Journal of Herpetology, Vol. 36, No. 3, pp. 356–366, 2002 Copyright 2002 Society for the Study of Amphibians and Reptiles Phylogenetic Relationships among the Salamanders of the Bolitoglossa macrinii Species Group (Amphibia: Plethodontidae), with Descriptions of Two New Species from Oaxaca (Me´xico) GABRIELA PARRA-OLEA,1 MARIO GARCI´A-PARI´S,2,3 AND DAVID B. WAKE2,4 1Museum of Comparative Zoology, Harvard University, Cambridge, MA 02138, USA, and Instituto de Biologia, UNAM AP 70-153, CP 04510, Ciudad Universitaria, Me´xico, D.F. 2Museum of Vertebrate Zoology, 3101 Valley Life Sciences Building, University of California, Berkeley, California 94720-3160, USA 3Museo Nacional de Ciencias Naturales, CSIC, Jose´Gutie´rrez Abascal 2, 28006 Madrid, Spain ABSTRACT.—The Bolitoglossa macrinii species group (restricted geographically to southern Oaxaca and southwestern Guerrero, Me´xico) is a monophyletic assemblage of five species, B. macrinii, Bolitoglossa riletti, Bolitoglossa hermosa, Bolitoglossa oaxacensis sp. nov., and Bolitoglossa zapoteca sp. nov. DNA se- quences totaling 1164 base pairs for the mitochondrial genes 16S ribosomal RNA and cytochrome b were analyzed to generate a phylogenetic hypothesis for the relationships within the group. Our hypothesis is in agreement with previous morphological and allozyme analyses. Divergence within the group is great (to 18.6% for cytochrome b, to 7.0% for 16S rRNA) and two clades are well supported: one including B. riletti, B. hermosa,andB. zapoteca and the other including B. macrinii and B. oaxacensis. These clades likely diverged beginning in mid-Miocene times. We describe two new species, B. oaxacensis, for inland Oaxacan populations previously assigned to B. macrinii,andB. zapoteca for the easternmost populations of the clade. The new species are diagnosed by less interdigital webbing, distinctive coloration (each with subdued or no white spotting but differing from each other in pattern and hue) and by extensive differentiation in allozymes (B. oaxacensis) and mitochondrial DNA sequences (both species). The new taxa are the third and fourth species of Bolitoglossa endemic to the State of Oaxaca. RESUMEN.—El grupo de Bolitoglossa macrinii (limitado geogra´ficamente al sur de Oaxaca y el suroeste de Guerrero, Me´xico) es un conjunto monofile´tico constituido por cinco especies, B. macrinii, Bolitoglossa riletti, Bolitoglossa hermosa, Bolitoglossa oaxacensis sp. nov., and Bolitoglossa zapoteca sp. nov. Se analizaron secuencias de ADN con un total de 1164 pares de bases de los genes mitocondriales 16S ARN ribosomal y citocromo b con objeto de generar una hipo´tesis filogene´tica sobre las relaciones de los miembros del grupo. Nuestra hipo´tesis concuerda con ana´lisis previos de morfolo´gicos y aloenzimas. La divergencia dentro del grupo es alta (hasta el 18.6% en el citocromo b, y hasta el 7.0% en el 16S rRNA). En nuestros ana´lisis existe apoyo para dos clados: uno que incluye a B. riletti, B. hermosa,yB. zapoteca, y otro que incluye a B. macrinii y B. oaxacensis. Estos dos clados probablemente comenzaron su divergencia a mediados del Mioceno. Se describen dos especies nuevas, B. oaxacensis, que incluye las poblaciones del interior de Oaxaca previamente asignadas a B. macrinii,yB. zapoteca que integra las poblaciones ma´s orientales del clado. Las especies nuevas se diferencian por su membrana interdigital reducida, su coloracio´n distintiva (ambas sin moteado blanco, o con e´ste casi borrado, aunque difieren entre sı´eneltonoyelpatro´n general de coloracio´n), y por su clara diferenciacio´n aloenzima´tica (B. oaxacensis) y de ADN mitocondrial (ambas especies). Los nuevos taxa son la tercera y cuarta especie de Bolitoglossa ende´micas del Estado de Oaxaca. The Bolitoglossa macrinii group is one of the tail base morphology, the group was included most readily diagnosed and distinctive clades in the ‘‘beta’’ section of Bolitoglossa (Wake and within the large genus Bolitoglossa (Wake and Lynch, 1976). Papenfuss et al. (1983) noted, how- Lynch, 1976; Papenfuss et al., 1983). All mem- ever, that the beta tail structure (Wake and Dres- bers of this clade have unusual osteological ner, 1967) of these salamanders is not that typ- characters: weak premaxillary bones, often with ical of other beta Bolitoglossa but involves large incomplete frontal processes, and small pre- individual variation and intraindividual asym- maxillary teeth, even in adult males. Mental metry, thus calling into question the value of the glands are absent in males (Papenfuss et al., tail structure character for diagnosing a beta 1983). Based on the presence of a complicated section. The large Bolitoglossa morio clade that occurs on the opposite (southeast) side of the 4 Corresponding Author. E-mail: wakelab@uclink4. Isthmus of Tehuantepec may be the closest rel- berkeley.edu ative of the Bolitoglossa macrinii clade, but rela- NEW BOLITOGLOSSA FROM OAXACA, ME´XICO 357 tionships within Bolitoglossa remain poorly un- (mtDNA) derived from 15 specimens, including derstood. Members of the two clades share sim- samples from throughout the geographic range ilarities in skull structure (Wake and Brame, of the B. macrinii group. These represent five in- 1969; Papenfuss et al., 1983), and a similar gen- group taxa from 10 localities (for detailed map eralized external morphology, with hand and see fig. 1 in Papenfuss et al., 1983) and three foot webbing varying from moderate to exten- outgroups. Many of these samples were used in sive. the allozymic studies of Papenfuss et al. (1983). The B. macrinii group at present includes Localities of origin, museum collection numbers three described species, all restricted to the Si- and GenBank accession numbers are given in erra Madre del Sur in southern Me´xico, north- Table 1. Genomic mtDNA was extracted from west of the Isthmus of Tehuantepec. Bolitoglossa small amounts of frozen tissue, fresh tail tips, macrinii (Lafrentz 1930) occurs along the Pacific or protein extracts using NaCl following a pro- slopes of the Sierra Madre del Sur in Oaxaca, in tocol modified from Miller et al. (1988). Frag- the vicinity of San Miguel Suchixtepec and San ments of 647 base pairs, corresponding to co- Gabriel Mixtepec. Bolitoglossa riletti Holman dons 7 (part)-223 (part) of the Xenopus cyt b 1964 also occurs along the Pacific slopes of the gene (Roe et al., 1985), and of approximately 517 Sierra Madre del Sur in Oaxaca but further to bp of the 16S gene, corresponding to positions the west, near Putla. Bolitoglossa hermosa Papen- 2510–3059 in the human mitochondrial genome fuss, Wake, and Adler 1983 may be restricted to (Anderson et al., 1981), were amplified via the low elevations along the drainage of the Rı´o polymerase chain reaction (Saiki et al., 1988), us- Atoyac in Guerrero, although there is an uncon- ing the primers MVZ 15 and MVZ 18 (Moritz firmed record of its occurrence at high elevation et al., 1992) for cyt b, and 16Sar and 16Sbr (Pal- (Adler, 1996). Despite the low frequency of cap- umbi et al., 1991) for 16S. PCR reactions con- ture of specimens, and the narrow geographic sisted of 38 cycles with a denaturing tempera- ranges of the species, the group is well studied ture of 92ЊC (1 min), annealing at 48–50ЊC(1 with respect to morphology and allozymes (Pa- Њ penfuss et al., 1983). min) and extension at 72 C (1 min) in a Techne PHC-1 thermocycler. PCR reactions were run in Specimens examined from south of Sola de ␮ Vega, in an inner valley of the Sierra Madre del a total volume of 25 l, using 0.6 units of Taq Sur of Oaxaca, differ from typical B. macrinii in polymerase (Cetus) in tubes containing 0.5 coloration, interdigital webbing, and allozymes, pmol of each primer, 0.75 mM dNTPs, and 1.5 suggesting that they might represent a different mM MgCl2 in a pH 8.4 buffer with 50 mM KCL species (Papenfuss et al., 1983). These authors and 10 mM Tris HCl (final concentrations). Both chose not to describe a new species because of heavy- and light-strand primers were used for the possible existence of geographically inter- PCR amplifications and sequencing. mediate populations that might establish genet- Double-stranded templates were cleaned us- ic continuity with typical B. macrinii across the ing MicroSpin S-300 HR columns (Pharmacia mountains to the south. Recent fieldwork along Biotech). Four ␮l of double-strand product were the Pacific slopes of the Sierra Madre del Sur used as the template for cycle sequencing reac- uncovered a population that is closer geograph- tions in 10 ␮l total volume with the Perkin-El- ically to typical B. macrinii than to the Sola de mer Ready Reaction Kit௢ to incorporate dye- Vega populations, permitting a test of the pre- labeled dideoxy terminators. Thermal cycling vious hypothesis. was performed using standard conditions. Cy- Another population from the vicinity of Quie- cle sequencing products were purified using golani, the easternmost locality for the group, ethanol precipitation and separated by electro- was assigned tentatively to B. macrinii by Pa- phoresis on a 6% polyacrylamide gel using an penfuss et al. (1983), although freshly collected ABI 377 DNA sequencer (Applied Biosystems). specimens were not available. Recently, we have Partial sequences of cyt b were read from both obtained specimens representing these eastern strands and aligned to each other by eye in the populations and have incorporated these speci- program Sequence Navigator௢ version 1.0.1 mens in our new analysis. (Applied Biosystems). The resulting partial 16S In this paper, we use partial sequences of the sequences were checked and aligned using mitochondrial genes 16S rRNA (henceforth 16S) CLUSTAL in the program Sequence Navigator௢ and cytochrome b (henceforth cyt b) to generate version 1.0.1 (Applied Biosystems). Computer- phylogenetic hypotheses and to evaluate previ- generated alignments were refined by eye and ous hypotheses of geographic structure and tax- by comparing them to published secondary onomy within the B.