Hyla Regilla Research Articles

Social Interaction among Chorusing Pacific Tree Frogs, Hyla regilla

A shift of call timing by Hyla regilla males in response to playback of recorded calls with various fixed repetition rates supports the hypothesis that alternate calling by chorusing males is true social interaction and not an artifact of body temperature. The observed patterns of phase shifting, inhibition of simultaneous calling and spatial distribution of individuals within the chorus support the hypothesis that social interaction serves to minimize call interference among neighboring males. This appears to be a strategy to maximize each male's chances of attracting a mate.

'Handedness' in the Pacific tree frog (Hyla regilla)

The jumping directions of 24 individual Pacific tree frogs (Hyla regilla), in response to repeated presentations of a model predator, were recorded. The mean jump angle was 70° from the frog's initial bearing regardless of whether the jump was to the left or the right. There was a slight bias within the sample towards left jumps, and most frogs had longer right than left hindlimbs. Some individual frogs jumped preferentially to the left, indicating the existence of 'handedness' in this species.

Structure and function of the amphibian follicular epithelium during ovulation.

Low concentrations of cytochalasin B (CCB) are known to inhibit ovulation in the frog, Hyla regilla. Examination of amphibian thecal cell ultrastructure reveals filaments (average diameter 71 A) arranged in bundles parallel to the surface of the oocyte. These filaments are often associated with hemidesmosome-like plaques on the basal plasmalemma, While individual filaments appear unaltered morphologically by CCB (1-5 microgram/ml), their organization into bundles, apparent relationship to the hemidesmosomes, and the highly contorted configuration of the thecal cells after oocyte expulsion, suggest that a nonmuscular contractile system residing within the follicle plays a fundamental role in ovulation. Our data suggest that the flattened epitheloid thecal cells shorten all axes that run parallel to the oocyte surface via filament bundle contractions, while they remain tightly bound together by macular attachment plaques. These cells thus increase in height to become cuboidal-low columnar in shape; the area covered by the base of each is greatly reduced. As this "thecal sac" decreases in size, the compression generated by the contractile mechanism forces the oocyte through the enzymatically weakened apex of the follicle and ovulation results.

Immunological Detection of Convergent Evolution in the Frog Anotheca Spinosa (Hylidae)

Maxson, Linda R. (Department of Genetics and Development, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801) 1977. Immunological detection of convergent evolution in the frog Anotheca spinosa (Hylidae). Syst. Zool. 26:72-76.-Protein evolution is primarily a divergent process, proceeding independently of organismal or morphological evolution (Wilson et al., 1974). This makes proteins ideal tools for detecting cases of convergent anatomical evolution. A probable instance of convergent evolution, involving the monotypic genus Anotheca, was detected by comparative studies of serum albumin. On the one hand, Anotheca albumin is shown to be as similar to the albumins of several North American Hyla as the albumins of the North American Hyla are to one another. On the other hand, Anotheca albumin is very different from the albumins of the marsupial tree frogs, as different as are those of Hyla. Phylogenetic analysis of the albumin data indicates that Anotheca is phyletically a member of the subfamily Hylinae. Thus, its placement in the Amphignathodontinae was due to convergent morphological evolution in Anotheca to resemble the other genera of marsupial tree frogs. [Serum albumin; phylogenetic analysis; Hylidae.] The tree frog family Hylidae is composed of approximately 500 described species, with about 30 new species being described each year. The 32 currently recognized genera are collected into four subfamilies, based on morphological characters and life history (Duellman, 1970): Hylinae, Amphignathodontinae, Hemiphractinae, and Phyllomedusinae. Anotheca spinosa (a monotypic genus found only in Central America and Southern Mexico) is currently assigned to the Amphignathodontinae, along with the marsupial tree frogs, on the basis of cranial osteology and external morphology (Duellman, 1970). However, Anotheca differs from other amphignathodontine frogs in having less specialized breeding habits and in having a diploid chromosome number of 24, as do most Hylinae. In three of the remaining six amphignathodontine genera where karyotype analysis has been performed, Gastrotheca and Fritztana species have each shown diploid numbers of 26 and 28, and Flectonotus had a diploid number of 30 (Duellman, 1970; Bogart, 1973). These facts led Duellman and others to wonder whether Anotheca's anatomical similarity to the marsupial tree frogs might not have resulted from convergent evolution. An earlier study of hylid albumins (Maxson and Wilson, 1975) indicated the albumin of Anotheca to be more similar to that of Hyla regilla (a hyline tree frog) than it was to the albumins of the marsupial tree frogs (Tables 1 and 2). Indeed, Anotheca albumin was as close to Hyla regilla albumin as was the albumin of numerous other North American Hyla species (Maxon and Wilson, 1974). Since albumin evolves at a rather steady rate in frogs (Maxson et al., 1975), it appeared likely that upon phylogenetic analysis, Anotheca would prove to share a common ancestor with the Group 1 assemblage of' hyline lineages (Maxson and Wilson, 1975 and Fig. 1). MATERIALS AND METHODS Plasma and phenoxyethanol preserved muscle tissues from representatives of 60 species of hylid frogs were used as sources of albumin. Voucher specimens are deposited in the Museum of Vertebrate Zoology, University of California, Berkeley. Albumin was purified from plasma and

The time–temperature relation of embryonic development in the northwestern salamander, Ambystoma gracile

A field and laboratory study on temperature-related embryonic development of Ambystoma gracile was made on a population from northwestern Washington. Natural spawning began in the beaver pond during early March, and the duration of embryonic development (stages 1 to 46) was about 62 days. Average water temperature in the pond during embryonic development was 8.5 °C (range, 4.4 to 14.3 °C). The laboratory data of embryonic development at constant temperatures show that the limits of temperature tolerance are about 5 to 22.5 °C. Rate of development was measured by determining time required to develop from first cleavage (stage 2) to gill circulation (stage 37); representative rates are 12.7 days at 20 °C, 27 days at 12 °C, and 89 days at 7 °C. Embryos of A. gracile have the slowest rate of development when compared with embryos of four other species of Ambystoma (maculatum, mexicanum, tigrinum, and jeffersonianum) and with embryos of three Pacific Northwest frogs (Ascaphus truei, Rana aurora, and Hyla regilla).

Osmotic and Ionic Regulation in Amphibians

Osmomineral regulation in four species of anurans from diverse habitats was studied under aquatic conditions. Ascaphus truei is aquatic, Rana pipiens is semiaquatic, Hyla regilla and Bufo boreas are terrestrial. The rate of osmosis is faster in terrestrial forms than aquatic forms. In μl cm⁻²h⁻¹ mean values were A. truei = 4.0, R. pipiens = 4.8, H. regilla = 5.6, and B. boreas = 16. Higher values reflect higher skin permeability to water and a higher osmolality of body fluids. Plasma Na⁺ is closely regulated at about 100 mM, which is over 100 times more concentrated than the pond water these species encounter in nature. Unidirectional fluxes of sodium were measured with 22Na or 24Na. At a given bath concentration of Na⁺ exchange rates were higher in the terrestrial species. Influx of Na⁺ displays saturation kinetics with increasing bath Na⁺ concentration. Over 90% of the influx is thermodynamically active. Compared with terrestrial species, aquatic species are characterized by high affinity-low capacity transport systems. Sodium efflux consists of renal and diffusive components. No exchange diffusion of Na⁺ was found. Renal efflux of Na⁺ was lower in aquatic species than terrestrial species (A. truei = 0.15, R. pipiens = 0.10, H. regilla = 0.35, B. boreas = 0.26 μM 10 g⁻¹h⁻¹). The integumentary loss of Na⁺ is a function of the transepithelial electrical potential difference which is a function of bath Na⁺ concentration. Body fluids become more electropositive to the bath by about 35 mV per decade increase in bath Na⁺ concentration. The constant field equation accurately predicts the increase in integumentary Na⁺ effux with increasing bath Na⁺ concentration. The most aquatic species, Ascaphus, had the lowest integumentary efflux (5.2 nM cm⁻²h⁻¹). Bufo, a terrestrial form, the highest (41 nM cm⁻²h⁻¹). All species maintained chloride balance in dilute baths in the face of an unfavorable electrochemical gradient. Chloride influx, measured with 36Cl, is predominantly carrier mediated, consisting of exchange diffusion and active transport in all species but B. boreas, in which exchange diffusion was absent. Eflux of Cl⁻ was lowest in Ascaphus and highest in Bufo. Renal loss was lower in aquatic species than terrestrial species (A. truei = 0.12, R. pipiens = 0.07, H. regilla = 0.22, and B. boreas = 0.16 μM 10g⁻²h⁻¹). The efflux was further partitioned into diffusive and exchange diffusive components. Permeability of the skin to Cl⁻, based on diffusive efflux, was lower for aquatic Ascaphus (0.4 × 10⁻⁸ cm s⁻¹) than for terrestrial Bufo (5 × 10⁻⁸cm s⁻¹). While anurans appear to possess common basic mechanisms for osmomineral regulation in freshwater, significant adaptive modifications have evolved which may affect distribution of species.

Spacing and calling in Pacific tree frogs, Hyla regilla

Calling male Pacific tree frogs space out more than if they were distributed at random on the available calling sites. When new frogs are added to an area that is already occupied by some calling males, fewer additional frogs call than in a control situation where frogs are added to an empty area. This shows that behavioral interactions between frogs limit the number that call in a local area. A further experiment showed that the mating call played through loudspeakers is sufficient to inhibit additional frogs from calling. We conclude that calling leads to spacing out, which in turn limits the density of calling frogs in a local area.

Protein Variation in Several Species of Hyla

Case, S. M., Haneline, P. G., and M. F. Smith (Museum of Vertebrate Zoology, University of California, Berkeley, California 94720) 1975. Protein variation in several species of Hyla. Syst. Zool. 24:281-295.-Allelic variation at fourteen loci coding for ten enzymes was studied in seventeen populations of Hyla regilla and in H. eximia, H. wrightorum, H. cadaverina, and H. euphorbiacea. Heterozygote deficiencies were found at several loci in several populations of H. regilla and possible reasons are suggested. The proportion of polymorphic loci and average individual heterozygosity were typical of other vertebrates with some exceptions: (1) the Lee's Camp, Oregon population; (2) the Santa Cruz Island population; (3) the three San Diego Co. populations; (4) the Sierra San Pedro Martyr population; and (5) the three populations in southern Baja California. Measures of genetic similarity (S) and genetic distance (D), as well as patterns of variation in allele frequencies at PGI and IDH-2, suggested three groups within Hyla regilla. These were: (1) the four Oregon populations; (2) the three central California populations; and (3) the ten southern California and Baja populations. The proportion of major alleles shared indicated Hyla eximia and H. wrightorum, are very closely related and that H. wrightorum is not a subspecies of H. regilla; H. eximia and H. wrightorum are also closely related to H. euphorbiacea. Hyla regilla and H. cadaverina are more closely allied to each other than they are to the other three forms. A strong correlation (r = 0.93) between genetic relatedness as measured by immunological comparisons of albumin and that measured by electrophoretic studies of many proteins was found, although immunological techniques cannot be used to study taxa which are very closely related (D = 0.25 or less). [Proteins; genetics; Hylidae] Since the pioneering work of Lewontin & Hubby (1966) attention has been focused on the amount and significance of genic variation in local populations. Subsequently electrophoresis has been used to study various aspects of local population genetics and the relationships among species (see Selander & Johnson, 1973 for a review). A number of electrophoretic studies of anurans have been conducted (Dessauer & Nevo, 1969; Gartside, 1972; Guttman & Wilson, 1973; Maxson & Wilson, 1974a; Platz & Platz, 1973; Rogers, 1973a and b; Salthe, 1969; Salthe & Nevo, 1969). The scope of these works is wide, ranging from an analysis of variation at a few loci as a method of assessing amounts of introgression to a survey of variation at a large number of loci in an effort to determine the overall level ' California Academy of Sciences, San Francisco, California; Present address: Department of Amiphibians and Reptiles, Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts 02138. of genic variation and to examine genetic relationships between two or more forms. No detailed study of the latter type has been done with any members of the genus Hyla. The main purpose of this study was to determine the amount of genic variation within and the genetic distance between populations of Hyla regilla. This is of particular interest since this species ranges from British Columbia to the tip of Baja California and has been divided into ten subspecies by Jameson, et al. (1966) on the basis of a discriminate analysis of morphological characters. Maxson & Wilson (1974b) have compared the serum albumins of eight of these subspecies using the method of microcomplement fixation. Electrophoretic estimates of genetic distance can provide an independent method for detecting subgroups within this wide-ranging species. In addition, several other members of the Hyla eximia group, in which H. regilla

An Analysis of the Mechanisms of Mating Call Discrimination in the Frogs Hyla regilla and H. cadaverina

Evaluation of the role of frequency spectrum, duration, temporal pattern of pulses and pulse form components in call discrimination behavior of H. regilla and H. cadaverina showed that pulse repetition rate is the only necessary component for effective discrimination. In each species the frequency of best hearing matches the dominant frequency of the call which optimizes detection of the calls but does not effect discrimination. MANY species of Anura maintain genetic integrity through ethological premating reproductive isolating mechanisms. This is largely accomplished by the ability of females to discriminate between mating calls of homospecific males and those of other sympatric species. Thus, encoding of species specific information in such calls and its analysis by the auditory system is essential. Straughan (1973) indicated that call discrimination by females was well established in some groups of frogs (i.e. Hylidae), but that the mechanisms of discriminatory behavior were not fully understood. Evidence from studies on neurophysiology and evoked vocal behavior of males of Rana catesbeiana indicated that the important component of the call was the dual peak power spectrum which was matched by a dual band pass frequency filter in the ear involving the amphibian and basillar papillae. Simultaneous stimulation of both papillae produced neurophysiological perception to correct vocal stimulus (Capranica, 1965). However, trials of female discrimination (Loftus-Hills and Littlejohn, 1971) indicated that the most important component for Litoria ewingi and L. verreauxi was pulse repetition rate. They argued that temporal components other than pulse repetition could not be encoded species specific information but they did not deal experimentally with each component. Generalization from these results are thus restricted to calls of the same basic structure.

Embryonic temperature adaptations of the pacific treefrog, Hyla regilla

1. 1. The embryonic temperature adaptations of the Pacific treefrog, Hyla regilla, from southern California and western Washington are described. Each population represents a different subspecies, and both belong to the “thermal group” of H. regilla. 2. 2. In western Washington the water temperatures of breeding pools during embryonic development may range from 0·5 to 20°C, and embryonic development requires about 3 weeks. 3. 3. The embryonic temperature tolerance of Washington embryos is 6–28°C and California embryos, 6–29·6°C. 4. 4. The developmental rate-temperature curves cross with Washington embryos faster at low temperatures and California embryos faster at high temperatures. 5. 5. Development of California embryos at high temperatures (29–31·5°C) shows inhibition of developmental rate and much variation in tolerance. There may be selection for increased heat resistance in this population. 6. 6. Compared to many cold and warm adapted frogs of North America, H. regilla seems generalized in its embryonic temperature adaptations. It is a eurytopic treefrog, one found in a wide variety of habitats that are thermally diverse.

Mate selection in Pacific tree frogs

DARWIN1 suggested that, in many animal species, females select mates and that some male courtship displays and secondary sex characters are a product of this sexual selection. Although the importance of sexual selection is generally acknowledged2, only a few studies have shown both that females prefer certain males and also that this preference is a function of male display behaviour, for example, studies of polygamous birds3 and fruitflies4. We have observed that in the Pacific tree frog (Hyla regilla), females mate preferentially with certain males, and that this preference is related to differences between males in calling behaviour.

Lactate Dehydrogenases of the Treefrog Hyla regilla: Subunit Homologies and Genetic Variation

Five major forms of lactate dehydrogenase were detected in most specimens of Hyla regilla examined by electrophoresis. They were designated M4, MaH, M2H2, MH3 and H4 on the basis of electrophoretic migration, thermal stability, immunological properties, tissue distribution and catalytic properties. An electrophoretic search for allelic variation at the loci coding for the M and H subunits was made with extracts prepared by soaking toes in a phenoxyethanol solution. A total of 414 individuals, representing 4 subspecies and 9 populations, from Oregon and California were screened. The search revealed 10 presumptively heterozygous individuals: 4 at the Ldh-M locus, 5 at the Ldh-H locus and one at both loci. As the presumptive heterozygotes appeared to produce approximately equal amounts of the variant and wild-type subunits, each locus was judged to be single (rather than duplicate).

Convergent morphological evolution detected by studying proteins of tree frogs in the Hyla eximia species group.

Protein studies have uncovered an apparent case of convergent evolution among North American tree frogs. The species Hyla eximia and Hyla regilla are so similar in external morphology that the "wrightorum" subspecies is assigned by some authorities to H. eximia and by others to H. regilla. Yet microcomplement fixation experiments show that "wrightorum" albumin, though virtually indistinguishable from authentic H. eximia albumin, differs as much from H. regilla albumin as from albumins of species outside the genus Hyla, such as Acris crepitans. The morphological resemblance of "wrightorum" to H. regilla is thus probably due to convergence.

Induction of sperm release in frogs by mammalian gonadotropin-releasing hormone

A single subcutaneous injection of a synthetic mammalian gonadotropin-releasing hormone (LH/FSH-RH) stimulated a rapid release of sperm (spermiation) from the testes of the intact treefrog Hyla regilla; the median effective dose was between 35 and 75 ng. The rate of response was dose-dependent, ranging from about 5 to 30 min. Even large injections of RH failed to stimulate spermiation in hypophysectomized frogs, but 100 ng RH was effective after transplantation of a pituitary into these animals. Thus, the action of RH is mediated by the frogs pituitary. These data confirm the lack of species-specificity in the hypothalamic gonadotropin-releasing factor.

Luteinizing hormone (LH) in the reptilian pituitary gland

The luteinizing hormone (LH) activity of pituitary glands from three orders of Reptilia was studied by means of the induction of ovulation in vitro from the ovaries of the frogs Hyla regilla and Xenopus laevis. Representatives from several families including both suborders of Chelonia (turtles) and several Crocodilia had relatively high concentrations of LH activity in the pituitary. In contrast, ovulation could not be induced with pituitaries from any of the Squamata (snakes and lizards) tested, although these glands had high levels of gonadotropins in other bioassays. Thus, there appears to be a major dichotomy between Squamata and other reptiles either in the levels at which LH is stored in the gland, in the structure of the LH molecule, or in whether LH exists. The lack of bioassayable LH in snakes and lizards distinguishes them from all other orders of tetrapods examined. Previous cytological studies of the reptilian pituitary indicated a regional distribution of LH-cells (luteotropes), but measurement of LH in different regions of the pars distalis from three families of turtles revealed a uniform distribution of this hormonal activity.

Latitudinal effects on metabolic rates in the frog, Acris crepitans: seasonal comparisons.

In his review of latitudinal effects on the physiological properties of animal populations, Vernberg (1962) points out that a variety of physiological differences occur among latitudinally separated populations of some species. The differences are of considerable interest both to the comparative physiologist and to the ecolo gist, since they provide insight into the relative roles of longer term genetic changes in populations versus shorter term physiological changes of organisms in their adaptation to varying climatic regimes. Differences in whole body oxygen consumption which are apparently genetically fixed have been reported for a variety of organisms. Where these differences have been reported, they are such as to suggest that latitudinal compensation in metabolism for temperature occurs. In general, northern populations have a higher rate of oxygen consump tion than southern populations of the same or related species when determined over a wide range of temperatures (Bullock, 1955 ; Prosser, 1955 ; Vernberg, 1962; Tashian and Ray, 1957 ; Hutchison, Whitford and Kohl, 1968). Several investigators have compared the metabolic rates of latitudinally sepa rated populations of the same species of anuran. Tashian and Ray ( 1957) corn pared northern and southern subspecies of Bufo boreas and found no significant dif ferences between them. Jameson, Taylor and Montjoy ( 1970) however, report a great deal of variability in metabolic rates among populations of the frog, Hyla regilla. Dunlap (1972) reports that for warm acclimated Acric crepitans, South Dakota frogs have higher metabolic rates at low temperatures than do Texas frogs. When comparing latitudinally separate populations one would hope to dis tinguish between short-term, environmentally induced variations in metabolic patterns and those that are genetically determined. However, the occurrence of marked seasonal changes in the metabolic rates of intact frogs which has been reported for a number of species, e.g., Rana fusca (Krogh, 1904); R. esculenta (Stangenberg, 1955); R. pipiens (Fromm and Johnson, 1955); R. temporaria (Dolk and Postma, 1927); Acris cre/,itans (Dunlap, 1969); Hyla regilla (Jameson et al., 1970), can complicate the interpretation of comparative studies. That these changes are not due to short-term thermal acclimation to seasonal differences in temperature is evidenced by the fact that recent investigators who have reported the seasonal changes have compared animals acclimated at the same

Tests for neuroplasticity in the anuran retinotectal system

Mapping of retinotectal projections in the tree frog Hyla regilla was carried out by both behavioral and electrophysiological recording techniques following tectal ablations, with and without optic nerve regeneration. Scotomata produced by unilateral and bilateral half tectum ablations and by unilateral rectangular midtectal excisions were found to remain essentially unaltered in all cases through recovery periods up to 334 days. Similarly, electrophysiological mapping of the rostral half tectum separated by Gelfilm implants from the caudal tectum for up to 191 days yielded a normal rostral visual field. The stability of the retinotectal projection in adult Hylidae observed in these experiments contrasts with the plastic readjustments obtained in young goldfish in which the retinotectal system is still probably growing and presumably capable of field regulation. The results are taken to support the original explanatory model for developmental patterning of retinotectal connections in terms of selective cytochemical affinities between retinal and tectal neurons.

Induction of spermiation in anurans by mammalian pituitary gonadotropins and their subunits

Tests with highly purified ovine FSH and LH in three species of anurans show that the spermiation response in these amphibia does not discriminate between the two pituitary gonadotropins: FSH and LH are essentially equipotent. Similar results are obtained with both hypophysectomized and intact frogs. Less purified gonadotropin preparations (NIH) give more variable results: LH is considerably more potent than FSH in Rana pipiens, but FSH is about twice as potent as LH in Hyla regilla and Eleutherodactylus coqui. There is a slight synergism between FSH and LH. However, the use of trypsin to inactivate LH and the use of antiserum to block FSH confirmed that the activity of each purified gonadotropin is an intrinsic property of the molecule. The relative potencies of the highly purified gonadotropin preparations in the anuran spermiation test differed markedly from those observed with the same preparations in mammalian and reptilian assays. In anurans, the relative potency of purified LH was about 20 times greater than in other systems, whereas, the relative potency of the highly purified FSH was only about 1 5 that observed in the other tests. The two individual subunits of LH had little activity in the frogs (ca. 2.5% of LH) and there was slight difference in their potencies. In contrast the β subunit of FSH was relatively potent (about 25% of native FSH), but the α subunit was inactive (<3%). In all three species of anuran, a crude extract of anuran (bullfrog, R. catesbeiana) pituitary glands was as potent as the most active purified mammalian hormone.

The water relations of the tailed frog, Ascaphus truei, and the pacific treefrog, Hyla regilla

1. 1. The tailed frog, Ascaphus truei, is a stenotopic anuran found only in or near swift, cold mountain streams, whereas the pacific treefrog, Hyla regilla, is found in a wide variety of habitats and is much more terrestrial. 2. 2. H. regilla loses body water at a slower rate than does A. truei. 3. 3. H. regilla tends to absorb water more readily from a moist substrate than does A. truei. 4. 4. H. regilla has a significantly greater tolerance to desiccation than does A. truei. 5. 5. A. truei has a greater preference for moisture than does H. regilla. The tailed frogs showed the greatest preference for water, while the Pacific treefrogs seemed to show the greatest preference for wet sand. 6. 6. These observed differences correlate well with the differences between the respective habitats of H. regilla and A. truei.

The thermal relations of the tailed frog, ascaphus truei, and the pacific treefrog, Hyla regilla

1. 1. The tailed frog is a stenotopic anuran found only in cold mountain stream habitats, whereas the Pacific treefrog is found in a wide variety of habitats which are thermally diverse. 2. 2. The critical thermal maximum (CTM) for Hyla regilla is significantly above that of Ascaphus truei at all acclimation temperatures. 3. 3. The incipient lethal temperature (ILT) for H. regilla is about 9°C higher than the corresponding value for A. truei. 4. 4. A truei tends to select a lower temperature in a thermal gradient than does H. regilla. 5. 5. These observed physiological and behavioral differences correlate well with the differences between the habitats of these two anuran species.