Speciation in the Rana pipiens Complex. Department of Biological Sciences, Illinois State University, Normal, Illinois 61761

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1 AMER. ZOOL., 13:73-79 (1973). Speciation in the Rana pipiens Complex LAUREN E. BROWN Department of Biological Sciences, Illinois State University, Normal, Illinois SYNOPSIS. The long held view that leopard frogs (Rana pipiens complex) are a single widely distributed species is not correct. Five sibling species are currently recognizable. These findings have important implications on the use of leopard frogs in experimental research. INTRODUCTION In choosing an animal for use in experimental research there are a number of factors that need to be evaluated. A reliable body of knowledge about the animal's taxonomic status is certainly one of the most important requirements. Leopard frogs of the Rana pipiens complex are among the most widely used animals for experimental research in the United States, but there has still been much controversy in regard to their taxonomy. The object of this paper is: (1) to review the single species hypothesis that has been the predominant view of leopard frog taxonomy in vogue since the 1940's; (2) to review new evidence that the R. pipiens complex consists of a number of sibling species; and (3) to discuss the implications of these new developments on experimental research involving leopard frogs. DEVELOPMENT OF THE SINGLE SPECIES CONCEPT In the earlier part of this century several workers (e.g., Dickerson, 1906; Kauffeld, 1937; Stejneger and Barbour, 1933, 1939; Wright and Wright, 1933) recognized two or more species of leopard frogs (e.g., R. brachycephala, R. pipiens, R. sphenocephala). Other workers (e.g., Boulenger, 1920; Kellogg, 1932) regarded all leopard frogs in North America as belonging to a single wide ranging species. In 1944, J. A. Moore published the first comprehensive I thank J. R. Brown, J. L. Frehn, J. P. Bogart, and W. F. Blair for critically reading the manuscript. monograph on the taxonomy of leopard frogs of Eastern North America. A number of morphological characters were examined (body proportions, pigmentation, secondary sex characters) and many measurements were made. Although some significant differences were found between different populations, overlap in characteristics was common and he found it difficult to distinguish previously described forms (R. brachycephala, R. pipiens, R. sphenocephala). Moore (1944) thus concluded that only a single widely distributed species of leopard frog occurred in Eastern North America and that this species should be known as R. pipiens. The results of artificial laboratory hybridizations involving leopard frogs from a number of localities were summarized by Moore (1946). When northern frogs (Vermont, Wisconsin, New Jersey) were crossed with southern frogs (Louisiana, Texas, central Florida, southern Florida), the hybrids showed morphological defects and slower rates of development. The most pronounced morphological defects and high mortality rates were in crosses between frogs from populations that were separated by the greatest geographical distances (Wisconsin X Texas, Vermont X southern Florida). In a later series of crosses (Moore, 1947), frogs from Vermont were hybridized with animals from Mexico (Nuevo Leon and San Luis Potosi). Hybrids of Mexican females X Vermont males were equivalent to hybrids of Texas females X Vermont males in degree of abnormality, but hybrids of Vermont females X Mexican males had even more severe defects. Almost all 73

2 74 LAUREN E. BROWN embryos died in Vermont X Mexico crosses. Moore (1946, 1947) thus concluded that there was a north-south latitudinal gradient. He postulated that when the parents were from adjacent localities along the gradient, the hybrids would be normal. However, as the north-south distance between the parental populations progressively increased, there was also a progressive increase in hybrid abnormality. Embryonic temperature adaptations of leopard frogs from different localities were summarized by Moore (1949). Embryos from northern localities were more resistant to lower temperatures than embryos from southern localities, while southern embryos tolerated higher temperatures better than northern embryos. Embryos from Louisiana were the exception; they had the higher upper tolerance limit characteristic of southern frogs and the lower tolerance limit characteristic of northern frogs. In rate of development, northern embryos were faster than southern embryos at lower temperatures, whereas southern embryos were faster than northern embryos at higher temperatures. In temperature coefficient of embryonic development, southern frogs were greater than northern frogs. Northern populations had greater egg diameters than southern populations with the exception of Mexican eggs which had greater diameters than eggs from all other localities. Moore (1949) pointed out that the north-south differences in temperature adaptations of leopard frogs were comparable to the difference in temperature adaptation between northern-adapted species and southern-adapted species. Further artificial hybridizations (Moore, 1950) involved females from Vermont with males from high elevations in Colorado (8,300 feet) and Costa Rica (3,700 feet). Vermont X Colorado hybrids were normal and Vermont X Costa Rica hybrids had only minor abnormalities. Moore (1950) concluded that the relative normalcy of these crosses was due to similar adaptations of the frogs at all three localities to cold temperatures. Volpe (1954) compared temperature tolerances and rates of development of embryos from Wisconsin and Mexico (Tamaulipas). Mexican embryos were better adapted to high temperatures than Wisconsin embryos, whereas Wisconsin embryos were better adapted to low temperatures than Mexican embryos. There was also a high level of genetic incompatibility when frogs from the two areas were crossed none of the hybrid embryos survived. Ruibal (1955) reported an altitudinal cline in leopard frogs from Mexico in regard to embryonic developmental rate and certain morphological traits. Laboratory crosses of Mexican highland X Mexican lowland' frogs, and Vermont X Mexican lowland frogs produced abnormal offspring. In contrast, Vermont X Mexican highland hybrids were the least abnormal. It was thought that the highland populations were derived from lowland populations, and that high altitude frogs evolved convergently to Vermont frogs in regard to morphology. Although other supporting studies were completed, the above papers form the main nucleus of the single species hypothesis. In summary, this hypothesis involves three primary contentions: (1) The leopard frogs of North America all belong to a single widely distributed species (JR. pipiens) that shows considerable variation; (2) adjacent populations along a north-south gradient are genetically compatible, but as the north-south distance between populations increases, there is a progressive increase in their genetic incompatibility; and (3) there are north-south, and high altitudelow altitude clines in embryonic temperature adaptations. Moore (1957) thought that there was a common genetic basis for north-south genetic incompatibility and north-south differences in temperature adaptations. He also concluded (Moore, 1946, 1957) that the extreme northern and southern populations possessed sufficiently developed reproductive isolating mechanisms that they could act as distinct species if the intermediate populations were eliminated. These views were predominant for many years and were virtually unchal-

3 SPECIATION OF LEOPARD FROGS 75 lenged until relatively recently. NEW EVIDENCE THAT THERE ARE SEVERAL SIBLING SPECIES OF LEOPARD FROGS The first important clue that the single species hypothesis might be incorrect was provided by McAlister (1962). He found three types of leopard frogs in Texas that differed in certain morphological characteristics. One of these "morpho-units" was found in eastern Texas, another occurred in northern Texas, and the third was distributed in central, southern, and western Texas. Most significant was the discovery that mating calls of frogs from Travis County (central Texas) recorded at 21 C had pulse rates twice as fast as frogs from Hardin County (eastern Texas) at 21 C. McAlister (1962) suggested that changes in selection pressure were responsible for differences in the "morpho-units" but he did not specify that his studies had any taxonomic implications. Post and Pettus (1966) were able to distinguish two types of leopard frogs in eastern Colorado on the basis of five contrasting morphological characters. One type was referred to as the "DF complex" (signifying discontinuous dorso-lateral folds) while the other was called the "CF complex" (indicating continuous dorso-lateral folds). There was no evidence of clinal variation or intergradation between the complexes and they were not found to occur sympatrically. It was concluded that gene flow between the complexes was greatly reduced or lacking. Polymorphism in the electrophoretic mobility of hemoglobins in leopard frogs from the coastal plain and Piedmont of Maryland was reported by Gillespie and Crenshaw (1966). For hemoglobin type Hb I there were two electrophoretically different forms (Hb Is, Hb If) that were interpreted as forming an allelic system. "Coastal Plain" and "Piedmont" populations were significantly different in the phenotypic frequencies of these alleles. The Hb Is allele was fixed in the "Piedmont population," but had a low frequency in the "Coastal Plain population." Two alleles of another system, Hb Hi (Md.) and Hb Ilf (Md.), were present in the "Piedmont population" but absent in the "Coastal Plain population." There were also certain morphological differences between the "Piedmont" and "Coastal Plain" populations. These results, plus the occurrence of a 30- mile wide hiatus between the populations, lead Gillespie and Crenshaw (1966) to suggest that there was little gene flow between "Coastal Plain" and "Piedmont" leopard frogs. Sympatry of the "DF complex" and "CF complex" was reported by Post and Pettus (1967) in an area five miles wide in Pueblo County, Colorado. The breeding seasons of the two complexes were not found to overlap in 1966 and 1967 (Post and Pettus, 1967) and no natural hybrids were observed. Undoubtedly the most significant contribution to the understanding of the systematics of the R. pipiens complex was made by Littlejohn and Oldham (1968). They recognized four different types of leopard frogs (designated "northern," "southern," "eastern" and "western" call types) in the West-Central United States. Mating calls (an isolating mechanism of considerable importance in anurans Blair, 1964) of the call types differed markedly in call duration, pulse rate, pulse duration, and pulse rise time. Males of the call types could usually be distinguished morphologically by (1) presence or absence of vestigial male oviducts, and (2) discontinuous or continuous dorsolateral folds. The call types were largely allopatric, but sympatry was found at eleven localities in Texas. Only a small number of natural hybrids was recorded. The "eastern call type" was found in eastern Texas, eastern Oklahoma and Missouri; the "western call type" was distributed from northern Texas, north into Kansas, and eastern Colorado; the "southern call type" occurred in central, western, and southern Texas; and the "northern call type" was found in Colorado and South

4 76 LAUREN E. BROWN Dakota. Littlejohn and Oldliara (1968) took an important step in suggesting that the four call types were different species and that the single species hypothesis was erroneous. However, formal taxonomic names were not given to the call types by Littlejohn and Oldham (1968) because topotypic calls of formerly described species in the complex were unavailable (this is still true at the present time), and data on interactions of call types were lacking west of Texas and Colorado, and over most of Eastern North America. The three "morpho-units" recognized by McAlister (1962) in Texas are equivalent to the "western," "eastern" and "southern" call types of Littlejohn and Oldham (1968). The "western call type" is also the same as the "DF complex" of Post and Pettus (1966, 1967) in Colorado, while the "eastern call type" corresponds to the "Maryland Coastal Plain populations" of Gillespie and Crenshaw (1966). The "CF complex" of Post and Pettus (1966, 1967) in Colorado, and the "Maryland Piedmont population" (Gillespie and Crenshaw, 1966) are the same as the "northern call type" (Littlejohn and Oldham, 1968). Mecham (1968a, b) found two types of leopard frogs ("northern form" or "type" and "southern form" or "type") in Arizona and New Mexico that could be distinguished by several morphological characters. The two forms were sympatric at three localities in the White Mountains region in eastern Arizona. Only one natural hybrid was identified from that area. Laboratory crosses (Mecham, 1968&) indicated that the "southern form" was very incompatible with "R. p. berlandieri" ( "southern call type" of Littlejohn and Oldham, 1968). Reproductive isolation between Mecham's "southern form" and "northern form" was indicated by differences in breeding seasons, mating calls and types of habitat occupied. He thus concluded that they were different species. Mecham's "northern form" is the same as the "northern call type" of Littlejohn and Oldham (1968), the "CF complex" of Post and Pettus (1966, 1967), and the "Piedmont population" of Gillespie and Crenshaw (1966). Mecham's "southern form," however, had a distinct call (Mecham, 1968&) and this type of leopard frog had not been reported by previous workers. Differences in embryonic temperature tolerances and rates of embryonic development were observed by Purcell (1968) using the "northern type" from Arizona, the "southern type" from Arizona, "R. p. berlandieri" from Texas and the "plains type" (= "western call type" of Littlejohn and Oldham, 1968) from Texas. Various artificial hybridizations among these types indicated considerable incompatibility between "R. p. berlandieri" and the other types. Hybrids that did not have "R. p. berlandieri" as one parent were relatively normal in development. Purcell (1968) found that genetic compatibility was not dependent on similar temperature adaptations, which was in contrast with the formerly hypothesized correlation between these factors (Moore, 1946, 1957). Purcell (1968) also gave evidence for differences in breeding seasons between the "northern" and "southern" forms in Arizona, and between "R. p. berlandieri" and the "plains type" in western Texas. Salthe (1969) examined leopard frogs from numerous localities and found twelve alternate forms of lactate dehydrogenase (HLDH) that could be distinguished electrophoretically or immunologically. Geographical variation was complex, but the distributions of the variant lactate dehydrogenases were, in general, correlated with the distributions of the call types of Littlejohn and Oldham (1968) except that the "northern" and "western" call types could not be distinguished from one another. Laboratory crosses of "R. p. berlandieri" (= "southern call type" of Littlejohn and Oldham, 1968) with other members of the R. pipiens complex and R. pipiens species group produced hybrid embryos that in many cases had pronounced defects (Mecham, 1969). It was thus concluded that "R. p. berlandieri" was definitely a species

5 SPECIATION OF LEOPARD FROGS 77 distinct from other members of the R. pipiens complex and R. pipiens species group. Mecham (1969) pointed out that the Texas frogs used by Moore (1946) in his artificial hybridization studies were from Monahans, which falls within the range of "R. p. berlandieri." Furthermore, the "northern type" (from Arizona) was genetically incompatible with "R. p. berlandieri" (Mecham, 1969). It is thus quite possible that the incompatibility observed by Moore (1946) between Texas and northern leopard frogs was a result of the Texas frogs being of a different species (see Cuellar, 1971). Mecham (1969) also reported a zone of sympatry between "R. p. berlandieri" and the "southern plains type" (= "plains type" of Purcell, 1968; = "western call type" of Littlejohn and Oldham, 1968) in western Texas. Cuellar (1971) provided further evidence of the distinctiveness of "R. berlandieri" (= "R. p. berlandieri" of Mecham, 1969; = "southern call type" of Littlejohn and Oldham, 1968). High incompatibility resulted in crosses of R. areolata X "R. berlandieri," whereas the "southern plains type" (=: "western call type" of Littlejohn and Oldham, 1968) and "R. sphenocephala" (= "eastern call type" of Littlejohn and Oldham, 1968) were quite compatible with R. areolata. Brown and Brown (1972) found three types of leopard frogs in Illinois. When the pulse rates, call durations, and pulse durations of the calls of these frogs were compared at the same temperatures with the mating calls of the call types of Littlejohn and Oldham (1968), it was found that the Illinois types were the same as the "northern," "western," and "eastern" call types. However the pulse durations of the "northern call type" in Illinois were shorter than those of the "northern call type" from Colorado reported by Littlejohn and Oldham (1968). Brown and Brown (1972) also showed the pronounced differences in the ealls of the three call types in a comparison over a narrow temperature range (call characteristics vary according to temperature). Each call type from Illinois (Brown and Brown, 1972) concurred respectively in diagnostic morphological characters with the call types of Littlejohn and Oldham (1968). Two sympatric localities were reported from Illinois and no natural hybrids were detected. All previous records of call types were from further west in the United States and the Illinois records represented considerable range extensions. Thus, the findings of three discrete call types in another geographical area that was in the middle of Moore's (1946) hypothesized north-south gradient, added weight to the evidence from the Western United States that the R. pipiens complex is made up of a number of species. Platz (1972) examined variations in electrophoretic properties of serum proteins (transferrins and albumins) and morphological characteristics of sympatric and allopatric populations of "R. p. berlandieri" and the "plains type" in western Texas. Only 12 out of 138 specimens from the contact zone proved to be natural hybrids. Some of these hybrids may have been back-cross individuals but there was no evidence of introgression. Morphology was of little use for detecting natural hybrids because eight specimens were initially designated on the basis of morphology as being of one or the other species before electrophoresis later demonstrated they were hybrids. Two animals that first appeared on the basis of morphology to be of one species, later proved to have albumins characteristic of the opposite species. These frogs were considered to be hybrids. Platz (1972) also reported that the zone of sympatry he studied (also mentioned by Purcell, 1968, Mecham, 1969, and Cuellar, 1971) in Mitchell and Coke Counties of western Texas was 56 km wide. Evidence that there is more than one species of leopard frog may be summarized as follows. There are five call types that differ markedly in characteristics of their mating calls. Premating isolation is also evident by differences in breeding seasons and habitat selection in some cases. Postmating reproductive isolation is well de-

6 78 LAUREN E. BROWN veloped between one call type ("R. berlandieri" or the "southern call type") and several other call types within the complex. The critical test of species differentiation is the maintenance of sympatry without excessive gene exchange. Sympatry between call types has been reported at eighteen localities in widely separated geographical areas (Texas, Colorado, Arizona, Illinois). A number of these records actually represent zones of sympatry. Natural hybridization is minimal or non-existent in the sympatric areas. There are morphological, physiological, and biochemical differences between the call types. However, there is inter-call type overlap in these characters and it is not always possible to distinguish the call types using these characteristics. We can thus conclude that the call types form a complex of sibling species (= "Morphologically similar or identical populations that are reproductively isolated."-mayr, 1963, p. 671). IMPLICATIONS OF THE NEW EVIDENCE Sibling species are common and found in all groups of animals (Mayr, 1963). Thus, the discovery of another complex of sibling species would hardly surprise many evolutionary biologists. However, the finding that leopard frogs are a complex of sibling species and not a single species is of special importance because, as Littlejohn and Oldham (1968, p. 1003) pointed out, the single species concept has "become widely used as a classical model for geographic speciation in which the effect of distance on rates of gene flow and the consequent development of reproductive isolation are shown." Moore's work (1944, 1946, 1949) has been frequently cited in biology texts and many biologists have thus been exposed to that view of evolution in their education. It is, therefore, of considerable significance that the single species hypothesis is no longer tenable. Brown and Brown (1972) pointed out that the discovery of several species of leopard frogs is also of considerable significance to research in experimental zoology. The majority of leopard frogs used by zoologists are purchased from biological supply companies. Commercial shipments are made up of frogs collected at many localities (Gibbs et al., 1971) and a shipment may contain more than one species. For example, last spring I examined a commercial shipment of leopard frogs to Illinois State University that contained three species. Experimental research has often involved segregating frogs into groups and applying different experimental conditions (e.g., different drugs or chemicals) to each group. Differing responses of the groups are analyzed to see if there is any correlation with the different experimental conditions. A cause and effect type conclusion is then often reached. One of the primary assumptions on which this type of research is based is that the experimental animals are reasonably similar genetically. However, there is no guarantee that this assumption is correct because the different groups of frogs studied may have included more than one species. Cause and effect conclusions may be invalid because differing responses of the different groups of frogs may be a result of interspecific genetic variation. Furthermore, the different groups may contain different numbers of species in different combinations (i.e., group I may contain the "northern call type"; group II may contain the "eastern," "western," and "southern" call types; group III may contain the "southern form" from Arizona and the "western call type"...). The use of leopard frogs for purely descriptive studies where different experimental conditions were not applied may also be subject to error because variation in descriptions may be due to interspecific genetic variation. Consequently, much of the previous experimental research involving leopard frogs is of questionable validity. Of course, under some experimental conditions all species of leopard frogs may react similarly. However, previously published studies would need to be repeated and expanded before these experimental conditions can be identified. It is therefore quite unfortu-

7 SPECIATION OF LEOPARD FROGS 79 nate that leopard frogs have been used so extensively in experimental research over such a long period of time. Even if an experimental zoologist is aware that there are several species of leopard frogs, he would likely encounter difficulty in correctly identifying each species in a commercial shipment (or even locally collected frogs) because of the lack of mating call data and the overlap in morphological characteristics. Thus, with the knowledge on hand about leopard frog speciation, one could come to the reasonable conclusion that these amphibians are not the best animals to use in experimental research at the present time. REFERENCES Blair, W. F Isolating mechanisms and interspecies interactions in anuran amphibians. Q. Rev. Biol. 39: Boulenger, G. A A monograph of the American frogs of the genus Rana. Proc. Amer. Acad. Arts Sci. 55: Brown, L. E., and J. R. Brown Call types of the Rana pipiens complex in Illinois. Science (Washington) 176: Cuellar, H. S Levels of genetic compatibility of Rana areolata with Southwestern members of the Rana pipiens complex (Anura: Ranidae). Evolution 25: Dickerson, M. C The frog book North American toads and frogs with a study of the habits and life histories of those of the Northeastern states. Doubleday, Page and Co., New York. Gibbs, E. L., G. W. Nace, and M. B. Emmons The live frog is almost dead. BioScience 21: Gillespie, J. H., and J. W. Crenshaw Hemoglobin variation in Rana pipiens (Amphibia: Anura). Copeia 1966: Kauffeld, C. F The status of the leopard frogs, Rana brachycephala and Rana pipiens. Herpetologica 1: Kellogg, R Mexican tailless amphibians in the United States National Museum. U. S. Nat. Mus. Bull Littlejohn, M. J., and R. S. Oldham Rana pipiens complex: mating call structure and taxonomy. Science (Washington) 162: Mayr, E Animal species and evolution. Harvard Univ. Press, Cambridge, Mass. McAlister, W. H Variation in Rana pipiens Schreber in Texas. Amer. Midland Natur. 67: Mecham, J. S. 1968a. Evidence of reproductive isolation between two populations of the frog, Rana pipiens, in Arizona. Southwest. Natur. 13: Mecham, J. S. 1968b. Studies on evolutionary effects of isolation in the Rana pipiens complex. Year Book Amer. Phil. Soc. 1968: Mecham, J. S New information from experimental crosses on genetic relationships within the Rana pipiens species group. J. Exp. Zool. 170: Moore, J. A Geographic variation in Rana pipiens Schreber of Eastern North America. Bull. Amer. Mus. Natur. Hist. 82: Moore, J. A Incipient intraspecific isolating mechanisms in Rana pipiens. Genetics 31: Moore, J. A Hybridization between Rana pipiens from Vermont and eastern Mexico. Proc. Nat. Acad. Sci. U. S. A. 33: Moore, J. A Geographic variation of adaptive characters in Rana pipiens Schreber. Evolution 3:1-24. Moore, J. A Further studies on Rana pipiens racial hybrids. Amer. Natur. 84: Moore, J. A An embryologist's view of the species concept, p In E. Mayr [ed.], The species problem. Amer. Assoc. Advan. Sci., Washington, D.C. Platz, J. E Sympatric interaction between two forms of leopard frog (Rana pipiens complex) in Texas. Copeia 1972: Post, D. D., and D. Pettus Variation in Rana pipiens (Anura: Ranidae) of eastern Colorado. Southwest. Natur. 11: Post, D. D., and D. Pettus Sympatry of two members of the Rana pipiens complex in Colorado. Herpetologica 23:323. Purcell, J. W Embryonic temperature adaptations of southwestern populations of Rana pipiens. M.S. Thesis. Texas Technological College, Lubbock. Ruibal, R A study of altitudinal races in Rana pipiens. Evolution 9: Salthe, S. N Geographic variation of the lactate dehydrogenases of Rana pipiens and Rana palustris. Biochem. Genet. 2: Stcjneger, L., and T. Barbour A check list of North American amphibians and reptiles. 3rd ed. Harvard Univ. Press, Cambridge, Mass. Stejneger, L., and T. Barbour A check list of North American amphibians and reptiles. 4th ed. Harvard Univ. Press, Cambridge, Mass. Volpe, E. P Hybrid inviability between Rana pipiens from Wisconsin and Mexico. Tulane Stud. Zool. 1: Wright, A. A., and A. H. Wright Handbook of frogs and toads The frogs and toads of the United States and Canada. Comstock Publ. Co., Inc., Ithaca, N. Y.

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