Occurrence of skin alkaloids in non-dendrobatid frogs from Brazil (Bufonidae), Australia (Myobatrachidae) and Madagascar (Mantellinae)

Tasloon, Vol. 22, No . 6, pp . 905-919, 1994 .

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OCCURRENCE OF SKIN ALKALOIDS IN NON-DENDROBATID FROGS FROM BRAZIL (BUFONIDAE), AUSTRALIA (MYOBATRACHIDAE) AND MADAGASCAR (MANTELLINAE) JOHN W. DALY,' ROBERT J . HIGHET2 and CHARLES W. MYERS3 'Laboratory of Bioorganic Chemistry, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20205, U .S .A ., 'Laboratory of Chemistry, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20205, U .S .A ., and 'Department of Herpetology, American Museum of Natural History, New York, NY 10024, U .S .A . (Accepted for publication 13 March 1984) J. W. DALY, R . J. HIGto3r and C . W. MYI3ts . Occurrence of skin alkaloids in non-dendrobatid

frogs from Brazil (Bufonidae), Australia (Myobatrachidae) and Madagascar (Mantellinae) . Toxicon 22, 905 - 919, 1984. - Several taxa of small frogs from the southern hemisphere contain alkaloids similar or identical to compounds previously known only from neotropical poison frogs of the family Dendrobatidae . Skin of the Brazilian toad Melanophryniscus moreinae (family Bufonidae) contains a new alkaloid 8-hydroxy-8-methyl(5'-hydroxy-2'-methyl-hexylidene)-1azabicyclo-[4 .3 .0]nonane (C Ha,NOJ, which is designated pumiliotoxin 267C. Such a structure is typical of the pumiliotoxin-A class of dendrobatid alkaloids . Melmophrynisew moreiroe contains smaller quantities of an alkaloid (C  HNO,) identical in chromatographic and mass spectral properties to the dendrobatid alkaloid allopumiliotoxin 323B. Allopumiliotoxin 323B and an isomer of 267C occur with unidentified alkaloids in skin of the Australian frog Pseudophryne semimarmorata (family Myobatrachidae) and also in the skin of the Madagascan frog Mantella aurantiarw (family Ranidae, subfamily Mantellinae) . In addition to new compounds, Mantella aurand= and M. madagascarienvis also contain other alkaloids (e .g. histnoniootoxm and pumiliotoxin B) that were known previously only in dendrobatid frogs . Such alkaloids have not been detected in a phylogenetically wide array of other anuran amphibians, and the dendrobatid alkaloids thus become an evolutionary enigma . Certain of these compounds may have arisen convergently from new biosynthetic pathways in several families of frogs, or these alkaloids may represent parallel expression of shared-primitive pathways that are unexpressed or lost in related frogs .

INTRODUCTION

elaborate a remarkable diversity of secondary metabolites that are often stored in cutaneous glands and released as a chemical defense against predators. In frogs these compounds include peptides, biogenic amines, bufogenins, tetrodotoxins and the dendrobatid alkaloids (Table 1). We suggest that the evolutionary development of amphibian defensive compounds falls into two categories . (1) A widespread naturally occurring active compound, such as a peptide (e.g. bradykinin, bombesin) or a biogenic amine (e.g. histamine, serotonin), is elaborated in excessive quantities for a secondary function of defense. Some toad toxins, for example, may have been secondarily elaborated from a physiological regulator of Na+-K'-ATPase, an enzyme crucial to sodium and water homeostasis (FLIER et al., 1980). (2) A new biologically active substance, frequently with a complex structure, is elaborated for the primary function of defense. An example is batrachotoxin, an extraordinarily toxic steroidal alkaloid that AMPHIBIANS

905.

J . W. DALY, R . J . HI(3HET and C . W. MYERS TABLE 1 . OCCURRENCE OF NOXIOUS COMPOUNDS IN FROG SKIN Family Ascaphidae Brachyeephalidae Bufonidae

Peptides

Novel classes Biogenic amines Serotonins Histamines Tyramines Catecholamines

+ +

+

Dendrobatidae

Discoglossidae Heleophrynidae Hylidae

+ + +

Hyperolüdae

+

Leptodactylidae

+ +

+

+

+

+

Myobatrachidae Pipidae Ranidae

+ + +

+ +

Rhacophoridae Rhinodermatidae

+

+

+

Tetrodotoxins (Btnrhycephahts) Bufodienolides (widespread), dehydrobufotenine (Blijo), pumiliotoxins (Mehmophryniww), tetrodotoxins (Atelopus) Batrachotoxins, pumiliotoxins, histrionicotoxins, gephyrotoxins, etc . (Dendrobates, Phyllobates) Dehydrobufotenine 0-sulfate (Acris) Trypargine (Kassina, Hylonbates) Dehydrobufotenine, spinaceamine (Leptodactyhts) Pumiliotoxins (Pseudophryne) Pumiliotoxins, histrionicotoxins, other alkaloids (Mantella)

+

'The serotonins include bufotenine, bufotenidine, O-methylbufotenine, etc. ; the histamines include N-methyl and N,N-dimethy lttistamine; the tyramines include candicine and leptodactyline; the catecholamines include epinine . Not included in the table are hemolytic proteins, reported from several species of Diseoglossidae, Hylidae, Pelobatidae and Ranidae (see list in DALY and WrrKoP, 1971) . Noxious substances seemingly have not been reported for the Centrolenidae, Leiopelmatidae, Pelodydidae, Pseudidae, Rhinophrynidae and Sooglossidae. A toxin of unknown structure is produced by Phrynomerus of the Microhylidae (JAEmR, 1971) . The Table is based on the following papers, and references therein : AKIzAwA et al., 1982 [frogs reported are now considered hyperoliids, not rhaeophorids] ; CEt and ERspAMER, 1966 ; Cm et al., 1968 ; DALY, 1982 ; DALY and WrrKoP, 1971 ; ERSPAMm, 1971 ; ERsPAMER and MELCHIoRIu, 1980, FLmR et at., 1980 ; HAnnnNN L, 1969 ; MICHL and KmSme, 1963 ; NAKAJIMA, 1981 ; Kim et al., 1975 ; SERBEN, 1982.

seems to be unique to dendrobatid frogs of the genus Phyllobates (MYERS et al., 1978; MYERS and DALY, 1983). Batrachotoxin is but one of over 200 novel alkaloids that have been isolated from various species of Dendrobatidae (Fig . 1), a family of frogs confined to South America and southern Central America (DALY, 1982; MYERS and DALY, 1983) . Some of the dendrobatid alkaloids have been given trivial names, but, because of their large number, it has been convenient to designate them with boldface numbers corresponding to their molecular weights, with code letters being added when necessary to distinguish compounds with the same isobaric molecular weight . In addition, the dendrobatid alkaloids are divided among a number of structural classes, including, for example, the steroidal batrachotoxins, the pumiliotoxin-A and allopumiliotoxin class (6-alkylidene-8methyl-8-hydroxyindolizidines and 6-alkylidene-7,8-dihydroxy-8-methylindolizidines), and the histrionicotoxins (1-azaspiro[5,5]undecan-8-ols) . No other family of animals produces such a diverse array of alkaloids as the Dendrobatidae, but we now report that

Fro . 1 . Fxoos of THE FASflt .Y DENDAoaAnDAE, Aaovr 1 .5 - 2 .41vmEs r.iFE size . A. Dendrobates Mstrionicus from northwestern South America, x 1 .7 (AMNH 102108) . This frog was black with spots of bright orange-red, but the species is highly variable (see MYens and DALY, 1976, color pl . 1) . B. Dendrobates pumilio from southern Central America, x 2.3 (AMNH 107122) . This specimen was nearly uniform reddish orange, but coloration is extraordinarily variable (MYEas and DALY, 1983, color pl .). C . Phyllobates terribilis from northwestern South America, x 1 .5 (AMNH 116721) . Usually golden yellow in color, this dangerously toxic frog produces large quantities of batrachotoxins (MYeas et al, 1978, color pl .) . D . Phyflobates hrgubris from southern Central America, x 2 .4 (AMNH 102247) . Black with gold stripes, this small frog produces trace amounts of batrachotoxins. From color transparencies by C W. Myers, American Museum of Natural hfhtory'(AMNH) .

FIG . 2. ALxALoiD-PRODUCING FROGS OF OTHER FAAIILIES, ABOUT 1 .8 TAO3S LIFE SIZE. A . Melanophryniscus moreirae, Bufonidae, from Brazil (AMNH 104066). Blackish brown above, bright red below . B . Mantella aurantiaca, Ranidae, Mantellinse, from Madagascar (AMNH 114042) . Golden yellow with concealed flash marks of bright orange on hindquarters . C . Pseudophryne semimarmorata, Myobatrachidae, from Australia (AMNH 111038) . Brown to bluish black above, orange, black and white below . D . Pseudophryne corroboree, Myobatrachidae, from Australia (AMNH 84046) . Gold with black stripes . From color transparencies by C. W. Myers (A,B D) and R . G. Zweifel (C), American Museum of Natural History (AMNH) .

Alkaloids in Frog Skin

909

certain of these compounds and additional new ones occur in a few members of three other families of frogs (Fig. 2). MATERIALS AND METHODS About 75 species of non-dendrobatid frogs, representing 10 families from five continents, were surveyed for alkaloids . All species were negative (Table 2) except for the following . Melanophrynisrm moreinae (Bufonidae) was collected by Myers and Daly in 1979 in the Serra da Mantiquerim 19 km. NW Itatiaia, 2320 m. elevation, State of Rio de Janeiro, Brazil. Pseudophryne semimarmorata (Myobatrachidae) from Gembrook State Forest, Victoria, Australia, was provided by R . G . Zweifel in 1981 . Specimens of Mantella aurandara and Mantilla madagascariensis (Ranidae, Mantelfinae), from Malagasy, were obtained in 1980-1981 from a commercial dealer and from J . D. Groves at the Philadelphia Zoological Garden . Voucher specimens are preserved in the amphibian collection of the American Museum of Natural History . Fresh skins were stored in 70-10044 methanol. Details of extraction, fractionation and chromatographic analysis of frog alkaloids are given in MYERs and DALY (1976) and DALY et al . (1978) . Specific methodology, including new and improved techniques, are indicated where appropriate in the following species accounts. The format for the physical and spectral properties of each alkaloid includes : (I) numerical designation (= mol . wt .) in boldface along with an identifying letter if needed; (2) empirical formula, (3) an R, value for thin-layer chromatography (silica gel, chloroform-methanol 9:1) ; (4) an emergent temperature (°C) for gas chromatography (see legend to Fig . 5 for conditions) ; (.S) the electron-impact mass spectrum, with intensities in parentheses, expressed relative to the base peak set equal to 100; (6) comments on chemical properties and taxonomic occurrence in frogs . Structures of alkaloids are presented in Fig . 3 . RESULTS

Melanophryniseus moreirae

Skins (wet weight 9 g) from 52 specimens of this small Brazilian toad (Fig . 2A) were extracted with methanol and then with water. Subcutaneous injection in 20 g white mice of the methanol extract (equivalent to 50 mg wet weight of skin) caused locomotor difficulties, salivation, convulsions and death in 10-13 min. Injection of the aqueous extract (equivalent to 100 mg skin) had no significant effect . The methanol extract was fractionated by partition first between 50°16 aqueous methanol and chloroform. The chloroform fraction was extracted into 0.1 N HCl, which was then rendered basic with ammonia and back extracted into chloroform to yield an alkaloid fraction . Thin-layer chromatography and combined gas chromatography - mass spectrometry of the alkaloid fraction revealed the presence of two alkaloids (Figs 4 and 5). High resolution mass spectrometry was used to determine empirical formulae . Chemical ionization mass spectrometry with ammonia and then with deuteroammonia provided data on exchangeable hydrogens. Perhydrogenation was used to determine number and location of double bonds. Phenylboronation was used to detect vicinal hydroxy groups (TOKUYAMA et al., 1984). Separation of the alkaloids by rotary centrifugal thin-layer chromatography (Chromatotron, Harrison Research, Palo Alto, . CA) with silica gel and chloroform-methanol solvent mixtures yielded 9 mg of,4he major alkaloid designated 267C and less than 300 pg of a minor alkaloid which appeared identical by gas chromatography - mass spectrometry to the known dendrobatid alkaloid, allopumiliotoxin 323B (Fig. 3). Nuclear magnetic resonance spectroscopy permitted a definitive formulation of the structure of 267C (Fig. 3), which, although ofAhepumili otoxin-A class of dendrobatid alkaloids, represents a previously unknown compound . The physical and spectral properties of the two alkaloids from Melanophryíüscus were as follows. 267C . C36H39NO3r 0.28, 190°, m/z 267 (16), 266 (7), 252 (5), 250 (2), 234 (3), 224 (7), 222 (9), 194 (12), 176 (5), 166 (100), 112 (8), 84 (18), 70 (75) . H3-derivative by reduction with hydrogen gas/palladium charcoal . Two exchangeable hydrogens by deuteroammonia chemical ionization mass spectrometry . No phenylboronide derivative .

910

J. W. DALY, R. J. HIGHET and C. W. MYERS TABLE 2 . GENERA OF FROGS FROM WHICH SKIN ALKALOIDS WERE NOT DETECTED

BUFONIDÀE

A telopus - 4 Bttfo - 5 Dendrophryniscw - 1

DENDROBATIDAE Colastethus - 4

HYLIDAE

Agalychnis - 1 Hyla - 1 Litoria - 4 Nyctimystes - 1 Phrynohyas - 1 Phyllomedusa - 1 smilisca - 1

HYPEROLIIDAE Hyperolius - 1 Kassina - 1 Leptopelis - 1

LEPTODACTYLIDAE

Adenomera - 1 Barycholos - 1 Crossodactylus - 2 Cyclorhamphus - 2 Edalorhina - 1 Eleutherodactylus - 5 Euparkerella - 1 Holoaden - 1 Hylodes - 4 Leptodactylus - 2 Lithodytes - 1 Megaelosia - 1

LEPTODACTYLIDAE (cont.) Panatelmatoblus - 1 Physalaemus - 2 Pleurodema - 1 Proceratophrys - 1 77toropa - 1

MICROHYLIDAE

Breviceps - 1 Microhyla - 1 Phrynomerus - 1

MYOBATRACHIDAE Cyclorana - 1 Limnodynastes - 1 Mixophyes - 1

PELOBATIDAE

Leptobrachium - 1

PIPIDAE

Xenopus - 1

RANIDAE

Hemisus - 1 Hylarana - 1 Phrynobatrachus - 1 Ptychadma - 1 Pyxicephahis - I Rana - 5

RHACOPHORIDAE Chiromantis - 1 Rhacophorus - 1

*Number of species examined shown after each genus . Fractionation, thin-layer chromatography and combined gas chromatography-mass spectrometry as described in DALY et al . (1978) .

[al R' +7.2° (0.8, CH,OH) . This compound has not been detected in some fifty species of dendrobatid frogs (DALY, 1982, unpublished results) . Proton magnetic resonance spectra (Nicolet NT 360 spectrometer at 360 MHz): 5.05 ppm (d, 9.9 Hz, H-10), 3.79 (d, 11 .9, H-5 eq.), 3.74 (m, H-14*), 3.06 (dd, J 8.7, 7.1, H-3 eq.), ca 2.4 (m, H-11 *), 2.35 (d, 11 .9, H-5 ax*), 2 .24 (m, H-3 ax), 2.15 (d, 16 .4, H-7 eq.), 2.11 (dd, 16.4, 0.8 Hz, H-7 ax*), 2.0 (br, H-8a), 1 .72 (br, H-1 and H-2*), 1 .35 (m, H-13), 1 .16 (d, 6.2, H-15*), 1 .13 (s, H-9), 0.99 (d, 6.4, H-16*) . Asterisks indicate that assignments were confirmed by double resonance (see assignments for pumiliotoxin 251D; DALY et al., 1980b; ToxuyAMA et al., 1984) . Carbon-13 magnetic resonance spectra (JEOL FX60 spectrometer): assignments are by comparison to pumiliotoxin 251D (DALY et al., l980ó; TOKUYAMA et al., 1984) and to 2hexanol (ROBERTS et al., 1970) and are consistent with an attached proton test (PATT and SHOOLERY, 1982): 134 .1 ppm (C-10), 130 .2 (C-6), 71 .7 (C-8a), 68 .3 (C-8), 68.11 (C-14), 54.4 (C-3), 53 .1 (C-5), 48.8 (C-7), 37.2 (C-13), 33.6 (C-12), 32 .0 (C-11), 24.3 (C-9), 23.5

Alkaloids in Frog Skin

91 1

H

323B

339A

,CH2 CH ?CH,

283A

Î95A

Fie . 3 . STRuerm OF AixArnm 267C FRom Melanophrynfçna moretrae AND oF Fra Af xALo ms FRom DwDROBATiD FRoas: PumuoToxiN B (323A), ALLopumn.loTom 323B. ALwpurmsoTOmv 339A, xfmoNicomm (2a3A) AND PumU.ioToamv C (195A) .

(C-15),23 .2 (C-1), 21 .7 (C-16),21.0 (C-2). The values assigned to the indolizidine carbons (C-1- C-11 and C-16) correspond to those reported for pumiliotoxin 251D within 0.2 ppm. Absence of exactly parallel substitution patterns results in somewhat larger discrepancies in C-12 - C-14 . 323B . C,9HNO,, 0.20, 22811 , m/z 323 (3), 306 (7), 210 (7), 209 (9), 182 (43), 114 (22), 70 (100). H,derivative . Three exchangeable hydrogens. No phenylboronide derivative . All properties of this minor alkaloid from Melanophryniscus appear identical to those of allopumiliotoxin 323B, which occurs in many frogs of the genus Dendrobates. The lack of a phenylboronide derivative is consonant with an axial 7-hydroxy group (TotcuyAmA et al., 1984). Biogenic amines such as serotonin, bufotenine and an unidentified hydroxyphenylalkylamine have been reported from species of Melanophryniscas (CEI and ERSPAMER, 1966; CEi et al., 1968). Melanophryniscas moreirae skin also contains relatively high levels of polar compounds which inhibit Na* - K*-ATPase (FLIER et al., 1980). Mantella aurantiaca Skins (wet weight 320 mg) from five specimens of this Madagascan frog (Fig. 2B) were extracted with methanol and an alkaloid fraction obtained. The skin had an unpleasant bitter taste. Subcutaneous injection in a 20 g white mouse of methanol extract

J . W . DALY, R . J . HIGHET and C . W .

91 2

MYERS

SOLVENT FRONT

O 267C 323B Origin-

0

0

A

B

®- 283A 0-283C 0 - 285A -3238 - 323A - 339A

267D 3238

@-323B

- Origin C

D

4.

FIG . REPREsETTCAnoNs oF siucA GEL THIN-LAYER cHRGMATGPLATEs oF ALKAinms FRGIN : (A) MeknrophryWscvs morehm (B) Mantdla aunantiaca; (C) Mantella mndagasrvntensis;

(D) Pseudophryne sendrnarmorata.

A sample of 10 pl of methanolic alkaloid fraction, corresponding to 10 mg wet-weight skin, was applied at origin and developed with chloroform :methanol (9:1) . After drying, iodine vapor was used to detect alkaloids . Spot intensities are depicted as follows : cross hatched, large amounts; hatched, moderate amounts ; dotted, small amounts . Designations of spots were based on comparison with R, values of authentic dendrobatid alkaloids or by extraction and mass spectral analysis for 267C and 267D . Some spots may represent more than a single alkaloid .

corresponding to 50 mg wet weight skin caused initially some locomotor difficulties, followed by complete prostration. Upon prodding, the mouse would move about briefly with hindlimbs nearly useless for locomotion. Recovery occurred after 90 min. Six alkaloids- were detected and. characterized by combined gas chromatography- mass spectrometry (Fig. 6). A thin-layer chromatoplate is depicted in Fig. 4. The major compound appeared identical to the dendrobatid alkaloid pumiliotoxin B (323A) (see Fig. 3). Trace amounts of an alkaloid with properties corresponding to allopumiliotoxin 323B were also present. Minor amounts of an alkaloid corresponding to allopumiliotoxin 339A (Fig. 3) were detected . The remaining three alkaloids (235C, 251G and 267D) had not been detected in dendrobatid frogs. One of these (267D) was isomeric with alkaloid 267C from Melanophryniscus, but eluted at a lower temperature on gas chromatography. The properties of the six alkaloids from Mantella aurandaca were as follows. 235C. C,,HnNO, 166°, m/z 235 (28), 234 (53), 220 (20), 162 (100, CH,6 N). H2derivative. One exchangeable hydrogen, apparently due to a hydroxyl group. This compound has been detected only in Mantella aurantiaca and Mantella madagascariensis.

Alkaloide in Frog Skin

91 3

257C

180

180 200 2~9 240 °C

FIG . 5 . ()As CHROMATOGRAM OF ALxALOws FROM Me1Mophryrtiscus Moreirae. Chromatography was with 2 MI of methanolic alkaloid fraction, corresponding to 2 mg of wet akin . A 1 .5% OV-1 column was programmed at 10°C per min to 280° from an initial temperature of 150° . Time of injection is indicated by the arrow . Programming was initiated after the methanol peak maximized . Detection was with flame ionization.

251G. C,,H,.N02, 178°, m/z 251 (26), 250 (45), 162 (100) . H,-derivative. Two exchangeable hydrogens. This compound is a hydroxyl congener of 235C'and has been detected only in Mantella aurantiaca. 267D. C,6H,9N0=, 178°, m/z 267 (13), 250 (10), 194 (16), 166 (100), 70 (80). H,derivative . Two exchangeable hydrogens. This alkaloid is an isomer of 267C and shows a very similar mass spectrum . The second hydroxy group must occur in the side chain, based on the mass spectrum . It seems likely that it will prove to be at the highly hindered C-11 position (see Fig. 3), to account for the elution at a relatively low temperature on gas chromatography. 323A . Pumiliotoxin B, C,,H,.N03, 225°, m/z 323 (17), 306 (7), 278 (11), 260 (5), 206 (18), 194 (32), 193 (20), 176 (10), 166 (78), 70 (100) . H,derivative . Three exchangeable hydrogens. Side chain phenylboronide derivative . All properties of this compound appear identical to those of the dendrobatid alkaloid pumiliotoxin B. 323B. C,9HNO,, 0.18, 273° . The properties of this trace alkaloid appear identical to those of the dendrobatid allopumiliotoxin 323B (see Melanophryniscus for properties). 339A . C,,H33N0 4, 0.08, 246°, m/z 339 (5), 322 (3), 294 (11), 209 (18), 208 (16), 192 (21), 182 (43), 114 (25), 70 (100) . H,-derivative. Four exchangeable hydrogens. Side chain

J. W. DALY, R. J . HIGHET and C . W . MYERS

91 4

B

A

196C. _207A 323A > 323E

11241E 235C 2698 283A . 285C

241 B 235C 267D

251O

f 160

180

2ÓO

220 240

2610 °C

160

180

2ÓO

220

240

°C

FIG. 6. GAS CHROMATOGRAMS OF ALKALOIDS FROM: (A) Mantella aurantiaca; (B) Mantella madagawariensis (FOR DETAILS SEE LEGEND TO FIG. 5) .

phenylboronide derivative . It would appear that this alkaloid is identical to the dendrobatid alkaloid allopumiliotoxin 339A (TOKUYAMA et al., 1984). Mantella madagascariensis A single skin (wet weight 70 mg) of this second Madagascan species was extracted and an alkaloid fraction obtained . Nine alkaloids were detected by gas chromatographic mass spectral analysis (see Fig. 6). Three of these (283A, 285A, 285C) were clearly histrionicotoxins (Fig. 3), formerly considered to be alkaloids unique to dendrobatid frogs . Interestingly, these three alkaloids occur in approximately the same proportions as the major histrionicotoxins in many populations of Dendrobates histrionicus (MYERS and DALY, 1978). A fourth alkaloid from M. madagascariensis was also one typical of dendrobatid frogs, namely allopumiliotoxin 323B. There were, in addition, three trace alkaloids, which also appeared to be compounds previously detected only in dendrobatid frogs. These were as follows: 195C, which had mass spectral properties and other characteristics of the decahydroquinoline alkaloid pumiliotoxin C (Fig. 3); alkaloid 207 (now to be designated 207A fide MYERS et al., 1984), which is a bicyclic tertiary amine, probably an indoliz idine; alkaloid 269B, a bicyclic secondary amine, perhaps a deoxyhistrionicotoxin . The last alkaloid often appears together with histrionicotoxins in dendrobatid frogs. Two of the alkaloids of M. madagascariensis had not been detected in dendrobatids : one was the major alkaloid (241B) of this mantellid species, while the other

Alkaloids in

Frog Skin

91 5

(235C) occurs also in M. aurantiaca. The properties of the nine alkaloids of Mantella madagascariensis are as follows. 195C. Pumiliotoxin C, CH"N, 156°, m/z 195 (5), 194 (6) , 152 (100), 109 (10) . Hoderivative . One exchangeable hydrogen . The properties of this trace alkaloid appear identical to those of pumiliotoxin C from dendrobatid frogs . 207A. CH,sN, 158°, m/z 207 (2), 206 (2), 138 (100). Hz-derivative. No exchangeable hydrogens. The properties of this trace alkaloid appear identical to those of 207A, a common dendrobatid alkaloid . 235C. C,.HNO. Properties of this minor alkaloid are presented under Mantella aurantiaca.

241B . C,6HasN, 167°, m/z 241 (15), 135 (45), 58 (100). Ha-derivative. No exchangeable hydrogen . This major alkaloid has not been detected in other frogs. It apparently lacks rings, but further supplies are required for adequate characterization . Alkaloid 241 (DALY, 1982) of Dendrobates occultator now becomes 241A with this addition . 269A. C,9HN, 203°, m/z 269 (2), 204 (100). H,o-derivative. One exchangeable hydrogen . The properties of this trace alkaloid appear identical to those of the dendrobatid alkaloid 269A . 283A . Histrionicotoxin, C,9HNO, 0.44, 205°, m/z 283 (15), 266 (12), 218 (53), 200 (22), 160 (25), 96 (100). The properties appear identical to those of histrionicotoxin from dendrobatid frogs. 285C. Allodihydrohistrionicotoxin, C,9HNO, 0.38, 207°, m/z 285 (9), 268 (7), 218 (5), 176 (20), 162 (16), 96 (100). The properties appear identical to those of allodihydrohistrionicotoxin. 285A. Isodihydrohistrionicotoxin, C,9HNO, 0.34, 211°, m/z 285 (18), 268 (8), 218 (12), 176 (32), 162 (20), 96 (100). The properties appear identical to those of isodihydrohistrionicotoxin. 323B . C,9HNO,, 0.18, 230° . The properties appear identical to those of the dendrobatid allopumiliotoxin 323B (see Melanophryniscus for properties) . Pseudophryne semimarmorata .

A single skin (wet weight 250 mg) of this Australian frog (Fig . 2C) was extracted with methanol and an alkaloid fraction obtained . Subcutaneous injection of methanol extract equivalent to 50 mg wet weight skin into a 20 g white mouse caused initial pain at site of injection, then severe locomotor difficulties, slowing of respiration, gagging, stretching and finally convulsions and death in 20 min. Two alkaloids were detected and characterized by combined gas chromatography -mass spectrometry (Fig. 7). A thinlayer chromatoplate is depicted in Fig. 4. The major alkaloid 267D was an isomer of 267C and appeared identical to 267D from Mantella aurantiaca. One alkaloid appeared identical to allopumiliotoxin 323B. There were, in addition, some alkaloids of unknown nature, with apparent molecular weights of 311, 313 and 315. Further supplies will be required for adequate characterization of these compounds. A report (HABERMEHL, 1965) that alkaloids in Pseudophryne corroboree (Fig. 2D) were of the samandarine class needs to be verified. In any case, samandarine-like alkaloids were not detected in P. semimarmorata . An unidentified toxic substance has been reported from species of Heleioporus, another genus of Australian myobatrachid frog (SOFTLY and NAUtN, 1975) . The properties of two of the alkaloids in P. semimarmorata were as follows. 267D. C,,H,9NO,, 0 .28, 178°, m/z 267 (18), 250 (14), 194 (9), 166 (100), 70 (78) . Two exchangeable hydrogens. The properties, including the mass spectrum, appear identical to

91 6

J. W. DALY, R. J. HIGHET and C. W. MYERS

267D

311, 313,315

323B I

160

I

180

I

200

I

220

I

240 °C

FIG. 7. GAS CHROMATOGRAM OF ALKALOIDS FROM Pseudophryne semimarmorata (FOR DETAILS SEE LEGEND TO FIG. 5) .

those of 267D from Mantella aurantiaca . Determination of the position of a side chain hydroxyl group will require further investigation . 323B . C,9H33NO3, 0.18, 230° . The properties appear identical to those of the dendrobatid alkaloid allopumiliotoxin 323B . DISCUSSION

Many of the alkaloids listed above appear to be identical to compounds previously known only from dendrobatid frogs, and at least two of the several new compounds appear to belong structurally to a class of dendrobatid alkaloids . The occurrence of shared alkaloids in diverse genera of frogs can be explained in several ways : (1) a shared alkaloid represents a uniquely derived character (synapomorphy) indicating that the frogs belong to a monophyletic lineage; (2) such an alkaloid represents a shared-primitive character (symplesiomorphy) that has been variously lost in diverging lineages ; (3) the alkaloids have been independently evolved in several lineages, whether by parallelism or convergence . Possibility number 1 can be eliminated with some confidence, since it implies that the several genera are more closely related with one another than with other frogs in their respective families . There is no morphological evidence presently available to indicate that Brazilian Melanophryniscus and Australian Pseudophryne are taxonomically misplaced in, respectively, the Bufonidae and Myobatrachidae, some genera of which lack

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dendrobatid alkaloids (Table 2) . The familial placement of Mantella has been controversial; the first named species was described as a Dendrobates in 1872 and Boulenger (1882) later created Mantella as a genus of the Dendrobatidae. However, the resemblance of Mantella to dendrobatids seems superficial, and in recent years it has been moved between the Ranidae and Rhacophoridae - most recently being considered as belonging to an endemic Madagascan subfamily (Mantellinse) of the cosmopolitan Ranidae (BLONDAERS-SCHLÜSSER, 1981 ; BUSSE, 1981). Frogs that produce dendrobatid alkaloids are usually brightly pigmented, but given the diversity of colors and patterns (Figs 1 and 2), these features are considered convergent and not evidence of relationship . Possibility number 2 (symplesiomorphy) cannot be dismissed out of hand, although it seems unlikely that production of primitive alkaloids would be retained so randomly within several radiating lineages that probably separated in the Mesozoic . However, the phylogenies of these groups are too poorly known and the biochemical survey is too incomplete for a proper evaluation of this hypothesis . Possibility number 3 (independent evolution) seems most likely to us, but without knowledge of biosynthetic enzymes it is futile to argue for a particular mechanism. Identical or similar alkaloids in distantly related frogs conceivably could arise de novo from a shared-primitive capacity for their production (parallelism). The best corroboration for parallel evolution would be the determination of identical biosynthetic pathways in different genera of alkaloid-producing frogs and identification of unexpressed or incomplete sets of enzymes in related frogs, although obtaining such data would be a formidable task . It is also conceivable that alkaloids have originated de novo from evolutionarily new biosynthetic pathways (convergence). The best corroboration for convergence would be the detection of fundamentally different biosynthetic pathways in different families of frogs; determination of identical pathways in a few unrelated species would be equivocal, since either parallelism or convergence could be evoked as a mechanism. Whatever the mechanism, it seems likely that the alkaloid allopumiliotoxin (323B) developed independently in the families Dendrobatidae (Dendrobates), Bufonidae (Melanophyrniscus), Myobatrachidae (Pseudophryne) and in the Ranidae (Mantellinse, Mantella) . If so, then the ability to form related alkaloid 267C and the isomeric 267D developed in Melanophryniscus (267C) and in Mantella and Pseudophryne (267D), but apparently not in dendrobatids . These alkaloids would be easily formed from a common dendrobatid alkaloid (pumiliotoxin 251D) merely by hydroxylation of the side chain, but this apparently does not occur in dendrobatids . Another related alkaloid, pumiliotoxin B (323A), is known from many dendrobatid frogs, but only from Mantella aurantiaca among the non-dendrobatids . This species of Mantella also forms the 7-hydroxy derivative of pumiliotoxin B, namely the dendrobatid alkaloid allopumiliotoxin 339A . The capacity to form dendrobatid alkaloids other than the (allo)pumiliotoxins appears to have occurred only in the mantellids, which form histrionicotoxins, probably the decahydroquinoline pumiliotoxin C, and other presently unclassified dendrobatid alkaloids (207A, 269A). Along with the ability to form dendrobatid alkaloids, some of the non-dendrobatids developed the capacity to form their own unique compounds, in particular alkaloids 235C, 241B and 251G of Mantella . Based on their mass spectral properties, these new compounds do not seem to belong to any of the known classes of dendrobatid alkaloids . Although the dendrobatid alkaloids seem to occur independently in several families of frogs, another group of alkaloids - the tetrodotoxins - provides a more striking

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J. W. DALY, R. J. HIGHET and C. W. MYERS

example of random phylogenetic occurrence in animals. These complex guanidinium alkaloids are found in an octopus (SHEUMACK et al., 1978), in certain marine snails (NARITA et al., 1981 ; NOGUCHI et al., 1981), in puffer and goby fishes (ELAM et al., 1977), in salamanders of the .genera Taricha and Notophthalmus (MOSHER et al., 1964; LEVENSON and WOODHULL, 1979) and in frogs of the genera Atelopus and Brachycephalus (KIM et al., 1975; SEBBEN, 1982). It is becoming evident that at least some novel defensive alkaloids may disappear when a poisonous species is raised under conditions of captivity. Tetrodotoxin is absent in hatchery-raised puffer fish (MATSUI et al., 1981) and perhaps in captive-raised newts of the genus Taricha (H. S. Mosher, personal communication) . The situation is still being studied in dendrobatid frogs, but batrachotoxin was not detected in second-generation Phyllobates terribilis (DALY et al., 1980x), although the toxin persists in the wild-caught parental frogs that have been living in captivity for a decade . These observations raise the possibility that symbiotic microorganisms or other environmental factors might play an important role in the synthesis of alkaloids during ontogeny . Clearly, there is much to learn about the evolutionary development of alkaloid production in animals. Acknowledgements - Fieldwork in

Brazil was conducted in collaboration with Dr P. E. VANZOLINI, MUSell da Zoologia, Universidade de 8áo Paulo. Recent fieldwork in South America has been supported by a grant to the American Museum from the Research and Developmental Laboratories of Astra Liikemedel AB, Sweden, and we thank Dr STIG AGURELL for his interest and support. For providing needed material from Australia and Madagascar, we are grateful to Dr RICHARD G. ZWEIFEL, American Museum of Natural History, and Mr JOHN D. GROVES, Philadelphia Zoological Garden .

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