—_ ene — mR Bh OMA Tae tOtaes mano a aeannean arate ee eee eee nat) ere had Ong (At Baba cress we HO Rad of 5 mer en Am * ho tend ‘ ee etm P cs ~ es . - ” hate C wee *. oo ~4 4 « gerne get A re = yes - 5% im er a » ri = : oo en PAA A COREE 6 A Om RRE AR te HE mm 6 os ad ya Ei? wee x a 7 ienteae aan nted on ok eta Ane) a Maes : hs pa . S “ er ee er tee re ee ees 7 a anne —_— of bc adap r : AD Ge teth intvarndtean. L-wpretiocaberveeictbaebeteiesetoitese aaa tnanaantid se 4 ~ : ae Senn ome. wane guneenn a ae Seeded wenn s me Os oan WORN. an Wrereererer eres vee nneete a4 - =~ ne -mhad taker t- rysagntastor 4-4 ” - wae eeraaas waenewm, 4 ee et ran 3 : mere pO wm a BP Be Bl ann CN Cor OB AN A Cae es sanncenaeoed Om hmmm anne — crs “tetas Nathan ne ee eS nee Serre era a, mate aeeed is oe AER ee ORE Mow © ton . 2 \ ™* 7 a cS Sek Ret RAPA Nie Ana Waren ORES en mente : arte tn te sae aoe + at ny OA OCR OM Ht we aan meenan co : ote ete ot rR 08 net ONE EMR Rhee PRONE HR tan ws ke Bi he D Sdeo oo ort rctrendlaetpont Tne. hl tatee deus eet pbeberdesseas 7 2 Fae. ee eee ee ee ee ee ones en ene rE ee err ee ee en etter amet Pee banner A 60bmsPhrwens 16. sn cemratimy we o48 tin bass wee atm & bs pe cesrerce ame - PED FOr eH eT Oe Henge earner os . ‘orate ooresnd. eretotn thee ae coee 7.4 20a tans Sener eee nun enhe t- wehemaere nas prepa De her ae 20 heen. Sec w mc heaghanonen ote RwOessin wees Oe enn ae ‘s saehecnine ne ~ eyes th-a-or—prethvary Pm eet A Me < 8. ss bene St tees ne neat auk me Bohetuen + . tm ewe etna en ie 28 OP eee Ne tele tS Heeekehem cee 9 tm oe ~ eorores <0 * hy rat watt NTE NORTE Ones Fae h Reems waste xe S ” pone ot caawe yt coe owe © SLIT tee tomer te font mee Re tome ee in ad, aod - ——ornaar. aoe Ba a nena, Sra. 0. hy aa een me Peas dont. ah as a ‘som ates =, ater ,. cs >. rs re mmm ta aetna ® was lew Soa sinnad . ‘ sate ome sa Reh Ose Marne. Fen bennennandeetalas tearm pew Cod ee eee oe = ee m ~s wt apne wre hee eens CeO aonen re ‘ *. Sen i - > . ope ane Pe se ors % : 5 ‘ a nm ate ~ eres whos - Rv detey — . , rs eemeesee dean es Se AAAS Rees ee eee asaeen Pesstne © + 00. 0e mabe? eens fom toh mist a Ae A Nene A NO RAD ENTERED SORT ORNATE MOR n rowan - - te err pale pig Se ota - - fase ++ Ai, ~ . Semee ats oy ae ete ee ~nonere ede a ineneprne Bctreredasaien eesbapeacoreeeas emia oem = . Ses god es tee : “s+ - — 2 + tema taser > cy nek NOTRE. . . Ce ee a a ' 7 cx . a as 7 —* ~ - ear 8 nee Sreiearar ean anasto mm ene bees eset te OOO + 9 nO oon OR ae Bhat OO 8G. e 8% amen OOOO ume 8 Rho tae « « PP eet 8 ohn oO ha BOE Ont Oe *~ ‘ aie ee 8 eaten. naam neha ate +4 a er er ee ee o Doe t eS ‘sound ae nen ene tte Sad mcwendise | oeaeateg uanomaann OA ny Me he the mem mn nk: saven SPN mek ge aR Re ngs Oe 170 S27 = iS >160 flavopictus (FL) 79 0 49 56 >120 132 =>176 125 =111 =) 14 bs! labyrinthicus (LB) 84 60 0) 33 162 vos >174 > 126 >110 >110 pentadactylus (PT) 64 49 37 160 > 160 >180 >127 ~112 > 3 bolivianus (BO) PO 105. | 120 th 2 0 ay >185 >78 87 >110 ocellatus (OC) SIs S140 147 ~118 77 0 >178 >125 ~135 > 160 fuscus (FU) >109° “108+ -=150 ~115 ~184 162 0 >150 >150 >110 notoaktites (NO) tee Te Fi =119 =175 vee 120 0 98 > 160 labrosus (LR) SOs bls 132 118 vee >185 >90 0 >85 podicipinus (PO) vee 131 ~150 >140 ~190 >140 >144 0 - = Comparison not carried out. Reciprocal Reactions If Mc’F analysis were a perfect measure of amino acid sequence difference, rather than an estimate ‘of such sequence differentiation, it would be pos- | sible to determine the ID between two species using ‘an antiserum to either species. For example, the | ID measured between two species, X and Y, should be the same whether using antiserum to X or anti- serum to Y. In practice, a deviation from perfect reciprocity (Maxson & Wilson, 1975) which av- 'erages 10%-15% is usually encountered in am- _phibian albumin studies (Heyer & Maxson, 1983; Maxson, 1984). This deviation is, in part, attrib- -utable to experimental technique and consider- ations of protein structure. The lower the devia- ‘tion from perfect reciprocity, the greater is the confidence that actual amino acid substitutions in the albumin protein are being measured. Recip- rocal tests are important in order to (1) draw phy- logenetic conclusions, (2) determine the confi- dence level that the experiments are actually measuring amino acid substitutions, and, as a con- sequence, (3) provide a framework for interpreting one-way tests. The reciprocal test data for 10 of the 11 species of Leptodactylus are presented in Table 1. The deviation from reciprocity of this matrix is very high and most IDs are very large, with several of the values approaching or exceeding the resolution of the technique (Maxson & Maxson, 1986). Ini- tially, we were very surprised by these large values. Most of the values indicate that the species studied had a common ancestor a very long time ago. Because of the generality of large values, we have not completed the data matrix for those tests that we confidently predict would result in large values but add no information regarding degree of sim- ilarity. A second observation is that among the 10 species tested only two closely related clusters of species are identified. All the other species are dis- tantly related to each other at about the same level of distance. The first cluster includes Leptodac- tylus fallax-flavopictus-labyrinthicus-pentadactyl- us; the second cluster is comprised of L. bolivi- anus-ocellatus. The first cluster species are all members of the L. pentadactylus species group; the second cluster pair are members of the L. ocel- latus species group. A third observation, for those cases where both reciprocal values are less than 100 IDU, is that considerable variation exists in the similarity of reciprocal values. At one extreme are the recip- rocal values for Leptodactylus fallax (84) and lab- yrinthicus (44), where the difference in values, 40 IDU, is almost as large as one of the values itself, 44 pu. In this instance, both antisera exhibit sig- nificant nonreciprocity in estimating 1D. The L. labyrinthicus antiserum underestimates values and the L. fallax antiserum gives overestimates. When the Sarich-Cronin (1976) correction for such non- randomness in reciprocity is applied, the estimates become 49 and 52 IDU, respectively. At another extreme is the pair of L. /abyrinthicus and pen- tadactylus, where the values (37 and 33 1pv) fall within experimental error of +2 IDU per test (e.g., experiments from same sample sources run on dif- ferent days). However, when correcting for the L. labyrinthicus antiserum, the two estimates are 41 and 33 1pu. This is no longer as ideal as the un- corrected values, but still not unreasonable for typ- ical albumin studies. If we examine comparisons only involving the MAXSON & HEYER: MOLECULAR SYSTEMATICS OF LEPTODACTYLUS 2 L. pentadactylus L. labyrinthicus 7 18 28 Soci s ae 82 EOCENE ' OLIGOCENE ' MIOCENE head BO Fao S020 10 Average ID four antisera in the pentadactylus group (fallax, flavopictus, labyrinthicus, pentadactylus) as pre- sented in Table 1, the percentage of deviation from reciprocity is 18.6. However, after correcting for nonrandomness, this value drops to a more typical 7.6%. Generally, the reciprocal values do not cor- relate as well as desired, indicating that the degree of noise is such that all results must be interpreted very cautiously. Production of antiserum to albumin from Lep- todactylus laticeps led to such disparate reciprocal values for several tests that we think it inappro- priate to discuss the relationships based on the available values for /aticeps. For example, /aticeps Ab tested against pentadactylus Ag, bolivianus Ag, and ocellatus Ag had values of 54, 70, and 75 1bu, respectively; whereas the tests of pentadactylus Ab, bolivianus Ab, and ocellatus Ab to laticeps Ag had IDU values of 105, 120, and 140, respectively. This kind of systematically asymmetrical reciprocity has been found in other studies as well (e.g., Sarich & Cronin, 1976; Maxson et al., 1985; Uzzell, 1982), although the precise reasons for the results are unknown at present. Phylogenetic Considerations Based on Reciprocal Data Examination of the reciprocal reactions in Table 1 reveals that only the members of the pentadac- tylus group (fallax, flavopictus, labyrinthicus, pen- tadactylus) and Leptodactylus labrosus have al- bumin distances close enough to be used in estimating a phylogeny. All of the other species have either overall larger distances or, in some cases, distances that could only be estimated as being greater than some large distance. Hence, we have estimated a tree only for the four members of the pentadactylus group and L. labrosus, amem- O EPOCH 1 L. fallax L. flavopictus L. labrosus Fic. 1. Phylogenetic relationships among members of the Leptodactylus pentadactylus group using L. labrosus as an outgroup. The scale indicates the av- erage immunological distance (ID) be- tween species and the geological epochs. ber of the fuscus group. Some of these data have been interpreted in an earlier study (Heyer & Max- son, 1982a). The additional antisera have not changed the conclusions of that earlier work, but have added some lineages not included at that time. The data used in constructing the phylogeny in Figure | are those in Table 1. The standard de- viation from reciprocity for the raw data is 15.6%; when the correction for nonrandomness was ap- plied, this dropped to 11.0%. Both the raw data and the corrected data were used to build phylog- enies according to the Maxson modification (Max- son, 1984) of the Wagner network described by Farris (1972). For both data sets, pentadactylus and /abyrinthicus were each other’s closest genetic relatives. The uncorrected data yielded a tree to- pology with flavopictus being the next lineage, but with a short branch length of only 3 1pu. The corrected data placed fallax closest to the first clus- ter, but the branch length was even less significant at 2.5 1pu. Thus we have presented in Figure 1 a topology that we think best interprets our data. There are three major lineages branching about the mid-Oligocene. The lineage leading to penta- dactylus and labyrinthicus branches about early Miocene and the lineage leading to /abrosus sep- arates from that, leading to the pentadactylus group in the Paleocene. The standard deviations (Fitch & Margoliash, 1967) for the tree presented, com- paring the raw and corrected input data, are 9.6% and 9.7%, respectively. One-Way Reactions Most of the data gathered in this study involve only one-way reactions of Ag samples against the antibodies raised to albumins of the 10 species shown in Table 1. A complete data matrix was FIELDIANA: ZOOLOGY TABLE 2. One-way immunological distances (ID) in the Leptodactylus melanonotus group. TABLE 3. One-way tests in the Leptodactylus ocel- /atus group. Antigens ip to L. podicipinus melanonotus Costa Rica >80 El Salvador >109 podicipinus Ybycui 0 Cordillera l El Tirol 4 wagnert Tapajos 35 deemed prohibitive, in terms of time invested, versus results anticipated in view of the large dis- tances. We chose to run all those reactions we predicted might show close values, together with a few reactions we did not predict would dem- onstrate close values. For any unpredicted results, further tests were performed in order to verify and/ or understand the results. By and large, the tests corroborated the four previously defined species groupings. We therefore present the results by | species groupings, followed by the results involv- ing Leptodactylus riveroi and silvinambus, then conclude with those tests which did not corrobo- rate the previously identified species groupings and/ or which we view as problematical. Leptodactylus melanonotus Group — Antiserum is available for L. podicipinus; the sample used for antibody production was obtained from frogs from Ybycui, Paraguay. Samples of other populations of L. podicipinus from Paraguay range from 1-4 IDU when tested by Mc’F (table 2). Leptodactylus podicipinus is more closely related to L. wagneri, with which it is inarginally sympatric, than it is to L. melanonotus (table 2). Leptodactylus podi- cipinus occurs in southern South America; L. wag- neri occurs broadly throughout Amazonia and the lowlands of South America east of the Andes; and L. melanonotus occurs in southernmost United States and from Mexico through Central America to Ecuador west of the Andes. Thus, the closest relative of podicipinus is its geographically closest form, wagneri. Based on morphology and distri- butions, we predict that when L. dantasi and pus- tulatus are tested (the remaining two species of the L. melanonotus group), they will show closer re- lationships with L. podicipinus and wagneri, with which they are mostly parapatric, than with the geographically distant L. melanonotus. Leptodactylus ocellatus Group—Several geo- graphic samples of L. bolivianus and ocellatus were ID measured with antisera to albumins of: Antigens bolivianus ocellatus bolivianus Venezuela 0 52 Venezuela, Bolivar (1) 3 49 Venezuela, Bolivar (2) 8 50 Venezuela, Bolivar (3) 4 47 Brazil, Acre 2 66 Brazil, Madeira 3 64 Peru 3 = ocellatus Brazil, Sao Paulo 77 0) Brazil, Minas Gerais ats 0) Brazil, Santa Catarina Bima 6 Uruguay “te zi Brazil, Madeira tee 26 Brazil, Purus vee 25 Brazil, Santarém vee 27 Brazil, Ceara (1) tee 26 Brazil, Ceara (2) see 30 tested against antisera to bolivianus and ocellatus. From all of the tests run (tables 1, 3, 6; LRM, pers. obs.), bolivianus and ocellatus are each other’s closest relatives, but the relationship is not espe- cially close, averaging about 60 1pu. There appears to be some population differentiation among the samples of bolivianus tested, but again the data should not be overinterpreted (table 3). Of partic- ular concern is the value of 8 1pu for the second sample from Bolivar State, Venezuela. This value exceeds experimental error and is twice that found for two other geographic samples from the state of Bolivar. With the exception of this one high value, all the remaining values suggest a relatively undifferentiated population structure for L. boli- vianus in South America. The geographic samples for L. ocellatus show a different pattern, with rel- atively small Ips among samples of L. ocellatus from Uruguay, southern and southeastern Brazil, and significantly higher distances for a series of samples from Amazonia and northeastern Brazil. The magnitude of these differences is consistent with specific differentiation of the two groups of populations, which are hereby so considered. Cei (1970) used Libby’s photronreflectometric technique to demonstrate that the serum of Lep- todactylus ocellatus from Argentina (Cordoba) dif- fered significantly from the serum of L. chaquensis (Tucuman, Argentina) and from a sample of another L. ocellatus group population from around Sao Paulo, Brazil, which Cei identified as L. mac- MAXSON & HEYER: MOLECULAR SYSTEMATICS OF LEPTODACTYLUS Ss TABLE 4. One-way tests in the Leptodactylus pentadactylus group. ID measured with antisera to albumins of: Antigens fallax flavopictus labyrinthicus pentadactylus fallax 0 62 44 36 flavopictus Brazil, Sao Paulo (1) 79 0 49 56 Brazil, Sao Paulo (2) air 0 ae 43 knudseni Venezuela, Amazonas 12 18 Venezuela, Bolivar 16 20 Brazil, Madeira 13 54 11 32 Brazil, Tapajos <7, 56 18 38 Peru wae 47 ity 29 labyrinthicus Brazil, Sao Paulo vee 4 35 Brazil, Ceara 84 60 0 33 pentadactylus Panama 64 49 37 0) Ecuador, Coastal (1) ss ore 5 Ecuador, Coastal (2) 8 Peru, Amazon vee vee 9 rugosus 24 =43 Sy 67 stenodema Brazil, Madeira ~l1l 61 38 51 Peru 5 ee 38 48 syphax 87 46 61 61 rosternum. Based on Gallardo’s (1964) revision, we would consider that our sample from Boracéia represented L. ocellatus and the samples from Amazonia and northeastern Brazil L. macroster- num. Thus, we are not certain whether Cei’s and our interpretations of L. macrosternum are the same. What is clear is that considerable biochem- ical evolution has taken place in this complex with very little accompanying morphological change. We would expect L. chaquensis to show closer relationships to our concept of L. ocellatus than to the Amazonian and northeastern Brazilian member of this species group. Leptodactylus pentadactylus Group—The re- sults of reciprocal and one-way tests (tables 1, 4) suggest that the Mc’F data corroborate a cluster of species previously defined on morphological bases (fallax, flavopictus, knudseni, labyrinthicus, pen- tadactylus; Heyer, 1979). There appears to be some variation within samples of L. pentadactylus. An additional sample run from coastal Ecuador since our previously published data (Heyer & Maxson, 1982b) does not corroborate our previous zoo- geographic statement regarding L. pentadactylus. We had previously stated that the Amazonian populations of L. pentadactylus were differentiated from the Middle American-coastal South Amer- ican populations. However, the current data do not suggest such a clear-cut pattern. Until further samples are evaluated, we conservatively interpret the samples to represent the same species, with no interpretation of patterns of differentiation among samples. The available data do not allow an unambiguous hypothesis of relationships among the members of this cluster. If only the reciprocal data are used, it is quite clear that Leptodactylus pentadactylus and /abyrinthicus are each other’s closest relatives. However, it is not clear whether L. knudseni is a closer relative of /abyrinthicus or pentadactylus, as one-way tests to knudseni give low and similar values to both fallax and labyrinthicus. An attempt to produce antibodies to knudseni to resolve this problem was unsuccessful. Based on larval and adult morphology, L. fallax is much more similar to flavopictus, knudseni, labyrinthicus, and pen- tadactylus than to stenodema. The immunological data conflict with the morphological data; they show a closer relationship between fallax and stenodema than between fa//ax and any other taxa in the pentadactylus group (table 4). Resolution of this conflict will require additional data. FIELDIANA: ZOOLOGY ; Leptodactylus fuscus Group— Most of the one- way tests run against fuscus, labrosus, and no- toaktites antisera give large ID values and do not demonstrate any particularly close relationships (table 5). Three examples that do suggest close relationships are therefore of interest. First, the geographic samples of fuscus tested against fuscus antisera prepared from individuals from Boracéia, Sao Paulo, Brazil, demonstrate close relationships relative to other tests against fuscus; however, the intraspecific tests do not make complete geograph- ic sense. The identical value of zero of samples from Boracéia and Paraguay is understandable, but the distances of 13-16 1DU to samples from Argentina (Tucuman), northeastern Brazil, and central Amazonia (Manaus) are greater than one would anticipate for intraspecific variation. Pre- viously reported values of fuscus from Argentina- Boracéia, Brazil (14) and Manaus-Boracéia, Brazil (0) (reported in Heyer & Maxson, 1982a) are in- correct in part. The Manaus-Boracéia test had in fact not been run; Heyer thought it had and con- cluded that the absence ofa value was a zero. Tests repeated from additional aliquots from the Ar- |gentine antigen yield a value of 16 1DU (within experimental error), but the Manaus-Boracéia dis- '|tance is also 16 as shown in Table 5. Leptodactylus \fuscus as currently defined would be an excellent candidate for detailed electrophoretic analysis. The second suggested close relationship is Lep- ‘| todactylus labrosus and ventrimaculatus. The sig- ‘|nificance of this relationship has been discussed elsewhere (Heyer & Maxson, 1982a). The third set -j|ofclose relationships is that between L. notoaktites and elenae (35) and L. notoaktites and mystaceus (11-20). These three species were, until recently (Heyer, 1978), considered conspecific; they are morphologically very similar and their distribu- tions are essentially parapatric. Based on overall morphological similarity, one of us (WRH) predicted that fuscus would show close relationships to camaquara, cunicularius, gracilis, and longirostris, of the species tested. The extremely large ID values argue against close re- lationships for these pairings, however. TABLE 5. One-way tests in the Leptodactylus fuscus group. ID measured with antisera to albumins of: Antigens fuscus labrosus notoaktites albilabris 76 66 <60 bufonius =196 >80 >74 camaquara 147 114 59 cunicularius 144 109 5i7 elenae 99 ~116 35 fragilis >100 122) 55 fuscus Boracéia 0 >150 >150 Paraguay 0 aia ose Argentina 16 NE Brazil 13 Manaus 16 gracilis 100 = 122 68 latinasus 90 : ~73 longirostris >144 92 mystaceus Madeira > 100 aie 20 Tapajos vee vee 17, Trombetas ne oe | mystacinus > 100 tee ~67 troglodytes 103 =84 ~94 ventrimaculatus =153 6 ~94 Leptodactylus riveroi and silvinambus — Lepto- dactylus riveroi antigen was tested against the anti- sera of bolivianus, fuscus, ocellatus, pentadactylus, and podicipinus. All of the tests resulted in very large ID values (table 6), indicating that L. riveroi is not closely related to any of the species tested. As the species tested represent the morphological diversity within the genus, it is likely that L. riveroi has no close relative within the genus. Leptodactylus silvinambus antigen was tested against the antibodies of bolivianus, fallax, fla- vopictus, fuscus, labyrinthicus, pentadactylus, and podicipinus. In this case, relatively low 1pu values were found between si/vinambus and fallax, and silvinambus and flavopictus (table 6). These results indicate that si/vinambus is related to some mem- bers of the L. pentadactylus group. Unusual/Problematical Data—Some of the re- TABLE 6. One-way tests comparing Leptodactylus riveroi and L. silvinambus to other species of Leptodactylus. ID measured with antisera to albumins of: Antigens FA FL LB PE BO Oc FU PO riveroi tee vee . =104 =180 ~90 ~169 > 130 silvinambus 1¢7 25 62 76 80 vee = 5] 130 Abbreviations are the same as in Table 1. MAXSON & HEYER: MOLECULAR SYSTEMATICS OF LEPTODACTYLUS 7 50% INTRASPECIFIC TESTS (N=19) 50% INTERSPECIFIC TESTS (N=179) PLEISTOCENE PLIOCENE MIOCENE OLIGOCENE sults that were unexpected or that indicated a problem with the antisera in estimating amino acid differences in the albumin proteins being com- pared have already been discussed. These include the conflicting morphological and immunological data on the relationships between Leptodactylus fallax and stenodema. The failure to produce an albumin antiserum to L. knudseni that yields con- sistent results may be due to a duplicated albumin locus in this lineage. Finally, the following results of tests run be- tween previously defined species groups are un- usual. Leptodactylus pentadactylus is clearly most closely related to other members of the pentadac- tylus group (table 4). However, pentadactylus anti- serum, when tested against antigens of fragilis, mystaceus (two geographic samples), and mys- tacinus, gave values of 38, 47, 53, and 58 IDU, respectively. These values are consistent with the values of pentadactylus with other pentadactylus group members, such as rugosus, stenodema, and syphax. We would have predicted that these one- way ID values should have been in the same range as the tests between pentadactylus and fuscus (115 IDU) and pentadactylus and labrosus (118 IDv). However, L. pentadactylus is the other low titer antiserum; it has consistently given somewhat lower ID values (see table 1), although these values are still lower than can be accounted for solely on the basis of reciprocals of pentadactylus to other species. The same kind of problem is evident in the antigen sample on melanonotus from El Sal- vador which was tested against L. pentadactylus antiserum; the test result was 36 IDU. The anti- serum of L. fallax gave very low ID values when tested against two different antigen samples of L. albilabris. Both tests gave values of about 12 IDU. This, together with the apparently unusually low IDU values observed between fallax and stenode- EOCENE Fic. 2. Histograms showing frequen- cy of pairwise immunological distance (ID) comparisons indicative of lineages diverging in the indicated geological ep- ochs. Top, intraspecific comparisons; bottom, interspecific comparisons. The Paleocene label includes presumed Pa- leocene divergences plus all older diver- gence estimates. PALEOCENE ma, indicates that, at best, the fa/lax values should be used cautiously. If it proves that the fallax anti- serum is not interacting in a uniform and pre- dictable manner with other Leptodactylus anti- gens, then all test results involving fallax anti- serum are suspect. Divergence Times in Leptodactylus Extensive studies of albumin evolution in di- verse vertebrates have indicated that albumin ac- cepts amino acid substitutions throughout the molecule at a stochastically regular rate (Maxson & Wilson, 1975; Maxson et al., 1975; Wilson et al., 1977). This rate for amphibians was estimated to be an average of 1.7 1pU per million years of divergence (Maxson et al., 1975). A calibration based on a more restricted but better dated fossil record for mammals corresponds to 1.8 IDU per million years of divergence (Wilson et al., 1977). Using these calibrations for all of the tests run with Leptodactylus, we arrive at some rather surprising conclusions (fig. 2). For intraspecific comparisons we see two dif- ferent patterns. Either populations of the same species are genetically indistinguishable, or allo- patric populations assigned to the same species have been reproductively isolated since as long ago as the Miocene. Examining the interspecific com- parisons, we find only 1% of the species compar- isons indicative of a Pliocene divergence. The re- mainder of the comparisons show moderate genetic divergence occurring from the Miocene through the Eocene, and most of the divergence dating to the Paleocene or earlier (the limits of resolution of Mc’F comparisons of albumin do not allow com- parisons between albumins which differ by over 35% in their sequence; this amount of change ac- FIELDIANA: ZOOLOGY ‘cumulates by roughly 100-120 million years of separation [Wilson et al., 1977]). In view of the large number of tests run with Leptodactylus, the trends are not likely to change with more com- parisons of additional species. Even if some of the values are incorrect, the noise is likewise negligible in terms of the summary presented in Figure 2. At least two explanations can be proposed for these data. First, albumin may be evolving at a much faster rate in Leptodactylus than in most other vertebrate lineages. Alternatively, the genus Leptodactylus, as presently constituted, is a very | old lineage with most species established since the |Paleocene and modest speciation occurring throughout the Eocene, Oligocene, and Miocene. A little additional speciation occurred in the Plio- cene, and essentially no major speciation events date to the Pleistocene. Two kinds of independent data—fossil and biochemical—can be brought to bear to distinguish between these two alternative explanations. Fossil Data—The fossil record for the genus could provide evidence on the age of some taxa and provide an independent calibration of albu- min evolution in this group. Unfortunately, as is true for most amphibians, the published fossil rec- ord of Leptodactylus is not sufficient to allow an |independent assessment for this taxon. | Biochemical Data—Cei and his colleagues have jinvestigated relations among varied species of | Leptodactylus, using comparisons of skin amines }and polypeptides as well as qualitative compari- | sons of serum proteins by means of precipitin anal- . | | yses. These approaches give some insight into as- | sociations of groups of species based on relative | similarities of small skin polypeptides (Cei & Er- |spamer, 1966; Cei et al., 1967) and patterns of | behavior with antiserum raised to whole serum (Cei, 1970). However, skin amines and polypep- tides are small molecules which appear to evolve rather rapidly and are not useful as general probes of relationships among relatively old taxa such as /anurans. The Mc’F analyses of albumin evolution |have the advantage over precipitin analyses, in | that the latter provide only a qualitative estimate ' describing some overall averaging of general sim- | ilarities of an unknown mixture of serum proteins. | The Mc’F analyses, on the other hand, have been | demonstrated to be an efficient estimator of amino acid differences between the species compared | (Maxson & Maxson, 1986), and it is this capability | that permits us to extrapolate time estimates from the ID measurements. At present the only relevant molecular study on Leptodactylus (besides MC’F analyses) is an anal- ysis of four species of Costa Rican Leptodactylus by starch gel electrophoresis (Miyamoto, 1981). While Miyamoto agreed with the species group assignments as used in this paper, it is difficult to use his data to support or refute the great age im- plied by the albumin studies. Because gel electro- phoresis can only detect the first amino acid sub- stitution causing a change in mobility, only relatively recently separated populations can be accurately diagnosed with gel electrophoresis due to the problem of muitiple substitutions (Maxson & Maxson, 1979). Thus, if the species are as old as the albumin data imply, electrophoretic data cannot refute the albumin results! However, even Miyamoto’s results show that over 80% of the loci scored between pentadactylus and melanonotus and between pentadactylus and bolivianus have fixed allelic differences, suggesting a long independence of lineages, such as the albumin data indicate. Thus, we suggest that the albumin data are the best available estimates of divergence time pres- ently available for Leptodactylus. We propose these data be given serious consideration until falsified. Conclusions One of our original research aspirations with molecules and Leptodactylus was to provide an exemplary showcase of molecular and morpho- logical evolution for a vertebrate genus. The MC’F albumin data do not allow the depth of interpre- tation we had anticipated at the outset. Most of the Mc’F albumin data corroborate the species groupings determined on the basis of other criteria (mostly morphological in nature). However, some MC’F tests suggest close relationships that cross the previously defined species groupings. The rather poor reciprocity seen with results of reciprocal tests indicates that there may be some problems with Leptodactylus albumins resulting in poor antisera or there may be multiple paralogous albumins in this genus, confounding the results. Although only single proteins were identified and purified, all al- bumins may not be homologous in this genus. Because of that possibility, we are hesitant to ac- cept blindly all of the 1p values. This noise in the Leptodactylus data also prevents us from unam- biguously resolving divergence events among closely related species (with moderate ID values), such as members of the Leptodactylus pentadac- tylus species cluster. The members of this cluster MAXSON & HEYER: MOLECULAR SYSTEMATICS OF LEPTODACTYLUS 2 (fallax, flavopictus, knudseni, labyrinthicus, pen- tadactylus) have a uniquely derived tadpole (Hey- er, 1979), defining this cluster as monophyletic, yet the branching sequences among its members can- not be unambiguously proposed due to the noise in our MC’F albumin data. The possible great age of most Leptodactylus species, together with the noise in the Mc’F albu- min data, lead us to the conclusion that MC’F al- bumin analysis is not the ideal choice for evalu- ating genetic relationships among most Leptodactylus species. Our studies have shown that there are some very interesting problems that should be pursued. Intraspecific variation in the widespread species L. fuscus and ocellatus should be studied in detail, probably with mitochondrial DNA analyses and/or electrophoretic techniques. Overall genetic relationships among Leptodactylus species should be examined using a more slowly evolving molecule than albumin, or by the direct sequencing of ribosomal genes. Acknowledgments Several individuals have gone out of their way to collect Leptodactylus albumin samples for us: J. E. Cadle, R. B. Cocroft, R. I. Crombie, H. Des- sauer, M. S. Foster, A. Gehrau, S. Gorzula, R. F. Laurent, R. W. McDiarmid, M. T. Rodrigues, N. J. Scott, Jr., R. A. Thomas, and L. D. Wilson. We very much appreciate the support these individ- uals have given our joint research projects. John E. Cadle and George R. Zug critically re- viewed this paper. Our studies are a result of field and laboratory work, and have been supported by the Museu de Zoologia da Universidade de Sao Paulo; National Science Foundation (grants DEB 82-01587 and BSR 83-19969); the University of Illinois (Department of Genetics and Develop- ment); and several sources at the Smithsonian In- stitution (Smithsonian Research Foundation; Fluid Research Award; Director’s Office, National Mu- seum of Natural History; and the Neotropical Lowland Research Program of the International Environmental Sciences Program). Literature Cited BENJAMIN, D. C., J. A. BERZOFSKY, I. J. EAsT, F. R. N. GURD, ET AL. 1984. The antigenic structure of pro- 10 teins: A reappraisal. Annual Review of Immunology, 2: 67-101. Cel, J. M. 1970. Relaciones serologicas entre los Lep- todactylus del grupo ocellatus-chaquensis de la cuenca chacoparanense y la forma macrosternum. Acta Zoo- logica Lilloana, 27: 299-306. CEI, J. M., AND V. ERSPAMER. 1966. Biochemical tax- onomy of South American amphibians by means of skin amines and polypeptides. Copeia, 1966: 74-78. Cel, J. M., V. ERSPAMER, AND M. ROSEGHINI. 1967. Taxonomic and evolutionary significance of biogenic amines and polypeptides occurring in amphibian skin. I. Neotropical Leptodactylid frogs. Systematic Zool- ogy, 16: 328-342. CHAMPION, A. B., E. M. PRAGER, D. WACHTER, AND A. C. WiLson. 1974. Micro-complement fixation, pp. 397-416. In Wright, C. A., ed., Biochemical and Im- munological Taxonomy of Animals. Academic Press, London. Farris, J.S. 1972. Estimating phylogenetic trees from distance matrices. American Naturalist, 106: 645-668. Fitcu, W. M., AND E. MARGOLIASH. 1967. Construc- tion of phylogenetic trees. Science, 155: 279-284. GALLARDO, J. M. 1964. Consideraciones sobre Lep- todactylus ocellatus (L.) (Amphibia, Anura) y especies aliadas. Physis, 24: 373-384. HEYER, W.R. 1969. The adaptive ecology of the species groups of the frog genus Leptodactylus (Amphibia, Leptodactylidae). Evolution, 23: 421-428. 1978. Systematics of the fuscus group of the frog genus Leptodactylus (Amphibia, Leptodactyli- dae). Natural History Museum of Los Angeles County Science Bulletin, 29: 1-85. 1979. Systematics of the pentadactylus species group of the frog genus Leptodactylus (Amphibia: Lep- todactylidae). Smithsonian Contributions to Zoology, 301: 1-43. HEYER, W.R., AND L. R. MAxSON. 1982a. Neotropical frog biogeography: Paradigms and problems. Ameri- can Zoologist, 22: 397-410. 1982b. Distributions, relationships, and zoo- geography of lowland frogs. The Leptodactylus com- plex in South America, with special reference to Ama- zonia, pp. 375-388. In Prance, G. T., ed., Biological Diversification in the Tropics. Columbia University Press, New York, 714 pp. 1983. Relationships, zoogeography, and spe- ciation mechanisms of frogs of the genus Cycloram- phus (Amphibia, Leptodactylidae). Arquivos de Zoo- logia, 30: 341-373. HEYER, W.R., AND W. F. PyBuRN. 1983. Leptodactylus riveroi, a new frog species from Amazonia, South America (Anura: Leptodactylidae). Proceedings of the Biological Society of Washington, 96: 560-566. Maxson, L. R. 1984. Molecular probes of phylogeny and biogeography in toads of the widespread genus Bufo. Molecular Biology and Evolution, 1: 345-356. Maxson, L. R., AND W.R. HEYER. 1982. Leptodactylid frogs and the Brasilian Shield: An old and continuing adaptive relationship. Biotropica, 14: 10-15. Maxson, L. R., R. HIGHTON, AND D. B. WAKE. 1979. Albumin evolution and its phylogenetic implications FIELDIANA: ZOOLOGY in the plethodontid salamander genera Plethodon and Ensatina. Copeia, 1979: 502-508. | Maxson, L. R., AND R. D. MAxson. 1979. Compar- ative albumin and biochemical evolution in pletho- dontid salamanders. Evolution, 33: 1057-1062. Maxson, L. R., D. P. ONDRULA, AND M. J. TYLER. 1985. An immunological perspective on evolutionary rela- tionships in Australian frogs of the hylid genus Cy- clorana. Australian Journal of Zoology, 33: 17-22. Maxson, L. R., V. M. SARICH, AND A.C. WILSON. 1975. Continental drift and the use of albumin as an evo- lutionary clock. Nature, 255: 397-400. Maxson, L. R., AND A. C. WiLson. 1975. Albumin evolution and organismal evolution in tree frogs (Hy- lidae). Systematic Zoology, 24: 1-15. Maxson, R. D., AND L. R. MAxson. 1986. Micro- complement fixation: A quantitative estimator of pro- tein evolution. Molecular Biology and Evolution, 3: 375-388. McCRANIE, J. R., L. D. WILSON, AND L. Porras. 1980. A new species of Leptodactylus from the cloud forests of Honduras. Journal of Herpetology, 14: 361-367. MryAmoTo, M.M. 1981. Congruence among character sets in phylogenetic studies of the frog genus Lepto- dactylus. Systematic Zoology, 30: 281-290. SARICH, V. M., AND J. E. CRONIN. 1976. Molecular systematics of the primates, pp. 141-170. Jn Good- man, M., and R. E. Tashian, eds., Molecular Anthro- pology. Plenum Press, New York. UzzeL., T. 1982. Immunological relationships of west- ern Palearctic water frogs (Salientia: Ranidae). Am- phibia-Reptilia, 3: 135-143. WIitson, A. C., S. S. CARLSON, AND T. J. WHITE. 1977. Biochemical evolution. Annual Review of Biochem- istry, 46: 573-639. MAXSON & HEYER: MOLECULAR SYSTEMATICS OF LEPTODACTYLUS it Appendix Specimens used for Mc’F analysis are listed. An LM (Linda Maxson) number ties into any additional data not listed here. Other abbreviations are for museum collections where specimen vouchers are deposited (museum number given) or will be deposited (no museum number given). Museum abbre- viations are per standardized list given in Copeia (1985: 802-832). Ab = antibody produced. Leptodactylus albilabris LM 29; Puerto Rico, Isla Vieques; USNM Leptodactylus bolivianus LM 1267-8; Brazil, Amazonas, Boca do Acre; USNM 202444—5 LM 1269; Brazil, Amazonas, Borba; USNM 202451 LM 1102; Peru, Madre de Dios, Tambopata Re- serve; USNM 247351, 247354 LM 524; Venezuela, Bolivar, S of Ciudad Bolivar LM 360; Venezuela, Bolivar, Ciudad Guayana; USNM 229778 LM 18; Venezuela, Bolivar, 28 km E El Palmar LM 1266; Ab; Venezuela, Sucre, Cumana Leptodactylus bufonius LM 417; Paraguay, Central, Villeta; USNM Leptodactylus camaquara LM 1275; Brazil, Minas Gerais, Serra do Cipo; USNM 217647 Leptodactylus cunicularius LM 1272-3; Brazil, Minas Gerais, Serra do Cip6; USNM Leptodactylus elenae LM 32-5, 62-3; Paraguay, El Tirol; USNM Leptodactylus fallax LM 10; Ab; Dominica, near Coulibistri; USNM 218253-—4 Leptodactylus flavopictus LM 1276; Ab; Brazil, Sao Paulo LM 1277; Brazil, Sao Paulo, Boracéia; USNM 209215 Leptodactylus fragilis LM 1289-90; Panama, Canal Zone, Gamboa; USNM 203650-1 Leptodactylus fuscus LM 1280; Argentina, Tucuman 12 LM 1279; Brazil, Amazonas, Manaus; USNM 202506 LM 1283; Brazil, Ceara, Santana do Cariri; USNM 216071 LM 1281-2; Ab; Brazil, Sao Paulo, Boracéia; USNM 209221-2 LM 12; Paraguay, El Tirol; USNM Leptodactylus gracilis LM 1285; Argentina, Tucuman Leptodactylus knudseni LM 1288; Brazil, Para, Parque Rio Tapajos; USNM LM 1348; Brazil, Rond6nia, Calama; USNM 202516 LM 1286-7; Peru, Madre de Dios, Tambopata Reserve; USNM LM 523; Venezuela, Amazonas, Tama-Tama LM 522; Venezuela, Bolivar, Ciudad Bolivar Leptodactylus labrosus LM 1295; Ab; Ecuador, Rio Palenque Biological Station; UIMNH 94604 Leptodactylus labyrinthicus LM 1351-2; Ab; Brazil, Ceara, Santana do Cai- riri; USNM 216079 LM 1296; Brazil, Sao Paulo, Assis; USNM 207674 Leptodactylus laticeps LM 1298; Ab; Argentina; UIMNH 94163 Leptodactylus latinasus LM 550; Argentina, Tucuman Leptodactylus longirostris LM 1302; Brazil, Para, Parque Rio Tapajos; USNM Leptodactylus melanonotus LM 1304; Costa Rica LM 548; El Salvador, Cuscatlan, El Sitio de los Hidalgo Leptodactylus mystaceus LM 1264; Brazil, Para, Parque Rio Tapajos; USNM FIELDIANA: ZOOLOGY LM 1263; Brazil, Para, Reserva Rio Trombetas; USNM LM 1305; Brazil, Rond6énia, Calama; MZUSP | Leptodactylus mystacinus LM 1306; Brazil, Sao Paulo, Fazenda do Veado; USNM 208092 Leptodactylus notoaktites LM 1316; Ab; Brazil, Parana, nr Sao Joao da ° Graciosa; USNM 217791-3 Leptodactylus ocellatus (= ocellatus) LM 1337; Brazil, Minas Gerais, Serra do Cipo; USNM LM 413; Brazil, Santa Catarina. Rio dos Cedros; USNM 243753 LM 1317; Ab; Brazil, Sao Paulo, Boracéia; USNM 209230 LM 1323; Uruguay, Maldonado, Sierra de Ani- mas; USNM 217801 Leptodactylus ocellatus (= macrosternum?) LM 1321; Brazil, Amazonas, Rio Madeira, Ma- nicoré; MZUSP LM 1319; Brazil, Amazonas, Rio Purus, Beruri; USNM 202512 LM 1326; Brazil, Ceara, Santana do Cariri; MZUSP LM 1327; Brazil, Ceara, Santana do Cariri; MZUSP LM 11; Brazil, Para, Santarem; USNM Leptodactylus pentadactylus LM 1332; Ecuador, Rio Palenque Biological Sta- tion; USNM LM 1333; Ecuador, Rio Palenque Biological Sta- tion; USNM LM 1328; Ab; Panama, Canal Zone; UIMNH 94165 LM 791; Peru, Amazonas, Rio Cenepa, Rio Huampami; USNM Leptodactylus podicipinus LM 25; Paraguay, Cordillera; USNM LM 26-7; Paraguay, El Tirol; USNM LM 61; Ab; Paraguay, Ybycui; USNM Leptodactylus riveroi LM 528-9: Venezuela, Amazonas, nr Tama- Tama Leptodactylus rugosus LM 1334; Venezuela, Bolivar, La Escalera; KU 181028 Leptodactylus silvinambus LM 19, 22-24; Honduras, Ocotepeque, Belen Gualcho and El Chagiiiton Leptodactylus stenodema LM 1335; Brazil, Amazonas, Rio Madeira, Res- tauracao; MZUSP LM 788: Peru, Amazonas, Rio Cenepa, Rio Huampami; USNM Leptodactylus syphax LM 1366x; Brazil, Minas Gerais, Serra do Cipo; USNM 218156 Leptodactylus troglodytes LM 1355; Brazil, Ceara, Santana do Cariri; USNM 216080 Leptodactylus ventrimaculatus LM 1356; Ecuador, Rio Palenque Biological Sta- tion; USNM Leptodactylus wagneri LM 1341; Brazil, Para, Parque Rio Tapajos; USNM MAXSON & HEYER: MOLECULAR SYSTEMATICS OF LEPTODACTYLUS 13 ee a er a - ae mee ae as : ty E oe a a, Pen Rs ee Se ie eo na a 7 bas ees ays ina u kee a on he ee Pe ee =o oe eG Lae o -_ ome ree, rh gee pe Rien a: ag Sats ee ee ee iar é ‘5 Te a ee eye oe ee oe ey a a yb ee eg tee . cere y. ae ee = ; o ae os i a oa een a oa - i . : . eee ee ee ea ie ae Save Ac ee a 7 a ee 7 5 = eo 2 ss = ao a re a4 [ aan Brad Vee rt me a 2 i aan oe ao Ee ae eg as a eee : a iP 7 = Jali oe ee ae ata Field Museum of Natural History Roosevelt Road at Lake Shore Drive Chicago, Illinois 60605-2496 Telephone: (312) 922-9410 £ ~~ 2 * - ‘p ae Seen Ts >a” 7 a - ad : = Core oe ieee ; ; i eee ot Pos Sateen on a a a See a _ le a ve ay ies Tigo ti A ee, 2 tes i : _ ee _ ; ae Tae os a = a - a - > eens ; ; mee ae 7 = am 7 oa SS 3 > ; ra - - oo 7 vee = : a ro 7 ee Ee eer LENA OS Bip mNE se oy URBANA SERIES $CHGO C001 -89 UNIVERSITY OF ILLINOIS- § 90.5FIN JELDIANA : ZOOLOGY. $ NEW 0-54 1988 sieve a CRT eR ~ Sara gerne ear oe a . vi