Natural Hybridization and Genetic Divergence Between the Toads Bufo boreas and Bufo punctatus

Natural Hybridization and Genetic Divergence Between the Toads Bufo boreas and BufopunctatusAuthor(s): Juliana H. FederSource: Evolution, Vol. 33, No. 4 (Dec., 1979), pp. 1089-1097Published by: Society for the Study of EvolutionStable URL: http://www.jstor.org/stable/2407469 .

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Evolution, 33(4), 1979, pp. 1089-1097

NATURAL HYBRIDIZATION AND GENETIC DIVERGENCE BETWEEN THE TOADS BUFO BOREAS AND

BUFO PUNCTATUS

JULIANA H. FEDER'

Museum of Vertebrate Zoology, University of California, Berkeley, California 94720

Received March 27, 1978. Revised March 23, 1979

Species of the toad genus Bufo are well known for their ability to produce viable hybrids under laboratory conditions (Blair, 1972). Additionally, many workers have documented natural hybridization in that genus (Blair, 1941, 1955; Volpe, 1952, 1960; Cory and Manion, 1955; Thornton, 1955; Fox et al., 1961; Guttman, 1967; McCoy et al., 1967; Zweifel, 1968; Brown and Guttman, 1970; Jones, 1973). A basic problem in field studies of hybridization is initial recognition of hybrid individuals. Often, intraspecific variation in morphology makes identification of hybrids difficult; that is, a hybrid may resemble an extreme morphological variant of one of the parental species. Electrophoretic tech- niques permit more direct analysis of an organism's genome than any known morphological approach and make possible demonstration of the genetic intermediacy of hybrids.

This study describes natural hybridization between two species of toads, Bufo boreas and B. punctatus, in a desert region of southwestern California. Morpho- logically these toads are strikingly distinct, being placed in different species groups by Tihen (1962) and Blair (1972). Bufo boreas (Fig. lc) is a large toad with elongate parotoid glands, a conspicuous light dorsal stripe and frequently, with extensive black dorsal spotting. Bufo punctatus (Fig. la) is smaller than B. boreas, has nearly spherical parotoid glands, lacks a

1 Current address: Committee on Evolutionary Biology, University of Chicago, Chicago, Illinois 60637.

dorsal stripe and has small red spots on its light dorsal surface. Neither species has prominent cranial crests.

The ranges of B. boreas and B. punctatus are parapatric (Fig. 2). Bufo boreas occurs from southern Alaska to northern Baja California, and eastward to the Rocky Mountains. It occupies diverse habitats, including high mountain mead- ows, desert springs, and the edges of lakes, ponds, rivers and streams. Bufo punctatus is distributed in parts of Texas, Oklahoma, the deserts of the southwestern United States, and southward into Mexico. It is found in semiarid to desert situations, especially in rocky canyons and arroyos.

Bufo boreas and B. punctatus occur together at the edges of their respective ranges at Darwin Canyon in the Argus Mountains of southeastern California (locality 1, Fig. 2). Although the ranges of the two species overlap in northern Baja California, they occupy different habitats within this region and are thus not sympatric. A permanent spring-fed stream in Darwin Canyon is the only known site of local sympatry. Discovery of toads intermediate in appearance between the two species at Darwin Canyon prompted a genetic analysis of the suspected hybridization, as well as a study of genic differentiation between the hybridizing species.

MATERIALS AND METHODS

Individuals representing both B. boreas and B. punctatus, as well as four hybrid individuals, were collected in Darwin Canyon, Argus Mountains, 5 km SW of Panamint Springs. Samples of B. punc-

1089



1090 JULIANA H. FEDER

a

cs b

FIG. 1. Bufo Punctatus (a), B. boreas x B. punctatus (b), and B. boreas (c) from Darwin Canyon. All photographs to the same scale. The bar indicates 25 mm.

tatus taken from outside the area of contact were also examined electrophoretically. All specimens are deposited in the Museum of Vertebrate Zoology, Univer- sity of California, Berkeley, California. The localities are indicated in Figure 2.

All toads were double pithed. Blood was collected from the aorta in heparin-

Bufo boreas

Sulfo punctktus

FIG. 2. Range of B. boreas (hatched area) and B. punctatus (stippled area). Hybridization occurs at locality 1. The localities are: 1: Darwin Canyon, Inyo Co., California; 2: Sabino Canyon, Pima Co., Arizona; 3: Boca de la Sierra (near Miraflores), Baja California Sur, Mexico; 4: 3.3 km W Agua Caliente, Baja California Sur, Mexico.

ized pipettes, and centrifuged to separate plasma and cellular fractions. Water sol- uble proteins were extracted by grinding aliquots of liver, heart, and skeletal muscle together with a volume of deionized water approximately equal to that of the tissue. Storage at -76 C and repeated thawing and refreezing of the tissue samples resulted in no change in mobility. Polymorphic loci were interpreted as systems of codominant alleles (Selander et al., 1971).



HYBRIDIZATION BETWEEN BUFO 1091

TABLE 1. Running conditions for electrophoresis.

Voltage Time Buffer type (V) (hr) Tissue Loci

Discontinuous Tris HCl pH 8.5 (1)* 250 1.5 hemolysate HB 1/ht/m** ADA

Discontinuous Lithium Hydroxide (2) 300 3 plasma ALB 1/ht/m GOT-1, GOT-2, LAP, PT-1

Discontinuous Tris Citrate (3 = Poulik) 250 2.5 1/ht/m ME, SOD Continuous Tris Citrate pH 8.0 (4) 130 3 1/ht/m ICD-1, ICD-2, MPI Continuous Phosphate pH 6.7 (7) 130 3 1/ht/m GPI, PEP, PGM Continuous Phosphate Citrate pH 8.0 (8) 100 5 1/ht/m GAPDH, MDH-1, MDH-2 Continuous Tris Citrate pH 7.0 180 3 1/ht/m a-GPD, LDH-1, LDH-2

* Numbers in parentheses following buffer type refer to buffer system of Selander et al., 1971. ** 1/ht/m = combined extract of liver, heart, and skeletal muscle. Continuous tris citrate pH 7.0 is buffer system c of Ayala et al., 1972.

Horizontal starch gel electrophoresis was carried out according to the tech- niques of Selander et al. (1971), Ayala et al. (1972) and Harris and Hopkinson (1976). Twenty-one presumptive genetic loci examined for electrophoretic variation were adenosine deaminase (ADA, E. C. 3.5.4.4), albumin (ALB), glucosephos- phate isomerase (GPI, E. C. 5.3.1.9), glu- tamate oxaloacetate transaminase (GOT-1 and GOT-2, E.C. 2.6.1.1) glyceraldehyde phosphate dehydrogenase (GAPDH, E. C. 1.2.1.12), glycerol-3-phosphate dehydrogenase (ax-GPD, E.C. 1.1.1.8), hemoglobin (HB), isocitrate dehydrogenase (ICD- 1 and ICD-2, E.C. 1.1.1.42), lactate dehydrogenase (LDH-1 and LDH-2, E.C. 1.1.1.27), leucine aminopeptidase (LAP, E.C. 3.4.11), malic enzyme (ME, E.C. 1.1.1.40), malate dehydrogenase (MDH-1 and MDH-2, E.C. 1.1.1.37), mannose phosphate isomerase (MPI, E. C. 5.3.1.8), muscle specific protein (PT-1), leucyl-ala- nine peptidase (PEP, E.C. 3.4.11), phos- phoglucomutase (PGM, E.C. 2.7.5.1), and superoxide dismutase (SOD, E.C. 1.15.1.1). Electrode and gel buffers, enzyme and protein assays and running conditions are summarized in Table 1. Three enzyme loci, ADA, GPI, and PEP, were stained using a 1%-agar overlay. Two additional systems were modified as follows: 1) 0.001 moles /3-mercaptoethanol and 0.0001 moles NAD were added to the phosphate citrate gels to demonstrate GAPDH activity, as recommended by Bruns and Gerald (1976); 2) 3.0 ml 0.1

molar MgC12 was substituted for MnCl2, which appeared to inhibit ICD-1 activity.

Electromorphs were compared using side-by-side alignment on a gel and were assigned values corresponding to their mobilities relative to the distance traveled from the origin by the common allele of B. boreas. Where two loci encoded an enzyme system (GOT, ICD, LDH, and MDH), the locus with bands migrating farther in an anodal direction was desig- nated 1; the other 2. All loci, except GOT- 2 and a single allele of GPI, migrated anodally.

Only loci in which the frequency of the common allele was 0.95 or less were con- sidered in calculations of percentages of polymorphic loci. Average individual heterozygosity was determined by direct count of the number of heterozygotes at all loci divided by the product of the number of individuals sampled from a population and the number of loci examined.

Nei's (1972) genetic distance, D, was calculated for all pairs of populations. Rogers' (1972) coefficient of genetic similarity, S, was also calculated to allow comparisons to other studies of allozyme variation in Bufo.

In order to ascertain the ploidy of the hybrid toads, karyotypes of those individuals were prepared using cells cultured from lung tissue.

RESULTS

Clear morphological differences exist between B. boreas and B. punctatus, with




TABLE 2. Variant frequencies at 17 loci polymorphic in B. boreas, B. punctatus and their hybrids. MDH- 2, GOT-2, ADA and LAP were monomorphic in all populations.

B. punctatus

B. boreas Hybrids 1 2 3 4 Locus Variant n = 25 n = 4 n = 25 n = 5 n = 6 n = 4

ICD-1 100 1.00 1.00 1.00 .95 1.00 1.00 84 .05

ICD-2 150 .13 100 .98 1.00 1.00 1.00 1.00 .87 50 .02

MDH-1 113 .50 1.00 1.00 1.00 1.00 100 1.00 .50

LDH-1 221 .50 1.00 .80 1.00 1.00 189 .20 100 1.00 .50

LDH-2 186 .50 1.00 1.00 1.00 1.0 100 1.00 .50

a-GPD 138 .36 .25 100 .64 .25 69 .50 1.00 1.00 1.00 1.00

SOD 120 .50 1.00 1.00 1.00 1.00 100 1.00 .50

MPI 100 1.00 .50 .10 .83 1.00 91 .30 .17 80 .50 1.00 .60

GPI 17 .50 1.00 1.00 .92 1.00 -100 1.00 .50 .08

PGM 100 1.00 .50 96 .50 1.00 1.00 .50 .50 79 .50 .50

PEP 113 .30 .13 .36 100 .70 .87 1.00 .70 1.00 .64 90 .30

GOT-1 2 70 .10 200 .44 .50 1.00 .90 1.00 1.00 100 .28 .13 14 .28 .37

ME 125 .50 1.00 1.00 1.00 1.00 100 1.00 .50

GAPDH 850 .50 1.00 500 .20 200 .80 1.00 1.00 100 1.00 .50

HB 108 .50 1.00 1.00 1.00 1.00 100 1.00 .50

PT-1 108 .50 1.00 1.00 1.00 1.00 100 1.00 .50

ALB 120 1.00 1.00 1.00 100 1.00 1.00 1.00

Percent polymorphism .142 0 .286 .143 .143 Average individual

heterozygosity .063 0 .095 .040 .071




TABLE 3. Nei's (1972) genetic distance (D), above the main diagonal; Rogers' (1972) coefficient of genetic similarity (S), below the main diagonal. Localities of B. punctatus are indicated in Fig. 2.

B. punctatus

B. boreas Hybrids 1 2 3 4

B. boreas .211 .911 1.011 .795 .922

Hybrids .692 .172 .277 .320 .288

B. punctatus 1 .369 .692 .105 .207 .156 2 .364 .639 .873 .115 .069 3 .441 .612 .808 .857 .052 4 .391 .621 .851 .899 .935

hybrids exhibiting intermediate morphology. The hybrids were all slightly smaller than adult B. boreas, and the parotoids were of intermediate size and shape. Dor- sal markings also exhibited characteristics of both species (see Fig. 1b); the median dorsal stripe was absent or reduced and the spotting patterns showed signs of both parental types.

Of the 21 loci examined for genetic variation in all individuals, 17 were polymorphic in one or more of the populations which were surveyed. Allele frequencies at these variable loci are found in Table 2. Bufo boreas from Darwin Canyon compared with all populations of B. punctatus examined in this study had different alleles at 11 loci: MDH-1, LDH-1, LDH-2, a-GPD, SOD, GPI, PGM, ME, GAPDH, HB, and PT-1. At five other loci (GOT-1, PEP, ALB, ICD-2, and MPI), certain populations of B. punctatus shared an allele with the Darwin Canyon B. boreas. At the remaining five loci, all individuals of both species and their hybrids were fixed for a single allele. At the Darwin Canyon locality the two species were com- pletely distinguished by 12 of 21 loci.

Values of Nei's genetic distance, D, and Rogers' genetic similarity, S, are found in Table 3. The D between Darwin Canyon B. boreas and B. punctatus is .911.

Genetic demonstration of hybridization is striking. Each hybrid individual was heterozygous for all 12 loci in which the two parental species at Darwin have different alleles. The frequency of variants at three additional loci (GOT-1, PEP, and ICD-1) in the hybrids was intermediate to

the allele frequencies of the parental species; for these loci, B. punctatus at Darwin is fixed for the common allele of B. boreas.

Genic differentiation, D, among the populations of B. punctatus is high for intraspecific comparisons. D ranges from .207 (between Darwin Canyon and Boca de la Sierra, Baja California) to .052 (between the two Baja California samples, from localities less than 10 km apart). Populations of B. punctatus exhibit great variation in allele frequencies. The Dar- win Canyon, California and Sabino Can- yon, Arizona populations share no alleles at two loci (ALB and GAPDH). Addition- ally, three loci (LDH-1, MPI, and GOT- 1) for which one allele is fixed at Darwin Canyon, are polymorphic in the Arizona population. Frequencies of the alleles which are absent at Darwin Canyon range from 0.10 to 0.40 in Sabino Canyon.

Levels of individual heterozygosity and percentages of polymorphic loci (Table 2) are low relative to other studies of genetic variation of Bufo (Rogers, 1973; Inger et al., 1974; Dessauer et al., 1975; Guttman, 1975; Matthews, 1975). The mean per- centage of polymorphic loci per population in all B. punctatus studied was 0.14 and the mean individual heterozygosity was 0.051. No variation whatsoever was found in the 25 individuals of B. punctatus from Darwin Canyon.

DISCUSSION

Morphological differences between B. boreas and B. punctatus permit easy iden-




tification of the two species, but the variable nature of external features of B. boreas makes detection of hybrids less certain than recognition of either parental species. In fact, one hybrid escaped detection on morphological grounds, and its proper identification was not noted until electrophoretic analysis. Corresponding to the morphological differences between these two species, the genic differentiation between B. boreas and B. punctatus is also great. The genetic distance between this congeneric pair of species is, in fact, higher than that reported among most species of a genus (see Ayala, 1975).

Although members of the genus Bufo have been studied electrophoretically for other purposes (e.g., to obtain variability estimates or to determine the degree of differentiation between intraspecific populations), there is only a single study of genic differentiation between two species in that genus. Rogers (1973) studied the genetic relationships between B. cognatus and B. speciosus, both members of the cognatus species group. Of the ten loci examined, five were fixed for the same allele in both species, three were polymorphic with the same allele common in both species, while only two were polymorphic with each species having a different allele in highest frequency. The two species examined in this study exhibit much greater differentiation than those Rogers studied. The hybridizing populations share no alleles at 12 of 2 1 loci. Bufo punctatus shares the common allele of B. boreas at only three others. Bufo boreas and B. punctatus are more distinct in morphology than are B. cognatus and B. speciosus and the species hybridizing at Darwin Canyon have been placed in different species groups by Tihen (1962) and Blair (1972). Thus, the large degree of genic differentiation is not unexpected.

Although hybridization between species as genically different as B. boreas and B. punctatus is not unique, neither is it common in vertebrates. Avise and Smith (1974) reported natural hybridization in Florida between two sunfish species, Le- pomis macrochirus and L. microlophus,

in which genic differentiation between the species was similar to that found in this study. Eight of 14 loci were fixed for different alleles in that case, while 12 of 2 1 were fixed for different alleles in the two species of Bufo in Darwin Canyon. In the lizard genus Cnemidophorus, diploid C. tesselatus have resulted from the past hybridization of C. tigris and C. septemvit- tatus (Davis and Selander, 1976). The genic identity (I of Nei, 1972) between the "parental" lizard species is .456, while I between B. boreas and B. punctatus is .402 (D = .911). In most other reported cases of vertebrate hybridization in nature, however, the parental species have been closely related (Patton et al., 1972; Hall and Selander, 1973; Hunt and Selan- der, 1973; Yang et al., 1974; Avise and Duvall, 1977; Szymura, 1977). Bufo boreas and B. punctatus are the most genically distinct terrestrial vertebrates reported to hybridize in nature. This study demonstrates that hybridization in Bufo can occur even between species differing greatly in their genic composition, with the hybrids surviving to adulthood under natural conditions.

In some instances of interspecific hybridization, hybrids display the alleles of only one of the parental species. Such repression of allelic expression has been attributed to the incompatibility of the regulatory genes and structural genes in hybrids (Whitt et al., 1973). Bufo boreas x B. punctatus hybrids, however, are en- tirely heterozygous at all loci which were differentiated in the parental species, sug- gesting compatible regulatory systems. Wilson et al. (1974) offered a mechanism by which hybrids may be produced between anurans with a large amount of genetic differentiation relative to other vertebrate groups. They suggested that regulatory and structural genes evolve at different rates, with much more variation in rates of regulatory gene evolution than in structural gene evolution. In particular, anurans may have undergone relatively little regulatory change. Thus, while B. boreas and B. punctatus exhibit considerable structural gene divergence, their regula-




tory genes may have differentiated only slightly. Relatively slow evolution of regulatory genes may account for the presence of viable hybrids in this instance.

Although natural interspecific hybridization in Bufo is known, hybridization between these two species at Darwin is surprising. A suite of reproductive isolat- ing mechanisms exist in this situation, including habitat preference differences, species-specific male mating calls, and differences in timing of reproduction in the two toads. By contrast, absence of introgression is not surprising as chromosomal imbalances effectively prevent backcrossing. The usual diploid chromo- some number in both B. boreas and B. punctatus is 22 (Bogart, 1972). One hybrid toad was karyotyped, and it had 33 chromosomes or three times the usual haploid complement. Most other interspecific hybrids in Bufo produced under laboratory conditions have also been shown to be 3N (Bogart, 1972). Gametes arising from triploid individuals are expected to have any number of chromosomes from the usual haploid complement to the diploid number. The triploid condition is probably highly unstable at sexual reproduction, and those triploid reptiles and amphibians which have been studied are unable to re- produce sexually (Uzzell, 1970; Maslin, 1971). Patton (1973) attributed lack of introgression in two species of pocket gophers also to chromosomal imbalances.

In the population at Darwin Canyon, all hybrids have been females, with small ovaries and no developed eggs; females of parental species collected at the same time have had large, egg-filled gonads. Other studies of natural hybridization in Bufo have also reported hybrids to be sterile, with atrophied gonads, and of only one sex. Volpe (1960) and McCoy et al. (1967) reported F1 hybrids to be sterile in crosses of B. fowleri x B. valliceps and B. woodhousei x B. punctatus, respectively. Rep- resentation of only one sex among the hybrids and the presence of nonfunctional gonads indicate that the genomes of the two species are only partially compatible. In none of these cases would the hybrids

contribute to genic exchange between the parental species.

In most well-studied cases, natural hybridization is attributed to recent ecological disturbances due to interference by agricultural or industrial man. At Darwin, hybridization between these two distantly related toads is occurring due to the pe- culiar narrow sympatry of B. boreas and B. punctatus. These species overlap in a desert oasis, which allows coexistence with nearly complete ecological isolation. Presumably, low levels of hybridization have been occurring for a considerable period, with no adverse effects on the parental species.

SUMMARY

Two toad species, Bufo boreas and B. punctatus, have parapatric distributions in western North America. They occur together, however, in a desert spring in southeastern California. Discovery of toads morphologically intermediate to the two species suggests that hybridization has occurred. Because intraspecific variation in morphology makes positive identification of hybrids difficult, starch-gel electrophoresis was carried out on 25 individuals of each parental species and four hybrids. The two species exhibit genic divergence corresponding to their morphological divergence. Nei's D between the species at the sympatric locality equals .911. This level of genetic differentiation is greater than any reported for a hybridizing pair of terrestrial vertebrates. In the two species, 12 of 21 loci are allelically distinct, two are polymorphic with the same alleles present, while the remaining seven are fixed for the same allele. All suspected hybrids are heterozygous at the 12 differentiated loci, with both parental alleles present.

ACKNOWLEDGMENTS

D. B. Wake, J. L. Patton, P. T. Speith, L. H. Throckmorton, R. J. Wassersug, and three anonymous reviewers offered suggestions on the manuscript. I profited from discussions with R. C. Stebbins, J. T. Mascarello, G. C. Gorman, and S. Y.




Yang. T. J. Papenfuss and C. Markmann aided by collecting specimens. G. M. Christman prepared Figure 2. Finally, I thank M. E. Feder for his aid during the field aspects of this work and for critically reading many versions of this manuscript.

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Natural Hybridization and Genetic Divergence Between the Toads Bufo boreas and Bufo punctatus