, Herpe'o!ogica, 38(2), 1982, 272-287 © 1982 by The Herpetologists' League, Inc. GENETIC DIFFERENTIATION AMONG PLETHODONTID SALAMANDERS (GENUS BOLITOGLOSSA) IN CENTRAL AND SOUTH AMERICA: IMPLICATIONS FOR THE SOUTH AMERICAN INVASION JAMES HANKEN AND DAVlD B. WAKE ABSTRACT: Art electrophoretic sUIVey of proteins from 14 populations representing eight species and two species groups of Central and South American Bolitoglossa examines patterns of intraspecific and interspecific genetic differentiation and the possible implications of these patterns for the question of the time of entry of plethodontid salamanders into South America. These species are very old, as evidenced by the great genetic differentiation among species (including presumed close cladistic relatives), the high degree of genetic subdivision within species, and the very high levels of genetic variation within individual populations. The fre­ quency ofpolymorphic loci and mean heterozygosity recorded for some populations, particularly the Costa Rican species B. subpalmata,may be the highest levels yet recorded fOJ; vertebrates. Cluster analysis based on degree ofgenetic relatedness reveals only a weak distinction between South American and Central American species; furthennore, many component lineages may predate the establishment of the continuous, pennanent Pliocene land connection between Paw nama and Colombia. Thus, the recent South American fauna likely comprises descendants of several lineages that independently entered South America. Lastly, a specimen of B. pandi collected recently in Colombia indicates that this species, which previously was known from a single specimen collected near the turn of this century and feared extinct, still survives. Key words: Amphibia; Caudata; Bolitoglossa; Allozymes; Biogeography; Electrophoresis; Panamanian Portal SALAMANDERS present a classic exam­ and Lynch, 1976). Bolitoglossa and Oed­ ple of a Holarctic distribution; eight of ipina, two genera distributed mainly in the nine extant families are found almost Central America, have representatives in exclusively in the temperate regions of South America, and the range of Bolito­ Europe, Asia, and North America. Only glossa extends as far as 17°S (Brame and the supergenus Bolitoglossa, which com­ Wake, 1963; Wake and Brame, 1966). prises seven genera ofthe highly derived However, while the South American sal­ I amander fauna is considerably richer family Plethodontidae, has penetrated tropical latihldes to any significant de­ than is generally acknowledged and oc­ gree, and only in the New World (Wake cupies an enormous geographical area June 1982]' HERPETOLOGICA 273 (including much of the Amazon basin), Bolitoglossa fauna is derived from sev­ I,1 fewer than 30 species are found in the eral lineages which separately invaded ~ entire continent, a number apparently no South America relatively early and in ad­ greater than that occurring in the small vance of a much more recent entry by neighboring country of Panama alone. Oedipina and the phalarosoma group of It is generally accepted that salaman­ Bolitoglossa. ders entered South America by dispersal Wake and Lynch (1976) subsequently from southern Central America, but the modified this hypothesis, suggesting that number of separate invasions of South as few as two large-scale movements are America and their timing are debated. sufficient to explain the present distri­ There are no fossils from tropical areas, bution of salamanders in South America. so it has been necessary to infer details The initial invasion from southern Cen­ of the South American ingression indi­ tral America, quickly following the clo­ rectly from two sources: (1) the relative sure of the Panamanian (Bolivar) Portal degree of morphological differentiation in the mid to late Pliocene, was made by of related groups living on either side of a morphologically generalized Bolito­ the former gap between what is now low­ glossa possibly resembling B. savagei of er Central America and northern South montane northern Colombia; an adaptive America, and (2) geologic evidence. radiation by descendants of this single Dunn (1926), in the first comprehensive stem lineage, producing the relatively discussion of this problem, suggested great morphological and ecological di­ that the trans-isthmian migration of versity that is displayed by the recent plethodontid salamanders occurred from South American fauna, was then evolved the late Miocene to the Pliocene, or as in situ. The second, and much more re­ much as 12-15 million years ago. Ideas cent, movement across the isthmian land of fixed continents prevailed in Dunn's connection by Oedipina, moving south, time, and his suggestion reflects then-ac­ and members of the B. phalarosoma and cepted dates of the establishment of a B. sima species-groups, moving north as land connetion between the two Ameri­ far as western Panama, would account for can continents. Darlington (1957) pro­ the present distribution of the remaining posed that salamanders entered South South American salamander element in America following a mid to late Pliocene the isthmian region. A third possibility­ land connection. Recent studies which and, to our knowledge, one not yet for­ incorporate views of continental drift es­ mally proposed-is that one lineage of timate a permanent land connection be­ Bolitoglossa and one of Oedipina en­ tween Panama and South America begin­ tered South America soon following clo­ ning 2 to 5 (Woodring, 1966), 5 to 7 sure of the Panamanian Portal. The for­ (Holden and Dietz, 1972), or approxi­ mer underwent considerable speciation mately 3 (Keigwin, 1978; Marshall et aI., and morphological diversification, the 1979) million years ago, thus supporting latter did not. the notion of a later, Pliocene crossing. Biochemical techniques have been Brame and Wake (1963) proposed mul­ used to analyze the effects of the estab­ tiple invasions of South America by sal­ lishment of the isthmian land connection amanders of several different lineages, on the distribution and diversification of beginning in the late Pliocene and ex­ marine groups, including fishes (Gorman tending into the Pleistocene. Their sug­ and Kim, 1977; Gorman et aI., 1976; Vaw­ gestion was based on the differing de­ ter et aI., 1980) and sea urchins (Lessios, grees ofmorphological similarity between 1979, 1981). These procedures are partic­ many South American taxa and their pre­ ularly valuable in providing an indepen­ sumed Central American relatives. Brame dent assessment of alternate hypotheses and Wake argued that most of the recent of phylogenetic relationship that are de- 274 HERPETOLOGICA [Vol. 38, No.2 84° 78° 72° 66° . '. 12° , , "- 6° t--l 100 km FIG. I.-Map showing collection localities of Bolitoglossa in southern Central America and northwestern South America. 1-5, B. subpalmata; 6, B. mannorea; 7, B. nigrescens; 8-9, B, adspersa; 10-11, B. valle­ cula; 12, B. pandi;-13, B. borburata; 14, "Soacha." rived from other sources, such as mor­ Bolitoglossa, and Oedipina, we cannot phology. Thus, we took advantage of the fully resolve the biogeographic ques­ recent collection of representatives of tions. Nevertheless, we can examine the several species of Bolitoglossa from genetic relationships among a number of northern South America-where most South and Central AmeriCan species as a salamander species generally are rare Or first step in identifying the origins of the otherwise difficult to obtain-to make entire South American salamander fauna. electrophoretic comparisons with conge­ Specifically, our goals are: (1) to deter­ ners from Panama and Costa RiCa. Our mine the, patterns of intraspecific and in­ South American sample includes repre­ terspecific differentiation in South Amer­ sentatives of the largest and most diverse ican and in Talamancan Central America, ofthe five species-groups of Bolitoglossa (2) to examine the possibility that the found on the continent; our Central South American species show a uniform American sample includes members of degree of genetic differentiation relative one of the five closely related species­ to those in Central America, as would be groups of BolHoglossa occuring from expected if all South American Bolito­ western Panama to Mexico. We hoped to glossa are derived from a single lineage identify implications of the biochemical with respect to Central American species, data for the biogeography of salamanders and (3) to estimate divergence times be­ in the isthmian region, but because we tween taxa in order to date the entry of lack samples of other species groups of salamanders into South America. ~--- -- _.. -- .__...._-- '._. __._...._....•_._._... 2 June 1982] HERPETOLOGICA 275 TABLE I.-Populations of Bolitoglossa included in electrophoretic analysis. Population Species Locality Sample size 1 B. subpalmata Volca.n Peas, Costa Rica 7 2 B. subpalmata EI Empalme, Costa Rica 11 3 B. subpalmata 22 km S of EI Empalme, Costa Rica 16 4 B. subpalmata 6 km W of Villa Mills, Costa Rica 4 5 B. subpalmata Villa Mills, Costa Rica 19 6 B. marmorea Voldm Chiriqui, Panama 16 7 B. nigrescens Valclin Chiriqui, Panama 3 8 B. adspersa Cmz Verde, Colombia 20 9 B. adspersa Guasca, Colombia 20 10 B. vallecula San Felix, Colombia 20 11 B. vallecula Santa Rosa, Colombia 1 12 B. pand; Alban, Colombia 1 13 B. borburata Rancho Grande, Venezuela 1 14 "Soacha" Soacha, Colombia 6 MATERIALS AND METHODS (Table 1). Our single specimen ofB. pan­ Three Central American and five .South di is only the second specimen of this American species