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~ CIassIfIcatIon and Description of Amphibians Commonly Uses fori Laboratory Research A. PHYLOGENY AND CLASSIFICATION Members of the class Amphibia are usually small animals characterized by smooth, moist, glandular skins without external scales. Fertilization is ex- ternal or internal. Eggs are laid in water or moist surroundings and have some yolk and several gelatinous envelopes. Most amphibians have an aquatic larva with metamorphosis to an adult form. Respiration takes place through simple lungs, gills, skin, and buccopharyngeal membranes. The skull is flattened, with fewer bones than fishes, and has two occipital condyles. The heart is three-chambered, and 10 pairs of cranial nerves are present. Amphibians are ectothermic. Although the class Amphibia contains approximately 3,500 species, relatively few are commonly used in laboratory studies. Only those species in which the literature indicates wide utilization in laboratory research in the United States are included below. Additional information on classifica- tion, distribution, identification, and life history may be found in Cata- logue of American Amphibians and Reptiles (Society for the Study of Amphibians and Reptiles, 1971 et seq.) or in Bishop (1947), Wright and Wright (1949), Conant (1958), Savage (1961), Stebbins (1966), Goin and Goin (1971), Blair (1972), and Porter (1972~. Anatomy is described in Ecker and Wiedersheim (1896~. The earliest known fossil amphibian (Ic~hyostega) was found in upper- Devonian freshwater deposits in Greenland and was probably descended from crossopterygian fishes. There are three living orders: 1. Order Apoda Slender vermiform burrowers with compact skull, no limbs or girdles and with greatly reduced eyes that are buried in the skin. Some species are viviparous. Fertilization is internal, and the male cloaca 12

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13 is modified to form a protrusible copulatory organ. Primitive genera have minute dermal scales imbedded in the skin. All living species belong to a single family, Caecilidae. Caecilians are primarily tropical forest dwellers and are not widely used in laboratory research. 2. Order Caudata Modern salamanders or urodeles usually have slim bodies and tails, four limbs (except in Sirenidae, which has forelimbs only and is sometimes placed in a separate order, Trachystomata), and a reduc- tion in slcull bones. 3. Order Anura Frogs and toads (salientians) are characterized by well- developed hind limbs and pelvic girdle adapted for jumping, fused head and trunk, webbed toes, 10 vertebrae and ribs reduced or absent. The term `'toad" is ambiguous; it is often used for the more terrestrial anurans, but is properly applied only to the family Bufonidae. The identification of amphibian larvae is often difficult. Orton (1952) . has provided a key to the genera of tadpoles in the United States and Can- ada, but few developmental stage keys have been published to encompass development through metamorphosis. Gosner (1960) has proposed a sim- plified table for staging anurans and reference is made below to develop- mental tables for those few species for which standardization tables are available. Juvenile amphibians also may be difficult to identify, although reference to identification manuals, such as those listed above, may be useful. B. DESCRIPTION OF SPECIES COMMONLY USED IN LABORATORY RESEARCH 1. Urodeles a. Ambystoma (2N = 28; ZW ~ - ZZ 6, but two triploid species are known) Species of this genus-the best known being the axolotl forms of the closely related speciesAmbystoma mexicanum and A. tigrinum (Smith, 1969) (Figure Beware widely distributed in North America. Some of the species show a proclivity toward neoteny: The larvae grow to large size (0.18- 0.30 m) (7-12 in.), retain their gills, and remain aquatic without develop- ing all of the adult characteristics (e.g., the axolotl forms noted above). With proper alterations of the environment, most of these neoteric forms *The committee gratefully acknowledges the photographic assistance of Gordon A. F. Dunn, Biomedical Graphic Communications, University of Minnesota, in making available Figures 1-12.

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14 _ .^ it_ It __ TV it_ FIGURE 1 The tiger salamander Ambystoma tigrinum in the usual adult form; they also exist in a neoteric form in the western United States. can be induced to metamorphose. Adding iodine to the water induces a faster metamorphosis since it is an essential component of thyroxine. When iodine is not present in the water, insufficient thyroxine is produced and metamorphosis is retarded. The well known axolotl form of A. mexicanum is found in Mexican mountain lakes and accounts for approximately 30 percent of urodeles used in laboratory research (Nace, 1970~. A. tigrinum is found in the western United States, particularly in lakes in Colorado, Montana, New Mexico, Texas, and Wyoming. The taxonomic history of the Mexican axolotl has been confusing. Many generic names (including Axolot, Axolotus, Philhydrus, Siredon, and Sirenodon) have been applied to this form. These names, however, are invalid as is the emendation Amblystoma put forward by Agassiz in 1846. This last generic name was rejected by the International Commission of Zoological Nomenclature (Smith, 1969~. Although some investigators have continued to use the in- correct "Amblystoma," the official name of the genus is Ambystoma. The adult tiger salamander A. tigrinum has an olive~yellow, yellow, or black ventral surface and a dorsal ground color of brown to black with olive or brownish yellow irregular light spots that extend well down the sides. In some subspecies the dorsal markings may form light vertical bars, but the pattern is highly variable. The spotted salamander A. macu- latum has round light yellow or orange spots on a dorsal black ground color and a slate-blue ventral surface; this species occurs from Nova Scotia and central Ontario south to Georgia and eastern Texas. Marble

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15 salamanders, A. opac'am, are characterized by silvery irregular cross- bands, gray in females and white in males, on a black background; the belly is black. This species occurs from New England to northern Florida and west to eastern Texas. Males of this genus can be recognized during the breeding season by the protuberant vent. The embryonic develop- ment of urodeles is usually staged according to Harrison's table for A. maculatum (see Rugh, 1965~. b. Notophthalmus viridescent (2W = 22; ZW ~ - ZZ d) The red-spotted newt (Figure 2) has a complex life history. The aquatic larvae transform into a bright to dull orange-red terrestrial eft stage and later transform into the olive-green aquatic adult. The term "newt" is generally applied to those salamanders that have a terrestrial stage ("oft") preceding the aquatic and mature adult form. In some areas, however, the oft stage may be omitted. The adult has a yellow venter with small black spots. All stages are characterized by red dorsal spots variable in number and position. Males are longer and have larger and longer vents, which may swell during the breeding season; the vent of females is small and conelike. In the past this species has been placed in the genera Tritun~s and Diem- ic~ylus, and reference in the literature may be found under all three names. This newt ranges from the maritime provinces of Canada to the Great Lakes southward to Florida and eastern Texas. Other species of the family Salamandridae sometimes used in laboratory studies include members of the genera Triturus (newts) from Europe and Asia, Taricha (newts) from the western United States, and Salamandra from Europe. ~74: ~ BY .u ..,, it_ 13-' f ,,~,~7 t~ . . ~ Or ~ - ., ,,,,' _ .~_ ha_ .~ FIGURE 2 Adult redisposed newt NotopAthalmus viridascens.

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16 c. Nectun~s maculosus (2N = 24; ZW ~ - ZZ d) The mudpuppy (Figure 3), like other members of the family Proteidae, is a neoteric aquatic form with gills and two pairs of gill openings. Adults range in size from 0.20 to 0.43 m (8-17 in.) and have a gray or rust-brown dorsum with indistinct scattered blue-black spots and a gray or dark belly with darker spots. Females have a simple vent; the vent of males has a wrinkled margin, crossed behind by a crescentic groove and containing two nipple-like papillae. Mudpuppies are found from western New England to Manitoba southward to northern Texas, northern Louisiana, and Tennessee. 2. Anurans a. Xenopus laevis (2~= 36; ZW ~ - ZZ d) The African clawed frog, or platanna (Figure 4), like other members of the family Pipidae, is stout-bodied and big-footed. Although highly aquatic, it is found in areas where ponds may dry out during long periods of low rainfall, and under such conditions may migrate overland to other bodies of water or may aestivate in mud burrows until rains return. The common name derives from the presence of small black curved claws on the inner three toes of the hind feet. The platanna is widely distributed in Africa, south of the Sahara Desert. A significant population derived from escaped animals now exists in southern California. Females have much larger vental flaps than those of males and are also larger in body En ~a. ad_ -Few _! Are ~ MA FIGURE 3 The mudpuppy Necturus maculates-a neoteric aquatic salamander.

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17 Hi_ __ .~ . ~ ~ an. ., FIGURE 4 The African clawed fro' Xenopus Levis. it_ id_ size. Males have a black spinulose nuptial surface on the inner arms and develop fingers during the breeding season. Developmental stages of the clawed frog were described by Nieuwkoop and Faber (19S6~. b. Rana catesbeiana (2N= 26; XX ~ - XY d) This species, the only anuran properly referred to by the common name '`bullfrog" (Figure S), is a member of the family Ranidae (true frogs). This family contains approximately 250 species, representatives of which are found on all continents. The bullfrog is the largest native anuran in North America, with a maximum snout-vent length of 0.20 m (8 in.~. It is green above and sometimes has gray or brown irregular markings. The venter is white and is often mottled with gray. There are no dorso- lateral folds on the trunk. Mature males have tympanic membranes about twice the diameter of the eye; in mature females these are ap- proximately the same size as the eye. Males have heavy, darkly pig- mented thumb pads in contrast to the more delicate, streamlined thumb of the female. This species is aquatic and is found from Nova Scotia west to Wisconsin and Nebraska southward through northern

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18 ~ _'' FIGURE 5 The bullfrog Rena catesbe'ane-the largest anuran native to North America. Florida and Texas. It has been widely introduced elsewhere, particularly on the West Coast and in Mexico, Cuba, Japan, and Formosa. c. Rana grylio (2N = 26; ~ Q - XY d) The pig frog (Figure 6) closely resembles the bullfrog and is sometimes supplied as "bullfrogs" by commercial dealers. This species can be dis- tinguished from R. catesbeiana by the full webbing to the tips of the toes and by the fourth toe that protrudes only a little beyond the adjacent toes; in the bullfrog, the fourth toe protrudes well beyond the other toes, and the toes are less fully webbed. Sexes may be distinguished as in R. catesbeiana R. Folio is an aquatic frog that ranges in the Coastal Plain from southern South Carolina to southeastern Texas and throughout Florida. d. Rana clamitans (2N= 26; XX ~ - XY d) The bronze frog (Figure 7) is highly variable in coloration and may have a green to plain brown or bronze back with darker dorsal spots or markings

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19 ~ ' FIGURE 6 The pig frog Rana grylio, sometimes supplied as the "bullfrog" by com- mercial dealers, closely resembles R. catesbeiana. FIGURE 7 The bronze frog Rana clamitans.

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20 _~ .~ ~ :~ hi_ ^\ .~ ~,~ b c 1 i! , I- ~ 5 ~0 d it' I'' FIGURE 8 Leopard frogs, Rane pipiens. (a) typical member obtained from north- ern United States; (b) Burnsi mutant of the leopard frog, obtained from Minnesota; (c) Kandiyohi variant of the leopard frog, obtained from Minnesota; (d) top: a "northem frog"; bottom: the more pallid "Mexican frog." Note that the spots be- tween the dorsolateral ridges of the northern frog tend to be circular in contrast to the transverse posterior spots in the corresponding area of the Mexican frog.

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21 and a white venter that often has vermiform markings or mottling. The upper lips are sometimes green, and males often have a yellowish throat. The prominent dorsolateral ridges end on the body. Sexes may be dis- tinguished as in R. catesbeiana. This species usually occurs in shallow water along streams, lakes, and springs and is abundant throughout much of its range in southeastern Canada and the eastern States. e. Rana pipiens (2N = 26; XX ~ - XY d) The leopard frog (Figure 8) is the most widely used anuran in laboratory research (Nace, 1970~. This is a highly variable species, perhaps a species complex (Littlejohn and Oldham, 1968; Pace, 1972; Brown, 1973; Platz and Platz, 1973), with wide differences in body size, coloration, and pat" tern. Local populations tend to differ, even between adjacent ponds. Typically, this species is brown or green with irregularly spaced dark spots on the dorsum between conspicuous dorsolateral ridges that extend to the groin. The dark spots are usually rounded and have light borders, but adjacent spots may merge. Southwestern forms tend to be light brown in color. Ordinarily, northern males have dark, thickened thumb pads, while southern males are so characterized only during the breeding season. The leopard frog is semiterrestrial and is found in shallow water habitats throughout its range; where protective cover occurs, it will often wander well away from water. The leopard frog occurs from southern Labrador to southern MacKenzie and eastern British Columbia southward through" out the United States and into Central America. Several forms are recognized by animal dealers and laboratory investi gators: 1. The "northern frogs" occur in the northern part of the range and generally fit the description often given for the subspecies R. p. pipiens. Adults range from 65 to 105 mm (2.5 - in.) in snout-vent length. 2. The "southern frogs" differ from the northern leopard frog by having a light spot in the center of the tympanum, a longer pointed head, and only a few dark spots on the sides. This form occurs from southern New Jersey to the Florida Keys westward to eastern Texas and northward in the Mississippi Valley to Missouri, Illinois, and Indiana. This form has been recognized as R. p. sphenocephala. 3. The Rio Grande leopard frog, R. p. berlandieri, which occurs from southern Nebraska into Central America, is characterized by a more pallid coloration, more warty texture, more fully webbed toes, and a tendency for the posterial dorsal spots to become transverse. It differs from other forms in its adaptation to more arid environments.

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22 4. "Mexican frogs" supplied by dealers, although often included in the above subspecies, may constitute a separate form. Such frogs have a much larger body size; have an average snout-vent length of 105 mm (4 in.), the maximum being about 130 mm (5 in.~; are dark brown; and have un- usually heavy hind limbs (Bagnara and Stackhouse, 1973~. 5. "Burns)" mutants occur in Minnesota and adjacent states and have no, or very few, dorsal spots. 6. "Kandiyohi" mutants appear to be mottled, because, in addition to the regular spotting pattern of this species, the ground color between the spots is invaded by dark pigment (McKinnell and Dapkus, 1973~. Both Bumsi and Kandiyohi have been incorrectly described as subspecies; instead they are single locus dominant mutants found in populations of the northern leopard frog. Melanistic forms, albinos and blues have also been reported from several locations throughout the range. Until a few years ago no attempt was made by amphibian supply com- panies to distinguish Rio Grande frogs (R. pipiens berlandieri) from other leopard frogs, and they were supplied as "on available basis" without any great attention being paid to their distinction. Size range, pattern, and coloration, as described above, can serve as rough indications of geographic origin, but users Would determine the collection point from the supplier. Developmental stages for all species have been described by Shumway (1940) for the embryonic and early larval period and Taylor and Kollros (1946) for the larval and metamorphic period. Life cycles of leopard frogs vary greatly between northern and southern portions of their ranges. The northern animals, which occur north of the line separating ice-free from ice-covered ponds or quiet streams in winter, lay their eggs in March or April. The progeny metamorphose 3 months later and hibernate through the winter with the adults on the bottom in water under the ice cover. Commercial collectors report movement of the frogs toward areas of water flow, presumably to regions of higher oxygen tensions. However, long periods are spent without much movement, fre- quently in large congregations. When water temperatures decrease from 4 to 2 C (39 to 36 F) in December, a shift occurs from lipid and protein utilization to carbohydrate utilization; a reverse change takes place in March when temperatures increase to 4 C (39 F). Under some circum- stances northern frogs may hibernate below the frost line in the mud of swamps or in soft soil, but the usual behavior pattern is to migrate from swamps and fields to lakes and quiet streams, a migration that is reversed in the spring. In place of hibernation, Mexican frogs are reported to aesti- vate during the hot months and to breed in January or February. Frogs from intermediate portions of the range may hibernate in swamps or soft

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23 ground or be active throughout the year, depending on the ambient tem- perature. Accordingly, frogs for reproductive studies must be handled quite differently depending on their origin. f. Rana palustris (2N = 26; XX Q - XY d) The pickerel frog (Figure 9) has an overall appearance similar to some leopard frogs, but may be distinguished by the square dorsal spots arranged in two parallel rows down the back and by the bright yellow or orange color on the concealed surface of the hind legs. The pickerel frog is found in a wide variety of habitats near cool water, but like the leopard frog has a tendency to wander. This species ranges from the Canadian maritime provinces to northern South Carolina westward to southeastern Texas and Wisconsin. ~ ~, ~-~. ~-~ - o~.~ .~, ~.~.~ 5iP~t~ a_ at_ _ ~ ,_ _ A , If i-.- ~;;4 'it' I_ ,, ~- - -~ . ~ _-_ FIGURE 9 Two rows of square-like spots on the dorsum and yellow limb under- surfaces distinguish this pickerel frog, Dana palustris, from leopard frogs similar in appearance.

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24 g. Rana sylvatica (2N = 26; XX Q - XY d) The highly secretive wood frog (Figure 10) is distinguished by the dark patches that form a mask extending from eye to ear. The general colora- tion varies from pink to brown or almost black. Dorsolateral folds extend along the sides to the groin. A denizen of moist woodlands, this frog may wander for considerable distances away from ponds and spends little time in the water except to breed. Wood frogs occur from the tundra of Lab- rador and Alaska south to the southern Appalachians with isolated popula- tions in Kansas, Colorado, Wyoming, Idaho, and the Ozarks. They hiber- nate burrowed in soil under the forest litter and are usually not encountered in numbers, except during mating periods in early spring. Pollister and Moore (1937) described stages of development from fertilization to oper- cular development. h. Bufo marinas (2N= 22; XX Q - XY d) The giant toad (Figure 11) may exceed 0.23 m (9 in.) in snout-vent length, although apparently not in North American populations, and is charac- terized by deeply pitted parotid glands that extend ventrally far down the body. This very large toad just enters the United States in southern Texas, -~;_ . an, ~ _] ~3 - ;;,. ~3 - ~*' FIGURE 10 The wood frog Rana sylvatica is recognized by the dark mask that ex tends posterior from its eyes to its ears.

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FIGURE 11 The giant toad Bufo marinas is found in southern Texas and southern Florida. but has been introduced into southern Florida where it is becoming fairly common. The secretions of the skin glands are toxic to many animals. Males have excrescences on the top of the first two fingers and on the inner side of the third finger and the inner palmer tubercle; these nuptial pads are particularly evident during breeding periods. Due to introductions by man, the giant toad is now almost pantropical. The natural home of this species was probably in the tropical and sub- tropical New World. Other species of Bufo, particularly the European B. bufo and the North American B. americanus and B. terrestns, have been used. i. Bombina orzentalis (2N = 24; XX ~ - XY d) The asiatic fire-bellied toad (Figure 12), a member of the family Discoglos- sidae, is known as suzu koeru or "tinkle-bell frog".in Japanese and is a counterpart of the European fire-bellied toad, B. bombina. The asiatic species is found along streams in mountainous areas in Manchuria and Korea. In nature this species has a highly variable color pattern ranging from a dull solid brown to bright green dorsum or various patterning of these colors, often overlaid with black markings. The ventral surface is generally red with a variable number of irregular black markings. In juve- niles, the underside is yellow and not marked with black. in laboratory

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qua Do -my Em-- ^~ ~ ~ hi ~- ~ ~-l - ' on If; -I FIGURE 12 The asiatic fire~bell;ed toad Bombina oriantalis is found along mountair' streams in Manchuria and Korea. raised adults, yellow is substituted for the red, which probably reflects nutritional factors. Mature females have enlarged abdomens and rounder snouts, and the dorsal "warts" are lower in profile than in the males. Ma- ture males have black pin-point spots (0.1-0.2 mm) (0.0039-0.0078 in.) on the apex of the rather pointed dorsal "warts," thickened forelimbs, and a black thumb pad. This species breeds in May in mountain springs or paddy fields. It hibernates in burrows or deep within talus slopes.