Zoology NEW SERIES, NO. 41 Molecular Systematics of the Frog Genus Leptodactylus (Amphibia: Leptodactylidae) Linda R. Maxson Department of Ecology, Ethology, and Evolution University of Illinois at Urbana-Champaign Urbana, Illinois 61801 W. Ronald Heyer Department of Vertebrate Zoology National Museum of Natural History Smithsonian Institution Washington, D.C. 20560 A Contribution in Celebration of the Distinguished Scholarship of Robert F. Inger on the Occasion of His Sixty-Fifth Birthday Accepted for publication February 10, 1986 February 29, 1988 Publication 1384 PUBLISHED BY FIELD MUSEUM OF NATURAL HISTORY O 1988 Field Museum of Natural History ISSN 001 5-0754 PRINTED IN THE UNITED STATES OF AMERICA Table of Contents List of Tables ABSTRACT............................... 1 1. Matrix of reciprocal immunological dis- INTRODUCTION........................... 1 tances among 10 species of Leptodactylus MATERIALSAND METHODS................ 2 ...................................... 3 RESULTSAND DISCUSSION................. 2 2. One-way immunological distances in the Reciprocal Reactions ................... 3 Leptodactylus melanonotus group ....... 5 Phylogenetic Considerations Based on Re- 3. One-way tests in the Leptodactylus ocel- ........................... 1 ciprocal Data ...................... 4 latus group 5 I One-way Reactions .................... 4 4. One-way tests in the Leptodactylus penta- 1 Divergence Times in Leptodactylus ...... 8 dactylus group ........................ 6 1 CONCLUSIONS............................ 9 5. One-way tests in the Leptodactylus fuscus j ACKNOWLEDGMENTS...................... 10 group ................................ 7 j LITERATURECITED ....................... 10 6. One-way tests comparing Leptodactylus i APPENDIX............................... 12 riveroi and L. silvinambus to other species of Leptodactylus ............... 7 I . List of Illustrations 1. Phylogenetic relationships among mem- bers of the Leptodactylus pentadactylus group using L. labrosus as an outgroup ... 4 2. Histograms showing frequency of pair- wise immunological distance compari- sons indicative of lineages diverging in the indicated geological epochs ......... 8 Molecular Systematics of the Frog Genus Leptodactylus (Amphibia: Lep todactylidae) Abstract 1 I More than three-quartersof the described species eastern Brazil and Amazonia are specifically dis- of the frog genus Leptodactylus were sampled and tinct from L. ocellatus from southeastern Brazil I analyzed using the quantitative immunological and Uruguay. I j technique of micro-complement fixation. Eleven albumin antisera to representatives of the four de- ! scribed species groups in this genus were compared Introduction i to one another and to albumins of all available species of Leptodactylus. The Neotropical frog genus Leptodactylus con- 1 The results of this analysis indicated enormous sists of over 45 species. Comparative morpholog- I albumin differentiation within the genus, suggest- ical and behavioral data (Heyer, 1969, 1979, and 1 ing that most species of Leptodactylus have been other revisions cited therein) indicate that these F established since the Paleocene with modest spe- species divide into four lineages. To test this hy- I ciation occurring throughout the Eocene, Oligo- pothesis we have been gathering micro-comple- cene, and Miocene. No evidence for Pleistocene ment fixation (MC'F)data on albumin evolution I speciation has been found. among Leptodactylus species since 1974. We ini- The four species groups of Leptodactylus defined tially anticipated that a few representative species on morphological and behavioral criteria are not samples would establish a molecular framework as clearly defined by this biochemical analysis. Al- to determine the relationships among the major bumins of species within each of the L. pentadac- lineages within the genus. Our early results indi- l tylus, L. melanonotus, and L. ocellatus groups are cated that the problem of defining relationships 1 more similar to one another than to members of within Leptodactylus was more complex than an- other groups. However, there is little evidence of ticipated. It is only now that we have sufficient close relationships among those members of the data from MC'F analyses to evaluate the utility of . L. fuscus group available for study. Leptodactylus this approach for delineating relationships within 1 riveroi is not genetically close to any of the refer- the genus. Because of Robert F. Inger's interest in i ence species and appears to represent yet another frog systematics, we offer this summary, warts and 1 lineage in this genus. Leptodactylus silvinambus all, as a token of our appreciation for his influence 1 has its closest relatives among members of the L. on herpetology. pentadactylus species group. Our initial interest was to determine if MC'F There is considerable intraspecific albumin dif- analysis would indicate genetic groups that would ferentiation in Leptodactylus bolzvianus, L. fuscus, correlate with the species groupings determined L. pentadactylus, and L. podicipinus. Populations from other data sources. These species groups and sometimes referred to as L. ocellatus from north- their key diagnostic features are: MAXSON & HEYER: MOLECULAR SYSTEMATICS OF LEPTODACTYLUS 1 1. The Leptodactylus melanonotus species group determining if albumin data would (3) shed light (5 species) on the relationships of L. riveroi and silvinambus a. Toes fringed to other members of the genus. b. Males with thumb spines, no chest spines c. No dorsolateral folds d. Eggs laid in foamy mass on top of water e. Larvae uniformly dark, labial toothrow anterior to beak entire Materials and Methods 2. The Leptodactylusocellatus species group (4- 6 species) The quantitative immunological technique of a. Toes fringed MC'F was used to compare albumins of the frogs b. Males with thumb spines, no chest spines of the genus Leptodactylus. Albumins were ob- c. Dorsolateral folds present tained from blood and phenoxyethanol extracts of d. Eggs laid in foamy mass on top of water muscle from most described species of Leptodac- e. Larvae uniformly dark, labial toothrow tylus. Antisera were prepared to purified albumins anterior to beak entire of 11 species, representing the four major de- 3. The Leptodactylus pentadactylus species scribed species groups: melanonotus group-L. group (1 1 species) podicipinus; ocellatus group-L. ocellatus, L. bo- a. Toes ridged in juveniles, free in adults livianus; pentadactylus group-L. pentadactylus, b. Males usually with thumb and chest spines L. fallax, L. jlavopictus, L. labyrinthicus, L. lati- c. Dorsolateral folds usually present ceps; fuscus group-L. fuscus, L. labrosus, L. no- d. Eggs laid in foamy mass on top of water toaktites. Collection and voucher information on e. Larvae mottled, labial toothrow anterior all specimens used in this study are indicated in to beak divided the Appendix. Some of the antisera used in this 4. The Leptodactylus fuscus species group (at study have been described earlier (Heyer & Max- least 23 species) son, 1982a,b; Maxson & Heyer, 1982). a. No fringes on toes All antisera were prepared and all MC'F analyses b. Males without thumb or chest spines were carried out according to established proce- c. Dorsolateral folds usually present dures (Maxson et al., 1979; Champion et al., 1974). d. Eggs laid in foamy mass in an under- Data are reported as immunological distance (ID) ground terrestrial incubating chamber units (IDW) which, for albumin, represent amino e. Larvae mottled, labial toothrow anterior acid differences in the two albumins being com- to beak divided. pared (Wilson et al., 1977; Benjamin et al., 1984). The mean rate of albumin evolution is such that Since these groups were initially defined (Heyer, 100 IDU accumulate for every 55-60 million years 1969), two species have been described that are that two lineages have been reproductively iso- intermediate. Leptodactylus riveroi is very similar lated from one another (Wilson et al., 1977). in overall appearance to L. wagneri, a L. mela- nonotus group member, but has a pair of dorso- lateral folds, as found in the L. ocellatus group. The call of L. riveroi is distinctive and unlike that of any other Leptodactylus species (Heyer & Py- Results and Discussion burn, 1983). Leptodactylus silvinambus, when de- scribed (McCranie et al., 1980), was associated The average titer (and slope) of the 11 antisera with the L. pentadactylus group by default, as it is 3600 (and 390). Although the averages are typ- was clearly distinct from members of the other ical of previous MC'F studies of albumin evolution three species groups. However, L. silvinambus is in vertebrates, three antisera had exceptionally low morphologically distinct from all other members titers of 1100 (L. fallax) and 1300 (L. laticeps, L. of the pentadactylus group. In addition to deter- pentadactylus). These low titers make it techni- mining whether MC'F analysis of Leptodactylus al- cally difficult to use these antisera, particularly at bumins would (1) substantiate the species groups IDS greater than 50 units. The remaining eight anti- summarized above, and (2) elucidate relationships sera had an average titer of 4500 and an average among the species groups, we were interested in slope of 390. FIELDIANA: ZOOLOGY Matrix of reciprocal immunological distances (ID) among 10 species of Leptodactylus. ID measured with antisera to albumins ok Antigens fallax (FA) Jlavopictus (FL) labyrinthicus (LB) pentadactylus (PT) bolivianus (BO) ocellatus