Hormonal induction of thumb pads and the evolution of secondary sexual characteristics of the Southeast Asian fanged frog,Rana blythii

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 279:587–596 (1997)

© 1997 WILEY-LISS, INC.

Hormonal Induction of Thumb Pads and theEvolution of Secondary Sexual Characteristics ofthe Southeast Asian Fanged Frog, Rana blythii

SHARON B. EMERSON,1,2* LARA CARROLL,1 AND DAVID L. HESS3

1Department of Biology, University of Utah, Salt Lake City, Utah 841122Division of Amphibians and Reptiles, Field Museum of Natural History,Chicago, Illinois 60605

3Division of Reproductive Biology and Behavior, Oregon Regional PrimateResearch Center, Beaverton, Oregon 97006

ABSTRACT The fanged frogs of Southeast Asia do not express most of the hormone-dependentsecondary sexual characteristics such as thumb pads that are common to other ranid frogs. At thesame point in the evolutionary history of the group that these androgen-mediated characteristicsare lost, male parental care first evolves. This behavior is often correlated with low androgenlevels. Prior work indicates that in one of the fanged frogs, Rana blythii, adult males have lowandrogen levels compared to North Temperate species of Rana. This leads to the question of whetherthese low androgen levels are related to the unusual male parental care and the lack of expressionof the thumb pad and other hormone-dependent secondary sexual characteristics in this species.We tested that hypothesis by examining the effects of exogenous dihydrotestosterone supplementson the expression of thumb pads in Rana blythii. Dihydrotestosterone injections appear to stimu-late the expression of the thumb pad in R. blythii. These results support the hypothesis that lowandrogen levels are involved in the loss of the thumb pad in R. blythii. This work provides anexample of how mapping characters on phylogenies can be a powerful approach for gaining in-sights into proximate physiological mechanisms of selection at the evolutionary level. J. Exp. Zool.279:587�596, 1997. © 1997 Wiley-Liss, Inc.

A group of Southeast Asian ranid frogs is char-acterized by an unusual suite of secondary sexualcharacteristics: fangs, enlarged heads, hypertro-phied jaw muscles, and male parental care (Boul-enger, ’20; Inger, ’66; Emerson and Berrigan, ’93).Some or all of these species also lack most of thesecondary sexual characteristics typical of maleranid frogs and known to be androgen-mediated:rough, keratinized nuptial pads, vocal sacs, en-larged forelimb flexors, and a male advertisementcall (Kelley and Pfaff, ’76, and references therein;Muller et al., ’69; Melichna et al., ’72; Schmidt,’83; Hannigan and Kelley, ’86; Sidor and Black-burn, ’94; Blackburn et al., ’95). Prior work ap-plying character optimization and mapping thegain and loss of these sexually dimorphic traitson a phylogenetic hypothesis for the group indi-cates that the evolutionary loss of the androgen-mediated secondary sexual characteristics mightbe, in part, a function of selection for male paren-tal care (Emerson and Berrigan, ’93; Emerson etal., ’93). Male parental care is related to low an-drogen levels in some vertebrates (Townsend and

Moger, ’87; Wingfield et al., ’90). The juxtaposi-tion of the loss of androgen-mediated secondarysexual characteristics with the evolution of maleparental care at the basal node of the clade sug-gests that the proximate mechanism throughwhich selection acted might have been lowerandrogen levels (Emerson et al., ’93). This hypoth-esis, that lower androgen levels have been in-volved in the evolution of sexual dimorphism andmale parental care in this group, was supportedby data showing that Rana blythii, one of thefanged frogs, has lower androgen levels than othertropical and temperate frogs that have been mea-sured (Emerson et al., ’93).

Contract grant sponsor: National Science Foundation, BSR-8822630, DEB-9317642; NIH, RR 00163, HD 18185.

*Correspondence to: Sharon B. Emerson, Department of Biology,University of Utah, Salt Lake City, UT 84112. Email: [email protected]

Received 21 January 1997; revision accepted 3 July 1997.

JEZ 883

588 S.B. EMERSON ET AL.

Another prediction derived from this hypothesisis that replacing androgen levels in Rana blythiiwould restore the expression of androgen-medi-ated secondary sexual characteristics such as thenuptial pad. This paper reports the results of suchan androgen replacement experiment. It also pro-vides an example of how mapping characters onphylogenies can be employed to gain insights intoproximate physiological mechanisms of selectionat the evolutionary level (Emerson, ’96).

MATERIALS AND METHODSExperimental rationale

In many frog species, breeding males developthickened patches of specialized skin on the thumbwhich are referred to as nuptial or thumb pads.These roughened areas are often keratinized andare usually visible to the naked eye. Comparativehistological work has shown that these externallyvisible thumb pads contain sexually dimorphic nup-tial glands (Thomas et al., ’93). For purposes of thispaper, frogs will be considered to lack nuptial padsif there are no externally visible, thickened patchesof rough, keratinized skin on the thumb.

Decades of experimental work (Lofts, ’84, andreferences therein) indicate that thumb padexpression is androgen-mediated in frogs. Tes-tosterone (T), dihydrotestosterone (DHT), and 11-ketotestosterone have all been shown to affect theexpression of nuptial pads (Wolf, ’39; Rastogi andChieffi, ’71; D’Istria et al., ’72; Iwasawa andKobayashi, ’74; Kelley and Pfaff, ’76; Izzo et al.,’82; Kanamadi and Saidapur, ’82, ’86; Kanamadi,’87; Lynch and Blackburn, ’95). More recently, an-drogens have been shown to affect nuptial glandexpression as well (Fujikura et al., ’88; Thomasand Licht, ’93; Epstein and Blackburn, ’97). An-drogen replacement experiments indicate that Tand DHT have much stronger effects than 11-ketotestosterone (D’Istria et al., ’72) and that DHTappears to be more effective than T (Izzo et al.,’82). One study (Rastogi et al., ’86) found a corre-lation between androstenedione (A) levels andthumb pad development and suggested that Amight be a major androgen controlling thumb padexpression. However, androstendione replacementexperiments on castrated frogs have never beendone to test that hypothesis.

Prior work indicated that R. blythii had low an-drogen levels relative to other breeding ranid andtropical frogs that had been examined (Emersonet al., ’93). Specifically, testosterone levels wereless than 50% and DHT levels were less than 30%

of those recorded for breeding Rana catesbeiana(Licht et al., ’83). Androstenedione levels were anorder of magnitude less than those reported forthe Mexican leaf frog (Rastogi et al., ’86), the onlyother frog for which androstenedione levels wereavailable. The low levels of androstenedione in R.blythii suggested that it was important to test ex-perimentally whether that hormone affected nup-tial pad expression. Accordingly, we began ourstudy by conducting an androgen replacement ex-periment testing the effects of A on the expres-sion of thumb pads in Rana pipiens. We chose thisspecies because R. pipiens is much more readilyavailable and easier to work with in the labora-tory than the Bornean R. blythii. We reasoned thatsince the same qualitative results to previous an-drogen replacement experiments with T and DHThad been obtained in tropical and temperate spe-cies across five different families of frogs (e.g., Kelleyand Pfaff, ’76; Izzo et al., ’82; Rastogi et al., ’86;Kanamadi, ’87; Harvey and Propper, ’97), the re-sults from R. pipiens could probably be extended,with reasonable confidence, to a congeneric species.Building on the results of this first experiment, wethen could use the most appropriate androgen inthe experiment to test the hypothesis that the lackof expression of the thumb pad in R. blythii is re-lated to low androgen levels.

Rana pipiens laboratory experimentThirty-two adult male Rana pipiens were ob-

tained from Charles Sullivan Co. (Nashville, TN)in November 1994. Frogs weighed between 22 and39 g. Animals were kept on a 14 h dark, 10 hlight regime at a temperature of 22 ± 2°C. All frogswere fed crickets two times a week. Animals werehoused separately in plastic shoebox containerswith moist paper towels on the bottom of the box.Elevating the box on one end created a standingpool of water approximately 1 inch in depth atthe other end of the box. Water was allowed to sit24 h before being used in the boxes. Containerswere cleaned twice a week.

Frogs were randomly sorted into five groups ofsix individuals each, except for the intact controlgroup which had eight frogs. Individuals of theintact control group did not have surgery or re-ceive androgen supplements. In all other groups,individuals were anesthetized by immersion in0.1% MS-222 (ethyl m-aminobenzoate methanesulfonic acid). In the sham-operated group, an in-cision was made in the lower abdomen, and eachtestis was exposed but not otherwise disturbed.Frogs in the remaining three groups were bilat-

ANDROGENS AND SECONDARY SEXUAL CHARACTERISTICS 589

erally gonadectomized after the abdominal inci-sion was made. The frogs were allowed a 4 weekrecovery period after surgery before receiving in-jections. All frogs were given 0.1% tetracycline intap water for 3 days after surgery.

The intact and sham-operated control groupsreceived no injections. A third control group (cas-trate, saline-injected) consisted of six castratedindividuals who received daily injections of frogRingers. Individuals of the remaining two groupsreceived daily injections of A (4-androstene-3, 17-dione) or DHT (5α-androstan-17β-ol-3-one). DHTwas chosen for comparison with A because previ-ous work had shown that DHT can control theexpression of thumb pads in frogs (e.g., Kelley andPfaff, ’76; Izzo et al., ’82) and it is more effectivethan T (Izzo et al., ’82). In this experiment, it canbe considered a positive control.

For experimental males, hormonal dose was 2µg hormone/g frog body weight, similar to valuesreported in studies from the literature (e.g., Izzoet al., ’82). Stock solutions were prepared by grind-ing up the steroid with gum arabic and suspend-ing the material in a small amount of 95% ETOH.Stock solutions were subsequently aliquotted intoplastic vials for daily use and frozen at –70°C. Analiquotted stock solution was prepared for injec-tion by bringing it up to the appropriate volumewith frog Ringers. All injections were intraperito-neal. The steroids and other chemicals were pur-chased from Sigma (St. Louis, MO).

After 21 days, blood samples were taken from allanimals by cardiac puncture. Blood collection wasdone with heparinized syringes. Frogs were thensacrificed by overdose with MS-222. Immediatelyafter death, the frogs were weighed, and the thumbsof the frogs were removed and placed in Histochoicetissue fixative (Amresco, Solon, OH). The bloodsamples were centrifuged within minutes of collec-tion, and the plasma was stored in liquid nitrogen.

Rana blythii field experimentThirteen frogs of the species Rana blythii were

obtained in February 1995 at the University ofMalaya Field Station (Ulu Gombak, Malaysia).Animals weighed between 50 and 250 g. Frogswere housed separately at ambient temperaturesin plastic aquaria under similar conditions tothose described above for Rana pipiens. Aquariawere housed in a field laboratory building thatconsisted of counters and a sink, with runningwater from the stream where the frogs had beencollected. Animal husbandry was the same as re-ported for R. pipiens.

The frogs were divided into size-matched con-trol (n = 5) and experimental groups (n = 8). Theexperimental animals (n = 8) were given intra-peritoneal injections of DHT daily for 14 days. Theadministered dose was 2 µg hormone/g bodyweight of frog. Control animals received no injec-tion. A saline-injected control group was not usedbecause we had a limited number of animals avail-able. Our lack of injection controls can be justi-fied in two ways. First, previous studies havedemonstrated no effect of injection on behavioraland/or physiological outcomes in similar experi-ments (Iwasawa and Koyayashi, ’74; Kanamadiand Saidapur, ’81, ’86; Lofts, ’84, and referencestherein; Rastogi et al., ’86; Kanamadi, ’87). Sec-ond, saline-injected animals control for the stress-ful effects of injection, and therefore this controlhelps explain negative results in an experimen-tal sample. We predicted, based on previous stud-ies, that our experimental animals would have aspecific androgenic response: increased thumb paddevelopment. There is no basis for thinking thatthe saline injection would induce thumb pad de-velopment or that the stress of saline injectionalone might inhibit development. Therefore, thistreatment was not necessary based on the designdeveloped from our original hypothesis. One con-trol and three androgen supplemented frogs diedover the course of the experiment. There was norelationship between the size of the frog and itslongevity.

At the end of the 2 weeks, blood samples weretaken from all surviving animals by cardiac punc-ture, and the animals were sacrificed. The thumbsand testes of all animals were fixed in Histochoice.Again, the blood samples were centrifuged withinminutes of collection, and the plasma was storedin liquid nitrogen.

Paraffin-embedded histological cross-sections ofthe thumbs of R. pipiens and R. blythii were pre-pared in order to measure thickness of the epi-dermal and glandular epithelial layers. In mostmale frogs, including R. pipiens, the nuptial padis best developed along the median surface of theprepollex (Duellman and Trueb, ’86). Accordingly,sections of this area were made in both R. pipiensand R. blythii. Ten sections were cut at the mid-point along the length of the pad (or comparablelength along the prepollex). Three measurementswere taken in each section at randomly chosenpoints along the midsection of the pad. The aver-age of these 30 measurements (ten sections, threemeasurements each) was used to calculate totalthumb pad height, height of the epidermis, and


height of the glandular epithelium. Measure-ments were done on a Nikon Labophot-2 micro-scope that had been fitted with a video cameraand interfaced with a Macintosh IIci computer.The image acquisition and manipulation applica-tion was NIH Image.

Histological cross-sections of the testes of R.blythii were prepared to confirm that the maleswere undergoing spermatogenesis and were repro-ductively active. This is necessary because themales of this species don’t call and don’t exhibitthe typical external morphological features suchas a thumb pad that would otherwise indicatebreeding state.

Radioimmunoassay techniquesSerum samples (10–160 µl) of R. pipiens and R.

blythii were analyzed by radioimmunoassay for5α-dihydrotestosterone (DHT), testosterone (T),androstenedione (A), and corticosterone (B) lev-els. Individual samples with plasma volumesgreater than 40 µl were assayed in two aliquots(20% and 80% of the original samples) taken af-ter the appropriate chromatographic fraction wasdiluted with a quantity of ethanol equal to thatof the original plasma. All samples for each hor-mone assayed yielded values. Corticosterone wasmeasured as an indicator of stress level of indi-vidual frogs (Houck and Woodley, ’95). We mea-sured corticosterone without further purification on1–3 µl of serum after diluting in EtOH to precipi-tate any potential serum-binding proteins. The an-drogens were estimated after extraction with etherand purification on Sephadex LH-20 microcolumnsas described previously (Emerson et al., ’93).

The serum steroid concentrations reported herewere estimated in single assays for each steroidto avoid between-assay variation. The intraassaycoefficients of variation for the assays were 8.8%(A), 3.7% (T), 9.2% (DHT), and 5.1% (B). Thesensitivity of the assays, defined as the concen-tration estimated at three times the standard de-viation of five zero dose points, were 1.8 (A), 6.0(B), 2.1 (DHT), and 7.0 (T) pg/tube. In all casesthese values were close to the value estimated forwater blanks run in parallel with the samples ineach assay. Recoveries following the extraction andpurification steps were 74.0% (A), 81.6% (DHT),and 85.6% (T).

Although the laboratory does not have pools offrog serum that are routinely assayed, it does havepools of male and female rhesus monkey serumwhich are run at two or three different volumesas quality control checks with each assay. These

pools are large enough that they can be used formultiple years. Additionally, the same antiserahave been in use for at least the past 5 years.Between-assay variation in the quality control se-rum pools in all assays has not exceeded 18% overthe last 4 years for all steroids routinely assayed.

Statistical analysisStatistical analyses were performed with the

JMP software program (SAS Institute, Inc., ’94).Hormone levels from the R. pipiens experimentwere log-transformed before analysis. Preliminaryanalyses indicated that there was no relationshipbetween hormone levels and body weight in ei-ther R. pipiens or R. blythii. A Tukey-Kramer HSDmultiple comparisons test was used to compareA, T, and B levels among groups. Values of DHThad unequal variances among treatments even af-ter log transformation. Consequently, DHT val-ues were compared among groups using WelchANOVAs. Due to the large number of compari-sons, we used a significance level of 0.01 to avoidobtaining significance by chance.

Thumb pad expression in R. pipiens was com-pared using a Tukey-Kramer HSD multiple com-parisons test. T-tests or a Welch ANOVA wereused to test for differences between control andexperimental frogs in the R. blythii field experi-ment, depending on whether the variances wereequal in the two groups. Significance level (P) wasset at 0.05.

RESULTSHormone levels in R. pipiens

Table 1 summarizes hormone levels for the dif-ferent treatment groups in the R. pipiens experi-ment. There were no significant differences inhormone levels of A, DHT, T, or B between theintact and sham-operated control groups. The cas-trate, saline-injected group had significantly dif-ferent T and DHT levels from the intact andsham-operated control groups, but A levels werenot significantly different. There were no signifi-cant differences in B levels between the castrate,saline-injected group and the sham-injected andintact control groups.

Frogs given DHT injections had DHT hormonelevels that were not statistically different fromthose of the intact and sham-operated controlgroups, but they were significantly different fromthose of the castrate, saline-injected frogs. Andros-tenedione-treated frogs had A levels that werestatistically different from those of the intact,


sham-operated, and castrate, saline-injected con-trol groups. Androstenedione-injected frogs alsohad T levels that were significantly different fromintact and sham-injected control groups as wellas the castrate, saline-injected and DHT-injectedgroups. There were no significant differences inB levels among any of the treatment groups orbetween treatment and control groups.

Thumb pad development in R. pipiensPreliminary analysis indicated that overall

height of the thumb pad was independent offrog body weight both within and between treat-ments (among treatments: F = 2.3, P > 0.13;within treatments: intact, control F = 3.02, P >0.55; sham-operated F = 0.66, P > 0.42; castrate,saline-injected F = 0.05, P > 0.81; A-injected F =7.1, P > 0.074; DHT-injected F = 3.02, P > 0.13).Therefore, thumb pad expression was compareddirectly across treatment groups. There was a sig-nificant effect of treatment on overall thumb padheight (ANOVA, F = 10.265, P < 0.0002). Sham-operated and DHT-treated frogs showed no sig-nificant differences from intact control frogs inthumb pad development (Fig. 1). The thumbpads of the castrate, saline-injected frogs werestatistically different from those of the intactand sham-injected controls and the DHT-treatedanimals (Fig. 1). The A-treated frogs had thumbpads that were statistically different from theintact control group but not the sham-injectedor DHT-treated groups.

Figure 2 plots the relationship between thethickness of the epidermal and glandular epithe-lial layers of the thumb pad for the frogs in theR. pipiens experiment. There is a significant posi-tive correlation between the degree of developmentof the two layers (r = 0.49, F = 11.3098, P < 0.002).A one-factor analysis of covariance with treatmentas the factor and glandular epithelium as the co-variate shows that DHT-treated frogs have alarger epidermal layer for a given glandular epi-thelium size than the other frogs (F1,27 = 4.835, P= 0.036) (Fig. 2). On the other hand, androstene-

dione-treated frogs showed a non-significant trendtoward a smaller epidermal layer for a given glan-dular epithelium size than most of the other frogs(F1,27 = 3.58, P = 0.06).

Hormone levels in R. blythiiTable 2 compares hormone levels of the intact

control and DHT-injected groups of R. blythii.Frogs given DHT supplements had significantlyhigher levels of DHT than the intact controls (t =9.683, P < 0.0002). There were no significant dif-ferences in B, A, or T levels between the groups(B: t = 1.474, P > 0.2005; T: t = 0.8566, P > 0.4308;T: t = 1.1446, P > 0.3693).

R. blythii given DHT injections had significantly

TABLE 1. Hormone levels in Rana pipiens (ng/ml)1

Group N A T DHT B

Control, intact 8 0.64 (0.13) 21.90 (2.20) 6.73 (0.82) 5.99 (0.70)Sham-operated 6 0.56 (0.15) 18.40 (2.04) 6.60 (0.58) 6.72 (0.81)Castrate saline injected 6 1.60 (0.92) 0.86 (0.25)2 0.58 (0.21)2 3.55 (0.79)Androstenedione 6 3.78 (0.80)2 10.00 (2.50)2 0.81 (0.17)2 5.60 (0.61)Dihydrotestosterone 6 0.57 (0.20) 1.48 (0.41)2 9.69 (1.20) 4.56 (0.73)1Mean and, in parentheses, SEM.2Significantly different than the intact and sham-operated controls.

Fig. 1. Comparison of thumb pad height in R. pipiens fromdifferent experimental groups. Vertical bars indicate standarderror of the mean (SEM).


lower DHT levels than R. pipiens given DHT (t =2.72, P < 0.029). Both the control and DHT-in-jected R. blythii had significantly higher B levelsthan those recorded for R. pipiens (t = 8.53, P <0.0000). However, there was no significant differ-ence in B levels between the control and DHT-injected R. blythii (t = 1.47, P > 0.20).

Thumb pad development in R. blythiiPreliminary analysis showed that there was no

effect of body size on the overall height of thethumb pad or on the epidermal and glandular epi-thelium portions separately (epidermis: F =0.1464, P > 0.713; glandular epithelium: F = 4.05,P > 0.083). Therefore, thumb pad expression wascompared directly between groups. The totalheight of the thumb pad was significantly greaterin frogs given DHT supplements compared to frogsin the control group (t = 2.7516, P < 0.03). Figure3 plots the size of the epidermal and glandularepithelial layers of the thumb pad in control frogs

and in those animals that had received DHTsupplements. Frogs given DHT supplements hada significantly thicker epidermis (t = 2.4717, P <0.04), and the external border of the epidermisshowed the beginning of ridges. In contrast, theexternal border of the epidermis of control frogs wassmooth. DHT-treated frogs tended to have a largerglandular epithelial layer as well, but the differ-ence from the control group was not significant (t =1.18, P > 0.2809). Both control and DHT-treatedfrogs had small, irregularly distributed nuptialglands located in the glandular epithelium.

DISCUSSIONAndrogen replacement experiments on R. blythii

suggest that elevated DHT levels produce a re-sponse in the development of the thumb pad tis-sue, supporting the hypothesis that low androgenlevels were involved in the evolutionary loss ofexpression of nuptial pads in the fanged frogs.These results are consistent with the priorwork demonstrating low androgen levels in field-sampled R. blythii as compared to North Temper-ate species Rana (Emerson et al., ’93). Recently,androgen levels were measured in the field for thefirst time in Occidozygous laevis, one of the sistergroup taxa to the fanged frogs (Emerson andBerrigan, ’93; Emerson and Hess, ’96). Whenmapped on a phylogenetic hypothesis of relation-ship, the results from the androgen replacementexperiment and the new data on androgen levelsconsiderably sharpen our understanding of theprobable physiological processes involved in theevolution of the loss of secondary sexual charac-teristics in the fanged frogs.

Figure 4 presents a cladogram of the relation-ships among the North Temperate species R.pipiens and R. catesbeiana, two member speciesof the sister group to fanged frogs, Rana limno-charis and Occidozyga laevis, and the fanged frogR. blythii. This phylogenetic hypothesis is basedon a 310 base pair sequence from the 12S riboso-mal RNA portion of the mitochondrial genome(Emerson, unpublished). Hormone levels for thevarious species, when known, are included. Thepoints on the phylogeny where the presence orabsence of thumb pads and the presence or ab-

Fig. 2. Regression line showing the relationship of epi-dermis to glandular epithelium development in the 32 frogsfrom the Rana pipiens experiment. Individual data pointsfor DHT-treated frogs (dht) and A-treated frogs (a) are shownby letters.

TABLE 2. Hormone levels in Rana blythii (ng/ml)1

Group N A T DHT B

Control 4 0.64 (0.44) 0.53 (0.38) 0.45 (0.20) 46.50 (7.08)DHT 5 0.13 (0.04) 1.51 (0.91) 5.17 (0.38) 30.30 (7.80)1Mean and, in parentheses, SEM.


ng/ml. Rana limnocharis has well-developedthumb pads and lacks parental care. Its andro-gen level is unknown. Occidozyga laevis also haswell-developed secondary sexual characteristicsincluding thumb pads and lacks male parentalcare, but the males have low androgen levels,around 6 ng/ml. These levels are similar to thosefound in R. blythii (Fig. 4). This suggests that lowandrogen levels may have actually evolved priorto the loss of expression of the androgen-medi-ated secondary sexual characteristics and acqui-sition of male parental care (Fig. 4). At the sametime, the response of thumb pad tissue to DHT in-jections in experimental R. blythii suggests that lowandrogen levels may still be one component in theevolutionary loss of expression of this secondarysexual characteristic. A second component may bea change in androgen receptor (AR) protein.

The masculinizing effects of androgen dependon the presence of a functional AR in addition toappropriate hormone levels (Fischer et al., ’95).Other work has established the presence of AR infrog thumb pad tissue (Delrio and d’Istria, ’73;Emerson, unpublished). One possibility, suggestedby the character optimization exercise, is that theloss of expression of the hormone-dependent sec-ondary sexual characteristics in the fanged frogsmay be related both to low androgen levels andsome shift in sensitivity of the AR in the tissues(Fig. 4). A similar scenario involving a shift in an-drogen receptor sensitivity has recently been sug-gested as the mechanism responsible for thesexual dimorphism of the larynx in Xenopus laevis(Kang et al., ’95). In this case, at the developmen-tal stage when masculinization of the larynx oc-curs, there are no differences in circulatingandrogen levels between males and females (Kanget al., ’95), but there are sex differences in ARmRNA expression and androgen binding (Fischerand Kelley, ’91; Kelley et al., ’89). Work is cur-rently under way comparing the sensitivity of theandrogen receptor in thumb pad tissue among R.pipiens, O. laevis, and R. blythii to test the hy-pothesis of an AR shift.

Androstenedione and thumb pad expressionAndrostenedione has not been commonly mea-

sured in amphibian anurans. Work on the Mexi-can leaf frog, Pachymedusa dacnicolor, suggestedthat A might be important in the control of thumbpad expression (Rastogi et al., ’86). The results ofthe R. pipiens experiment do not support thatidea. While A-injected frogs had bigger thumbpads than the castrate, saline-injected group,

Fig. 3. Graphs indicating the difference in degree of de-velopment of the epidermal and glandular epithelial layersof the thumb pad in DHT-injected and control Rana blythii.The vertical bars indicate the standard error of the mean(SEM). The difference between DHT and control frogs wassignificant (*P < 0.04) for the epidermis.

sence of male parental care first appear have beenindicated. The two temperate species of Rana havewell-developed thumb pads and lack male paren-tal care. They have androgen levels of around 30


Fig. 4. A cladogram showing the relationships among sixspecies of ranid frogs based on partial sequence of the 12Smitochondrial gene. Mean T (black bars) and DHT (stippledbars) levels for breeding males are indicated for taxa as avail-able. Hormone levels are from field-sampled animals exceptfor R. pipiens (Licht et al., ’83; Emerson et al., ’93; Emerson

and Hess, ’96; this study). The locations on the phylogenywhere secondary sexual characteristics are gained or lost areindicated by arrows. The hypothesized points at which an-drogen levels and AR sensitivity are thought to have changedare indicated in dots on the phylogenetic hypothesis.

those differences were not significant. And the A-injected group had significantly smaller thumbpads than the intact control group.

The A-injected animals had elevated T levels.

While not as high as the intact and sham-oper-ated control groups, T levels were significantly dif-ferent from the castrate, saline-injected group.(These elevated T levels might account for the par-


tial development of the thumb pad in the A-in-jected animals, as T has a well-established effecton thumb pad expression [Lofts, ’84, and refer-ences therein.]) Androstenedione-injected animalsalso had A levels that were significantly higherthan those of the intact and sham-operated con-trol groups. This suggests the possibility that theA-injected frogs were converting androstenedioneto testosterone. Since the testes had been removedin these animals, the most likely site of T synthe-sis would be the interrenal. However, it has re-cently been shown that the frog brain can alsosynthesize androgens (Mensah-Nyagan et al., ’96),and, in birds, brain estrogen production can el-evate plasma levels of this hormone (Schlinger andArnold, ’92, ’93).

ACKNOWLEDGMENTSWe thank Loree Harvey and Catherine Propper

for helping with techniques and discussing vari-ous stages of the work with us. Janice Ragsdaleassisted with the experiments. Alejandro Purguehelped in measuring thumb pad tissue. Yong HoiSen provided hospitality and logistical assistancein Malaysia. We thank the Malaysian governmentand the University of Malaya for allowing us todo research at their facilities. This research hasbeen supported by National Science Foundationgrants BSR-8822630 and DEB-9317642 to S.B.E.and by NIH RR 00163 and HD 18185 to D.L.H.

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Hormonal induction of thumb pads and the evolution of secondary sexual characteristics of the Southeast Asian fanged frog,Rana blythii