individuals whose transferrin and

hemoglobin patterns are illustrated in Figs. 1 and 2.)

B. americanus

B. hemiophrys B. houstonensis

B. microscaphus B. terrestris

a-g c h ?

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B. woodhousii

21? C with a gradient of 15 v/cm. Hemo- globin samples were subjected to electro- phoresis for 4 hr at 21? C with a gradient of 35 v/cm. Gels were sliced in half so that the two halves were mirror images of each other. One half of a hemoglobin gel was stained with orthodianisidine stain; the other half and one half of a transferrin gel were exposed to Buffalo Black NBR (Allied Chem- ical). Because of the possibility of differences in migration distances due to different lots of starch, slight differences in the pH of buffers, temperature fluctuation, etc., pro- teins could be analyzed only by comparing relative mobilities. These were calculated by measuring the distance each band migrated from the center of the fraction to the origin and dividing this number by the mobility of a human blood sample which was present on each gel. The mobilities of toad trans- ferrins and hemoglobins were compared to human transferrin C and hemoglobin A, respectively. Transferrins and hemoglobins were considered to have identical mobilities when the relative mobility of one was ? .005 of the other. Whenever there was doubt as to identity, the samples were rerun in adja- cent slots.

RESULTS

Transferrins.-No individual possessed more than two iron-binding components.

Transferrin with a relative mobility of .68 was observed in all 15 B. woodhousii from

North Bay, Cochrane, Ont., Canada Minn.

Minn.

3 miles E of Bastrop, Tex. Intervale Ave., Houston, Tex.

2 miles E of Virgin, Utah

Bay Co., Fla. Jasper Co., S.C. Jasper Co., S.C. McIntosh Co., Ga.

1 mile N of Mesa, Ariz. 3 miles E of Bastrop, Tex. Austin, Tex. St. George, Utah 18 miles N of Fredericksburg, Tex.

Mesa, Arizona. This component was also present in three animals from St. George, Utah, one from Bastrop, Texas, and one from Austin, Texas. Another, faster-moving, component with a relative mobility of .81 was present in four of the last five specimens. The transferrin at .81 was found in five other toads from Bastrop, Texas, and one other from Austin, Texas. Two of these Bastrop, Texas B. woodhousii showed a second band at .94. One animal from Fredericksburg, Texas, showed this faster- moving component plus another transferrin with a relative mobility of .56.

Two transferrins with relative mobilities of .40 and .53 were noted in one B. amer- icanus from Minnesota. Three bands were present in three combinations in the seven toads from Ontario, Canada. All had a

component at .81; one showed a slower- moving band at .68 and three others had a faster-moving transferrin at .94.

Eight of the nine B. houstonensis exam- ined had a component with a relative mobil- ity of .94. One had a second band at .87 and three had a slower-moving transferrin with a relative mobility of .81. The ninth individual had components at .8i and .73.

All B. terrestris sampled had a transferrin with a relative mobility of .73. However, two individuals had a second band at .61, one had a faster band at .81 and another had a band unique for the group at .85.

Ten of the 16 B. microscaphus tested had

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GUTTMAN-BUFO AMERICANUS GROUP

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Fig. 1. Individual transferrin phenotypes of the Bufo americanus group toads examined. Vertical axis indicates relative mobility; individuals sampled from each species are indicated by small letters along horizontal axis.

a transferrin with a relative mobility of .64. Two of these 10 toads also possessed a band at .86. Two others had a component at .68; a third pair of toads possessed a transferrin at .78. The other four animals possessed just the one band. The remaining individ- uals showed a component with a relative mobility of .61. Two of these animals pos- sessed a second band at .78; another toad showed a second band at .75.

The two transferrins noted in the one B. americanus from Minnesota had mobilities identical to those of two B. hemiophrys. The slower of these components (.40) was present in a third B. hemiophrys but this animal also showed a faster-moving band (.66). Four other individuals possessed only the more rapid of the two Minnesota B. americanus bands.

The transferrin phenotypes of the 77 animals are shown in Fig. 1.

Hemoglobins.-Five phenotypes were evi- dent from the 12 B. woodhousii examined. A toad from Bastrop, Texas had one hemo- globin component with a relative mobility of .94. The hemoglobin of five other speci-

mens from Bastrop, Texas, and one from Fredericksburg, Texas, migrated at a relative mobility of 1.00; the hemoglobin of two specimens from Austin, Texas migrated at 1.05; the hemoglobin of two individuals from Utah had a relative mobility of 1.08. One specimen from Utah had a band that was faster-moving than any other B. wood- housii sampled (1.14).

The hemoglobin of the one B. americanus examined migrated with a relative mobility of .97.

Five B. terrestris from South Carolina possessed the same two hemoglobin compo- nents, the slower one with a relative mobility of .87 and the faster one migrating at 1.05. One of two individuals from Georgia had two bands but the mobilities differed from those of the South Carolina specimens (.94 and 1.12). Three other B. terrestris possessed one hemoglobin each. One animal from Florida and the second toad from Georgia showed the same slow-moving band as the South Carolina individuals. The second Florida specimen had a band at .91.

The one B. hemiophrys examined pos-

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COPEIA, 1969, NO. 2

1.15-

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Fig. 2. Individual hemoglobin phenotypes of the Bufo americanus group toads examined. Ver- tical axis indicates relative mobility; individuals sampled from each species are indicated by small letters along horizontal axis.

sessed a hemoglobin with a relative mobility of .91.

Four B. microscaphus showed the same

component with a relative mobility of 1.00. Two B. houstonensis from Houston, Texas

were sampled. Their single component, with a relative mobility of .79, was the slowest-

moving of the americanus group. The hemoglobin patterns of the 29 animals

sampled are illustrated in Fig. 2.

DISCUSSION

It is evident from the data presented that transferrin and hemoglobin components are

polymorphic within species of the amer- icanus group. Thirteen molecular types of transferrin appeared in the 77 individuals. Other workers also noted intraspecific varia- tion in the blood proteins of Bufo. Variation was found in the serum proteins of B. amer- icanus and B. woodhousii (Brown, 1964), the

plasma proteins of B. woodhousii and B. valliceps (Fox et al., 1961), the transferrins and hemoglobins of B. regularis and B.

rangeri (Guttman, 1967), and the transfer- rins of B. terrestris (Dessauer et al., 1962).

The mechanism of inheritance of trans- ferrin is becoming increasingly well known.

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GUTTMAN-BUFO AMERICANUS GROUP

Studies of fish (Barrett and Tsuyuki, 1967; Koehn and Johnson, 1967; and others), toads (Fox et al., 1961), birds (Desborough and Irwin, 1966; and others) and mammals (Goodman et al., 1965; and others) indicated there are multiple, codominant transferrin alleles at a single locus. Thus an individual homozygous at the transferrin locus will usually possess a single transferrin band (see Ashton, 1965; Guttman, 1967 for other situations); a pair of bands usually indicates the heterozygous condition.

Although the adaptive value of particular hemoglobin combinations is known (Ingram, 1963; Manwell et al., 1963), available data provide no evidence to show that transferrin types differ in their ability to transport iron. However, several possible explanations such as balanced polymorphism, fluctuating en- vironmental conditions in a region and therefore differential selection, and inter- specific hybridization (Guttman, 1967) have been offered to explain protein polymor- phism. Interspecific hybridization appears to contribute to the variation within this species group and is discussed below.

B. woodhousii probably represents the earliest speciation from the americanus group ancestral type (W. F. Blair, 1962; and others). It is currently found in sympatry with all other members of the group except B. hemio- phrys. Fragmentation of the other species probably occurred later, with the nominal species, B. houstonensis, possibly being a post-Pleistocene isolate of B. americanus (W. F. Blair, 1963b).

Within the americanus group all species are genetically compatible and hybrids be- tween any two species are intermediate mor- phologically (W. F. Blair, 1963a). A. P. Blair (1941) stated that wherever any combination of B. americanus, B. terrestris or B. wood- housii occur together hybrids are to be ex- pected; this statement was later revised to include hybridization between B. woodhousii and B. microscaphus (A. P. Blair, 1955). Brown (1967) collected hybrids between B. woodhousii and B. houstonensis in central Texas. Morphological evidence of secondary intergradation between B. americanus and B. hemiophrys was presented by Henrich (1968). In the present study, a toad from Minnesota, identified as B. americanus by morphological criteria, possessed the same two transferrins as two B. hemiophrys from the state (Compare Fig. 1, B. americanus h and B. hemiophrys). All B. hemiophrys ex-

amined showed at least one of these two bands. These were not found in B. amer- icanus from the other locality sampled. The presence of B. hemiophrys type transferrins in a B. americanus may possibly be the result of hybridization and introgression although W. F. Blair (pers. comm.) believed these two species are allopatric there. If they normally occupy the same environment in Minnesota, selection for identical transferrins can be postulated. However, Breckenridge (1944) extended the range of B. americanus west- ward across Minnesota to the forest-grassland boundary. If the B. arericanus with the B. hemiophrys type transferrins was collected in the grassland, then its transferrin type may be explained by the same selection pressures as are evidently maintaining the B. hemiophrys transferrins. On the other hand, if the animal was found in the ecotone or the forest, common selection pressures can be excluded. Specific locality data are unavailable.

W. F. Blair (1957b) found that a structural analysis of the breeding calls of B. micro- scaphus and B. woodhousii showed that they interbreed in the Virgin River drainage area in southwestern Utah, and thought that introgression occurred. In four localities where no B. woodhousii were found, some apparent hybrids with B. microscaphus were noted; in the same number of localities the opposite situation prevailed (A. P. Blair, 1955). B. microscaphus was collected in the vicinity of Phoenix, Arizona, where B. wood- housii is known to occur (Riemer, 1955), and some B. woodhousii collected in New Mexico were thought to have B. microscaphus charac- ters (Gehlbach, 1956). In the present study two of the 16 toads identified as B. micro- scaphus possessed a transferrin found in 20 of 27 B. woodhousii sampled, including all B. woodhousii that could have been sym- patric with the former species. The hemo- globin of two toads that were intermediate in external appearance was the same as that of B. microscaphus while another pre- sumed natural hybrid had an intermediate hemoglobin (Guttman, unpubl.). All three individuals possessed transferrins character- istic of both species.

Hybrids between B. houstonensis and B. woodhousii possessed hemoglobins and trans- ferrins characteristic of both parents (Gutt- man, unpubl.). All seven B. houstonensis from Bastrop, Texas, and one of the two from Houston, Texas, had a transferrin

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COPEIA, 1969, NO. 2

band that was also present in two B. wood- housii from the former locality and in two natural hybrids. These two B. woodhousii and one from Fredericksburg, Texas, were the only toads of this species with this com- ponent. The transferrin typical of B. hous- tonensis that appeared in the B. woodhousii from Fredericksburg, Texas, was approxi- mately 130 miles from where it was possibly introduced into the species. The band was not noted in the B. woodhousii collected in Austin, Texas. Before gene flow over this great a distance can be postulated, B. wood- housii from intermediate localities must be collected and found to possess this band. An alternate explanation is that during the Pleistocene, when mesic conditions prevailed on the Edwards Plateau in Texas, the dis- tribution of B. houstonensis included the Fredericksburg region. If this situation existed, the two species may have interbred as they now do at Bastrop, Texas.

Explanations are not presently available for the remaining polymorphism. Within this species group, B. microscaphus possessed the greatest transferrin variability. Six molec- ular types of iron-binding proteins were present in the 16 animals sampled. Of these, only one could be attributed to interspecific hybridization. Five different hemoglobins and four different transferrins were present in the ten B. terrestris sampled. Transferrins were distributed in four patterns of one or two components each. Hunsaker et al. (1961) found that chromatographic differences in parotoid secretions of 10 B. terrestris from 10 locations were almost as great as the differences noted when they compared the three species of toads tested. In the present study, one of the four transferrin morphs present in B. woodhousii possibly resulted from introgression with B. houstonensis but none of the four hemoglobins noted were shared with any other members of the group. A total of three transferrins were noted in the blood of B. americanus and three others in the blood of the B. hemiophrys sampled; except for the two bands in the one B. amer- icanus from Minnesota, these were not shared. A. P. Blair (1941, 1947) showed that morphological characters of B. americanus from different populations varied; no two localities were alike. Intraspecific variation in serum antigens of B. americanus was demonstrated by Abramoff et al. (1964).

Samples of at least 50 individuals from each of many populations are necessary

before the natural variation within popula- tions and within species can be understood and statistically significant gene frequencies can be calculated. The author is presently examining blood proteins in a variety of populations of B. woodhousii and B. amer- icanus with the intention of answering some of the interesting questions raised in this preliminary paper.

ACKNOWLEDGMENTS

I am indebted to Dr. W. F. Blair for super- vising this research and supplying many of the animals. My wife, Ruth, was of constant assistance throughout the study. Much of this work was supported under NSF grant No. GB4128 in Environmental and System- atic Biology to the University of Texas.

LITERATURE CITED

ABRAMOFF, P., R. DARNELL, AND J. BALSANO. 1964. Serological relations of toad popula- tions of the Lake Michigan area. In: Taxo- nomic biochemistry and serology. C. A. Leone, ed., pp. 515-525. Ronald Press, New York.

ASHTON, G. C. 1965. Serum transferrin D. al- leles in Australian cattle. Austral. J. Biol. Sci. 18:665-670.

BARRETT, I. AND H. TSUYUKI. 1967. Serum transferrin polymorphism in some scombroid fishes. Copeia 1967(3):551-557.

BLAIR, A. P. 1941. Variation, isolation mech- anisms and hybridization in certain toads. Genetics 26:398-417.

1947. Variation of two characters in Bufo fowleri and B. americanus. Am. Mus. Novit. No. 1343, 5 pp.

. 1955. Distribution, variation, hybrid- ization in a relict toad (Bufo microscaphus) in southwestern Utah. Ibid. No. 1722, 38 pp.

BLAIR, W. F. 1956. Call difference as an isola- tion mechanism in southwestern toads (genus Bufo). Texas J. Sci. 8:87-106.

1957a. Mating call and relationships of Bufo hemiophrys Cope. Ibid. 9:99-108.

. 1957b. Structure of the call and re- lationships of Bufo microscaphus Cope. Copeia 1957(3):208-212.

. 1958. Mating call in the speciation of anuran amphibians. Am. Nat. 92:27-51.

1962. Non-morphological data in anuran classification. Syst. Zool. 11:72-84.

. 1963a. Intragroup genetic compati- bility in the Bufo americanus species group of toads. Texas J. Sci. 15:15-34.

. 1963b. Evolutionary relationships of North American Bufo: A progress report. Evolution 17:1-16.

BRECKENRIDGE, W. J. 1944. Reptiles and am- phibians of Minnesota. Univ. Minnesota Press, Minneapolis.

BROWN, L. E. 1964. An electrophoretic study of variation in the blood proteins of the toads, Bufo americanus and Bufo woodhousei. Syst. Zool. 13:92-95.

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1967. The significance of natural hybridization in certain aspects of the specia- tion of some North American toads (genus Bufo). Ph.D. thesis, Univ. of Texas, Austin.

DESBOROUGH, S. AND M. R. IRWIN. 1966. Addi- tional variation in serum proteins in Colum- bidae. Physiol. Zool. 39:66-69.

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Fox, W., H. C. DESSAUER, AND L. T. MAUMUS. 1961. Electrophoretic studies of blood pro- teins of two species of toads and their natural hybrid. Comp. Biochem. Physiol. 3:52-63.

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GUTTMAN, S. I. 1967. Transferrin and hemo- globin polymorphism, hybridization and in- trogression in two African toads, Bufo reg- ularis and Bufo rangeri. Comp. Biochem. Physiol. 23:871-877.

HEBARD, W. B. 1964. Serum-protein electro- phoretic patterns of the Amphibia. In: Taxo- nomic biochemistry and serology. C. A. Leone, ed., pp. 649-657. Ronald Press, New York.

HENRICH, T. W. 1968. Morphological evidence of secondary intergradation between Bufo

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HUNSAKER, D., R. E. ALSTON, W. F. BLAIR, AND B. L. TURNER. 1961. A comparison of the ninhydrin positive and phenolic substances of parotoid gland secretions of certain Bufo spe- cies and their hybrids. Evolution 15:352-359.

INGRAM, V. M. 1963. The hemoglobins in genetics and evolution. Columbia Univ. Press, New York.

KOEHN, R. K. AND D. W. JOHNSON. 1967. Serum transferrin and serum esterase polymorphisms in an introduced population of the bigmouth buffalofish, Ictiobus cyprinellus. Copeia 1967 (4):805-808.

KRISTJANSSON, F. K. AND C. G. HICKMAN. 1965. Subdivision of the allele TfD for transferrins in Holstein and Ayrshire cattle. Genetics 52: 627-630.

MANWELL, C., C. M. A. BAKER, AND W. CHILDERS. 1963. The genetics of hemoglobin in hybrids -I. A molecular basis for hybrid vigor. Comp. Biochem. Physiol. 10:103-120.

RIEMER, W. J. 1955. Comments on the dis- tribution of certain Mexican toads. Herpeto- logica 11:17-23.

SMITHIES, 0. 1959. An improved procedure for starch-gel electrophoresis: further variations in the serum proteins of normal individuals. Biochem. J. 71:585-587.

WITTLIFF, J. L. 1962. Parotoid gland secretions in two species groups of toads (genus Bufo). Evolution 16:143-153.

DEPARTMENT OF ZOOLOGY AND PHYSIOLOGY, MIAMI UNIVERSITY, OXFORD, OHIO 45056.

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