Copeia, 1983(4), pp. 1051-1073 Genetic Variation in a Dwarfed Lineage, the Mexican Salamander Genus Thorius (Amphibia: Plethodontidae): Taxonomic, Ecologic and Evolutionary Implications JAMES HANKEN Patterns of genetic relationship and taxonomic identity are examined in Thor- ius, an enigmatic genus of minute neotropical salamanders that represent the smallest tailed tetrapods. Data comprise an electrophoretic analysis of 16 protein loci in 69 population samples from 55 localities throughout the range of the genus in southern Mexico. Eight of the nine described species are genetically distinct: Nei's D commonly exceeds 0.9. There is no biochemical evidence of more than a single species at Zoquitlan, Pue., type locality of T. schmidti and T. maxillabrochus. Genetic differentiation between disjunct subspecies of T. pen- natulus is less than the mean pairwise value between described species, but exceeds that observed between some sympatric species pairs. Seven additional as yet undescribed species are identified, based either on their sympatric occur- rence with, or great genetic differentiation from, described species. Most species initially identified by electrophoresis are morphologically distinct. Electropho- retic data fail to resolve the taxonomic affinity of a few remaining populations, although they do provide clearcut and distinguishable hypotheses of relationship that can be tested using other sources, such as morphology. The 15 species constitute three faunal units: Veracruz and eastern Puebla; Sierra de Juarez, Oaxaca; southern and western Oaxaca, and Guerrero. Local endemism is observed within each region, in which sympatry involving two or three species is common. Species frequently demonstrate narrow elevational zonation and distinct habitat and microhabitat preference (e.g., arboreal vs ter- restrial). The distribution of four species on Cerro Pelon, Oax., is examined in detail. RADIATION of the lungless salamanders The morphology of Thorius is highly derived (Plethodontidae) in the New World trop- and unique among plethodontid salamanders ics rivals-in the extent and nature of morpho- (Hanken, 1980, 1982; Wake, 1966); indeed, be- logical diversity-that of any vertebrate taxon cause of the several novel attributes that belie of comparable rank. Comprising at least 11 gen- its affinities with the Plethodontidae, at one time era (Wake and Elias, 1983), neotropical pleth- Thorius was placed in its own (i.e., monotypic) odontid salamanders display a remarkable array family, Thoriidae (Cope, 1869). Ironically, the of structural and functional configuration, which morphology of Thorius, though readily distin- in many instances is associated with changes in guishing it from all other plethodontid genera, locomotion, feeding mechanics and ecology (Al- superficially appears highly conservative within berch, 1981; Lombard and Wake, 1977; Wake, the genus. This retarded past attempts to re- 1966). But perhaps the most intriguing trend solve even alpha-taxonomic relationships using in the group is phylogenetic size reduction, or morphological evidence and, as a result, spe- dwarfism. Reduced adult body size has evolved cialists familiar with the genus agree that, al- in several genera, which each contain at least though nine species are formally described, one species whose adult body size may approach taxonomic identities remain poorly known that of the smallest urodeles. By far the most (Wake and Lynch, 1976). (In fact, although ex- extreme example is the Mexican genus Thorius, ternal morphology is relatively invariant, inter- a small-size "specialist" in which adult body size nal morphology, particularly osteology, is high- may be as little as 13 mm SL (Standard Length: ly variable, including many characters that snout to posterior end of vent), making these distinguish species. But because of substantial salamanders among the smallest of all tailed tet- intraspecific variation, in most cases it has not rapods. been possible to reliably infer, a priori, taxo- ? 1983 by the American Society of Ichthyologists and Herpetologists 1052 COPEIA, 1983, NO. 4 nomic boundaries and relationships from mor- variation-known to affect most external char- phology.) acters (Freeman, 1977)-have not been taken Many recent taxonomic studies have benefit- into account when evaluating species differ- ted greatly from the advent of biochemical pro- ences. Osteological characters, with the excep- cedures that may be used to quantify molecular tion of the presence/absence of maxillary teeth differentiation, and thus affinity, among popu- and the relative size of premaxillary teeth in lations (Avise, 1974; Wake, 1981; Wilson et al., males, have been ignored. As a result, available 1977). This is particularly true for vertebrate species descriptions often are of little use in es- groups that previously have defied systematic tablishing the identity of specimens from any analysis by standard morphological criteria. but a type locality, and are sometimes unable Molecular differentiation often is large and may to provide unequivocal identification of addi- be used, by considering patterns of genetic re- tional specimens collected at type localities from lationship, to tease apart a complex of cryptic which more than one species has been de- species and delineate taxonomic boundaries. scribed. A brief summary of each species is pro- Plethodontid genera in which this approach has vided below. More detailed descriptions, and been used effectively include Batrachoseps additional references, are provided in Freeman (Yanev, 1978, 1980), Desmnognathus(Tilley et al., (1977) and Hanken (1980). 1978; Tilley and Schwerdtferger, 1981; Tilley, Thoriuspennatulus, type species for the genus, 1981); Plethodon (Duncan and Highton, 1979; was described by Cope (1869) based on seven Highton, 1979; Larson and Highton, 1978) and specimens from "Orivaza," Ver. This terres- Pseudoeurycea(Lynch et al., 1977). In the hope trial species is known from several localities in of gaining the necessary insight into species Veracruz in the vicinity (mostly to the south- boundaries which morphology alone had failed east) of Volcan Orizaba at elevations of 800- to provide, I performed an electrophoretic 1,200 m-elevations lower than those of any oth- analysis of protein variation and differentiation er species. Shannon and Werler (1955a) de- among populations of Thorius from throughout scribed a disjunct population from Volcan San its range in southern Mexico. I used allozyme Martin in the isolated Tuxtla Range of extreme evidence mainly in two ways: 1) identification southeastern Veracruz as T. p. narisinagnus.Both of sympatric, often cryptic, species, and 2) clus- subspecies have the distinctive circular nostril ter analysis of populations based on observed which is not seen in any other species of Thorius; levels of differentiation. Consideration of the they are distinguished by the slightly larger nos- pattern of local differentiation in a single species, tril in T. p. narismagnus and slight differences T. macdougalli, aided interpretation of the pat- in external coloration. terns seen among more distant allopatric pop- Thoriuspulmnonaris and T. narisovalis, two ter- ulations and the cluster analyses. The results restrial species from the montane forest of Cer- provide a hypothesis of genetic relationship and ro San Felipe, Oax., were described by Taylor taxonomic identity that may be used, together (1939). These species were distinguished by with morphology, to resolve taxonomic uncer- nostril dimensions (larger and more elongate in tainty and define the systematics of this previ- T. pulmonaris) and different elevational distri- ously intractable group. butions (about 2,000 m for T. pulinonaris vs 2,600-3,000 m for T. narisovalis). Taylor also SYSTEMATICS OF THORIUS suggested slight differences in microhabitats fa- vored by the two species: T. narisovalis was typ- Characters that have been used to distinguish ically found under the bark of fallen logs, the nine species of Thorius involve often slight whereas T. pulinonaris was found invariably in differences in external morphology (e.g., head leaf litter. and nostril shape, foot and digit proportions, Thorius dubitus and T. troglodytes were de- degree of interdigital webbing, trunk and tail scribed by Taylor (1941) from the mountains shape), external coloration and adult body size. above Acultzingo, Ver. Prominent characters Unfortunately, many of these characters, while used to differentiate these species were adult of potential use for distinguishing species, are body size (greater in T. troglodytes)and external susceptible to preservation artifact; such arti- proportions. These two terrestrial species also facts have not been considered in previous differed slightly in elevational distribution and species descriptions based on preserved mate- microhabitat preference: T. dubitus occurred at rial. Furthermore, both sexual and seasonal slightly higher elevations and was collected from HANKEN-SALAMANDER ISOZYMES 1053 under moss and other plants; T. troglodyteswas (McDiarmid, pers. comm.). In the Sierra de found under rocks. Shannon and Werler Juarez, Oax., from which only a single species, (1955b), however, later collected both species T. macdougalli, is described, the presence of two at the same elevation (3,050 m) near the type additional species, differing in adult body size locality. and external coloration, has been suspected Thorius minutissimnusand T. macdougalli were (Freeman, 1977; D. B. Wake, pers. comm.). described