46 HERPETOLOGICA [Vol. 33, No. 1

FERM, V. H., AND S. J. CARPENTER. 1968. Mal- SANDERS, H. 0. 1970. Pesticide toxicities to formations induced by sodium arsenate. J. tadpoles of the westem chorus frog Pseudacris Reprod. Fertil. 17:199-201. triseriata and Fowler's toad Bufo woodhousii FERM, V. H., A. SAXON, AND B. M. SMrTH. 1971. fowleri. Copeia 1970:246-251. The teratogenic profile of sodium arsenate in SOKAL, R. R., AND F. J. ROHLF. 1969. Biometry. the golden hamster. Arch. Environ. Health W. H. Freeman and Co., San Francisco. 22:557-560. SWIGGART,R. C., C. J. WHITEHEAD, JR., A. HAZELWOOD, E. 1970. Frog pond contaminated. CURLEY, AND F. E. KELLOG. 1972. Wildlife British J. Herpetol. 4:177-185. kill resulting from the misuse of arsenic acid HOOD,R. D., AND S. L. BISHOP. 1972. Terato- herbicide. Bull. Environ. Contam. Toxicol. 8: genic effects of sodium arsenate in mice. Arch. 122-128. Environ. Health 24:62-65. 1976 MATSUMURA, F. 1972. Current pesticide situa- Received: 16 June tion in the United States, p. 33-60. In: F. Accepted: 23 September 1976 Matsumura,G. M. Boush, and T. Misato [eds.] Environmental toxicology of pesticides. Aca- Department of Biology, Pan American demic Press, New York. University,Edinburg, Texas 78539, USA

STUDIES OF NEOTROPICAL OF THE GENUS , I: SYSTEMATIC STATUS OF PSEUDOEURYCEA UNGUIDENTIS

JAMES F. LYNCH, SUH YUNG YANG, AND THEODOREJ. PAPENFUSS

ABSTRACT: The systematic status of Pseudoeurycea unguidentis, an enigmatic Mexican plethodontid described by Taylor (1941), is reviewed in the light of morpho- metric and biochemical analysis of recently collected material. It is concluded that P. un- guidentis is a valid species which differs from , a superficially similar sympatric form, in numerous phenetic and genetic characters. Far from being conspecific, as suggested by an earlier investigator, these two salamanders are more different genetically than are most congeners in other groups.

SOME 35 yr ago, E. H. Taylor (1941) smithi, influenced Bogert (1967) to syn- described unguidentis from a onymize the two forms in his treatment series of plethodontid salamanders col- of OaxacanPseudoeurycea. lected in 1938 on Cerro San Felipe and Field parties from the Museumof Verte- nearby Cerro San Luis in central Oaxaca, brate Zoology,University of California,and Mexico. The type series of the new species the Smithsonian Institution visited Cerro (which subsequently was assigned to the San Felipe on four occasions in 1974 and genus Pseudoeurycea) was taken in local 1975 in conjunction with studies of the sympatry with Pseudoeurycea smithi, a salamanderfauna of the region. On two of closely similar salamander. Except for the these trips (January and November 1974) mention of one additional specimen of P. smithi was the only Pseudoeuryceaen- Pseudoeuryceaunguidentis by Taylor and countered,but in August 1974 and August Smith (1945), the species has not been 1975 one of us (TJP) obtained series of reported since Taylor's original description. what appeared to be a different species in The lack of fresh comparative material, sympatrywith P. smithi. Both of the latter combined with the close resemblance be- collections were made at the same locality tween P. unguidentis and the common P. along the crest of a gently sloping ridge

HERPETOLOGICA 33:46-52. March 1977 March 1977] HERPETOLOGICA 47

more vigorous in its attempts to escape and tended to occur off the ground more fre- quently, the loose bark of fallen logs being the favored microhabitat. The complex of morphological, ecological, and behav- ioral differences distinguishing the two Pseudoeurycea is reminiscent of that sepa- rating the slender, active, bark-dwelling Aneides ferreus from the more robust, sluggish, ground-dwelling Aneides flavi- punctatus where the two species are sym- FIG. 1.-Dorsal vi.ew of living adult P. mithi patric in northwestern California (Steb- *.XX.:ee.e::~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... bins, 1951; J. Lynch, personal observation). In his description, Taylor stated (1941: 61) that P. unguidentis differs from P. smithi ". . in having longer limbs ... and larger hands and feet. The tail is somewhat longer and narrower." Because these dif- ferences correspond to those we perceived in the Pseudoeurycea from Cerro San Felipe, we provisionally assigned the sec- (top) and P. unguidentis (bottom) a a. Vertical ond form to P. unguidentis. To test our bar = 25 mm. Photograph by James Hendel, hypothesis that two distinct species (as Scientific Photographic Laboratory, University of California,Berkeley. opposed to mere morphological variants) were collected, we employed two inde- pendent modes of analysis: multivariate covered with humid, fairly open oak- morphometrics and electrophoretic screen- forest. Based on several altimeter readings ing of isozymes. taken over a 2-yr period, the elevation of this locality is 3050 + 25 m. Taylor (1941: METHODS 57) gives the elevation of the type locality of P. unguidentis as "about 2200 m", but The specimens reported in this paper in view of the rarity of specimens of any are deposited in the collection of the Mu- species of Pseudoeurycea at such low ele- seum of Vertebrate Zoology, Berkeley. A vations in central Oaxaca, and given the sample of living salamanders was shipped restriction of the sympatric P. smithi to to Berkeley for biochemical analysis in areas above 2800 m (Bogert, 1967), we August 1974; other specimens were killed believe Taylor's figure to be in error. by immersion in dilute chloretone solution Relative to sympatric P. smithi (Fig. 1A), within a few hours of capture, fixed in the second Pseudoeurycea (Fig. 1B) ap- 10% Formalin, and later transferred to peared to us to have a somewhat more 70% ethanol for permanent storage. slender habitus, longer legs, and lighter Morphometric Analysis.-The morpho- lateral and ventral pigmentation. These logical analysis is based on a detailed ex- subtle differences appeared to be consis- amination of 49 preserved P. smithi and tent, even though the overall similarity to P. smithi in size, build, and pigmentation 21 P. unguidentis. Discriminant function was indeed striking. Collecting notes sug- analysis was employed to specify patterns gested the presence of behavioral and eco- of interspecific differentiation. The follow- logical differences as well. Relative to P. ing characters were considered (all mea- smithi, the second form was noticeably surements in millimetres): head length 48 HERPETOLOGICA [Vol. 33, No. 1

(HL), the distance from tip of snout to were dissected and samples of blood and center of gular fold; body length (BL), tissue were removed. Liver, kidney, spleen, the distance from center of gular fold to stomach, heart, and skeletal muscle were posteriorangle of vent; head width (HW), homogenized using the methods of Se- the distance across broadest part of head; lander et al., (1971), and extracts were tail length (TL), the distance from pos- stored at -76? C. Combinedtissue extracts terior angle of vent to tip of tail; coupling and hemolysate fractions of blood were ratio (CR), the sum of the lengths of subjected to horizontal starch-gel electro- right fore limb and right hind limb divided phoresis as described by Selander et al., by the axilla-groindistance; maxillary tooth (1971). The following gel buffer systems count (MT), the total number of teeth on were used: (1) tris-hydrochloricacid (pH the maxillae; and vomerine tooth count 8.5) for hemoglobin (Hb); (2) lithium (VT), the total number of teeth on the hydroxide for albumin (ALB), transferrin vomers. Standard length (SL), the dis- (TRF), glutamate oxalate transaminases tance from tip of snout to posterior angle (GOT-1 and GOT-2), and peptidases of vent, was substituted for charactersHL (PEPT-1 and PEPT-2); (3) discontinuous and BL in comparisonsinvolving adult P. tris-citrate (Poulik) for lactate dehydrog- unguidentis, most of which had been dis- enases (LDH-1 and LDH-2); (4) con- sected in the gular region for tissue re- tinuous tris-citrate (pH 8.0) for sorbitol moval. dehydrogenase(SDH), alpha-glycerophos- Discriminantfunction is a multivariatesta- phate dehydrogenase (a GPD), isocitrate tistical technique that defines the weighted dehydrogenases(IDH-1 and IDH-2), man- combinationof characterswhich best sepa- nose phosphate isomerase (MPI), and su- rates the members of two or more pre- peroxidedismutase (SOD); (5) continuous defined groups. Each individual is as- tris-citrate (pH 7.0) for leucine amino- signed group membership a priori; the peptidase (LAP), malate dehydrogenases discriminantprocedure then specifies the (MDH-1 and MDH-2), phosphogluco- optimal set of weighted character coef- mutases (PGM-1 and PGM-2), and phos- ficients that maximizes the ratio of be- phoglucose isomerase (PGI); and (6) tris- tween-groupto within-groupvariance. The maleic EDTA for 6-phosphogluconate reader is referredto Blackithand Reyment dehydrogenase(6PGD). (1971) for a general discussion of the Genetic interpretationsof allozymicvari- applicationof this and related multivariate ation are based on criteria elaborated by techniques to morphometricstudies, and to Selander et al. (1971). Estimates of het- Lynch and Wake (1975) for an example erozygosityare derived from actual counts involving plethodontidsalamanders. of presumedheterozygotic genotypes. Mean Because ontogenetic and sexual variation heterozygosity (H) is defined as the num- in plethodontid salamanders can obscure ber of heterozygotic genotypes recorded interspecific differences, we made the fol- in a sample divided by the product of the lowing separate comparisons: (1) adult number of individuals and the number of males, between species; (2) adult females, loci surveyed. Estimates of polymorphism between species; (3) juveniles (both sexes are based on the number of loci having combined), between species; (4) adult more than one allele divided by the total males vs. adult females, within species. The number of loci. This estimate is highly Smithsonian Institution's version of the conservative, as only loci showing more SPSS program for stepwise discriminant than one allele in our limited samples are analysiswas used in all computations. consideredto be polymorphic. The Rogers Electrophoresis.-Specimens of P. smithi coefficient (Rogers, 1972) is used as an (n = 26) and P. unguidentis (n = 21) estimate of interspecificgenic similarity. March 1977] HERPETOLOGICA 49

* Psmithi E PRunguidentis has a higher CR value (i.e., relatively longer limbs) than P. smithi. Juveniles 6A adult males a show less marked interspecific differentia- tion in CR; for the latter group HW is the A 4- most efficient single discriminator,P. smithi juvenilesgenerally having broaderheads. _I-I__I I II l 11-Ii MorphologicalComparisons. -6 -4 -2 0 2 4 Intraspecific -As summarizedin Table 2, the discrimi- nant function analysis was generally suc- cessful in distinguishingthe sexes in both 4 adult females species. In some instances the best sexual discriminators were not the best inter- specific discriminators. Among 14 adult -6 -4 -2 0 2 4 P. unguidentisthere were no misclassifica- tions as to sex, and SL and HW proved to be the best discriminators.Males tend to exceed females in overall size (SL), but 4_ juveniles have relatively narrowerheads. Of the 43 CF2- ,,,nn1 adult P. smithi examined, 38 (88%) were assigned to the appropriate sex on mor- -6 -4 -2 0 2 4 phological grounds, with HW, BL, and CR being the main discriminators.Relative Discriminant score to females, male P. smithi tend to have FIG. 2.-Histograms showing discriminantscores shorterbodies, a broaderhead, and longer for interspecific morphometriccomparisons of (A) legs. adult 8 8, (B) adult 9 9, and (C) juveniles in P. smithi and P. unguidentis. The units are stan- Regressionanalysis of individual charac- dard deviations relative to the mean for all cases. ters as a function of SL (see Lynch and The absence of interspecific overlap in the scores Wake, 1975, for explanation of methodol- for all three comparisons indicates that the dis- ogy) confirms that the characters desig- criminant function successfully separates the species in the three subgroups. nated most importantdiscriminants in the multivariateanalysis do show statistically significant differences between species or RESULTS between the sexes. As an example, there is a highly significant(P < .01)interspecific Interspecific Morphological Differentia- differencein the head width of adult males tion.-The discriminant analysis strongly projected to a common body size. How- supports our original judgment that two ever, some interspecificoverlap does occur distinct populations of Pseudoeurycea co- in this and other single characters,and the exist on Cerro San Felipe. Whether adult discriminant function analysis allows a males (Fig. 2A), adult females (Fig. 2B), much cleaner separationof species or sexes or juveniles (Fig. 2C) are compared, those than would be possible in any single-char- individually we visually assigned to the acter analysis. smithi and unguidentis groups can be dis- Electrophoretic Comparisons.-Sixteen no "mistakes" on tinguished with virtually proteins encoded by 22 presumptive loci of the six to seven morphological the basis Allelic frequencies for traits we utilized (Table 1). In inter- were examined. specific comparisons of adults of either variable loci, proportion of polymorphic sex CR contributes the highest percentage loci, and proportion of heterozygous loci of variation to the standardized discrimi- are given in Table 3. These data constitute nant function. Pseudoeuryceaunguidentis compelling evidence of profound genetic 50 HERPETOLOGICA [Vol. 33, No. 1

TABLE 1.-Summary of results of discriminant analysis of interspecific morphological differentiation in adult 8 8, adult 9 9, and juveniles (sexes combined) of Pseudoeurycea smithi and P. unguidentis. The absolute magnitude of each standardized coefficient is proportionalto the contributionof that vari- able to the discriminant function. For each comparison the most important coefficient is indicated by boldface type. See text for explanationof variables.

Discriminant coefficient ______9 9 Juveniles Variable raw standardized raw standardized raw standardized SL 0.216 1.287 -0.096 -0.589 HL -0.334 -0.313 BL 0.200 0.706 HW -0.325 -0.403 0.726 0.661 -1.284 -0.912 TL -0.059 -0.400 -0.048 -0.364 0.010 0.085 CR -17.767 -1.457 -17.290 -1.314 -9.110 -0.583 MT 0.053 0.300 0.077 0.406 0.010 0.053 VT -0.211 -0.830 -0.158 -0.686 -0.028 -0.157 No. and % a priori assignmentsto correct species 28 (100%o) 29 (100%) 13 (100%) Incorrect assignments 0 (0%) 0 (0%) 0 (0%)

differentiation between P. smitht and P. Pseudoeurycea.Six of the 12 remainingloci unguidentis. Of the 22 loci considered, 10 share at least some polymorphismsbetween (IDH-1, a GPD, MDH-1, LAP, GOT-1, species, although frequency differences LDH-1, LDH-2, Hb, ALB, TRF) share no may exist in 5 of these (6GPD, PGI, PGM- common alleles in our samples of the two 2, PEPT-1, GOT-2). The remaining six loci are fixed identically in the two species. Although our sample sizes are too small TABLE 2.-Summary of results of discriminant analysisof intraspecificmorphological differences to permit firm conclusions, genetic vari- between adult e a and adult 9 9 in P. smithi ability appears to be higher in P. smithi and P. unguidentis.See text and legend to Table than in P. unguidentiswhether the propor- 1 for futherexplanation. tion of polymorphicloci (0.50 vs. 0.18) or Discriminant coefficient the mean proportion of heterozygous loci P. smithi P. unguidentis per individual (0.09 vs. 0.04) is the cri- Var- iable raw standardized raw standardized terion.

SL 0.495 2.672 DISCUSSION HL -0.334 -0.384 BL 0.200 0.917 There can no longer be any serious HW -1.284 -1.105 -3.536 2.627 doubt that P. unguidentis and P. smithi TL 0.010 0.058 0.016 0.142 are distinct species. Even if the existing CR -9.110 -0.393 -4.086 -0.176 sympatrytest were MT 0.010 0.050 0.102 0.637 unavailable,the strong VT 0.028 0.083 -0.190 -0.430 genetic and morphological differentiation demonstratedin the present analysiswould No. and justify retention of full specific %assign- status for mentsto P. unguidentis. In fact, by conventional correct genetic criteria the affinity between the sex 38 (88.0%) 14 (100%) latter species and the sympatric P. smithi Incorrect is ratherlow for congeners. The computed assign- level of genic similarity (R = 0.42) is ments 5 (11.6%) 0 (00%) lower than that found by Hedgecock and March1977] HERPETOLOGICA 51

TABLE 3.-Alleles and frequencies in each poly- TABLE 3. (Continued) morphic locus in P. smithi and P. unguidentis. h is the proportion of individuals heterozygous at a Locus Allele P. smith-i h P. unguidentis h given locus. Hb a .019 Locus Allele P. smithi h P. unguidentis h b .981 c 1.000 MPI a .154 .038 0 b .846 1.000 .231 0 ALB a 1.000 b 1.000 IDH-1 a .019 0 b .981 o c 1.000 TRF a .904 .038 0 b 1.000 c .096 a GPD a 1.000 b 1.000 .115 0 0 0 Proportion 6PGD a .058 .550 of loci b .904 .450 polymorphic .500 .182 c .038 Proportion .192 .400 of loci PGI a .077 heterozygous .091 .041 b .923 .200 c .800 .154 .300 Ayala (1974) for three species of the sala- mandrid genus Taricha. Indeed, the two PGM-2 a .173 1.000 b .827 Oaxacan Pseudoeurycea fall toward the .192 0 lower end of the range of genetic similari- found in animal MDH-1 a .058 ties congeneric species b .673 (see review by Avise, 1974). Far from c .269 being mere morphologicalvariants, as sug- d 1.000 gested by Bogert (1967), or even incipient .500 0 species, P. unguidentis and P. smithi are LAP a 1.000 b 1.000 highly distinctive evolutionaryunits. 0 0 Work now in progress by two of the present authors (JFL and SYY) in collab- PEPT-1 a .025 b .675 oration with D. B. Wake suggests that low c .212 levels of genic similarity characterizemost d .788 .300 of the approximately25 species that com- .231 .150 prise the genus Pseudoeurycea. Because GOT-1 a .846 electrophoreticmethods are relatively in- b 1.000 sensitive when the populations being com- c .154 pared share few alleles (Avise, 1974, gives .231 0 0.4 as the approximatelower limit of genic GOT-2 a .038 1.000 similarity for meaningful electrophoretic b .962 .077 0 comparisons), allozymic data will have limited utility in our attempts to quantify LDH-1 a 1.000 evolutionaryrelationships across the entire b .975 c .025 genus. Webster et al. (1972) reached a 0 .050 similarconclusion with respect to the much LDH-2 a 1.000 larger lizard genus Anolis. On the other b 1.000 hand, electrophoreticmethods have proved 0 0 useful in establishing the evolutionarydis- 52 HERPETOLOGICA [Vol. 33, No. 1 tinctness of superficially similar popula- LYNCH,J. F., AND D. B. WAKE. 1975. System- tions, as in the present study, and in as- atics of the Chiropterotritonbromeliacia group (Amphibia: Caudata), with description of two sessing patterns of relationship within new species from Guatemala. Los Angeles Cty. species groups of large genera such as Mus. Contrib. Sci. (265): 1-45. Anolis (Yang et al., 1974). ROGERS,J. S. 1972. Measures of genetic simi- larity and genetic distance. Univ. Texas Publ. Future papers in this series will further (7213): 145-153. explore patterns of morphological,genetic, SELANDER, R. K., M. H. SMITH, S. Y. YANG, W. E. JOHNSON, AND J. G. GENTRY. 1971. Bio- and ecological variation within the genus chemical polymorphism and systematics in the Pseudoeurycea. genus Permyscus, I. Variation in the Old-field mouse (Peromyscus polionotus). Studies in Acknowledgments.-Wethank Martin Feder, Genetics VI. Univ. Texas Publ. (7103):49-90. JamesKezer, CharlesKimmel, and David Wake STEBBINS, R. C. 1951. of western for help in collectingthe animalson which this North America. Univ. California Press, Berkeley studyis based. The governmentof Mexicogranted and Los Angeles. collecting permits for 1974 and 1975. Cynthia TAYLOR, E. H. 1941. New plethodont salaman- Hillaryperformed much of the laboratorywork for ders from Mexico. Herpetologica2:57-65. the electrophoreticanalysis. CharlesRoberts pro- , AND H. M. SMITH. 1945. Summary of videdaccess to the SmithsonianInstitution Systems the collections of amphibians made in Mexico for computerwork. David Wake providedexpert under the Walter Rathbone Bacon traveling advice,and he and F. J. Ayala criticallyread the scholarship. Proc. U. S. Natl. Mus. 95:521413. manuscript. Fieldwork was supported by the WEBSTER, T. P., R. K. SELANDER, AND S. Y. Yang. Museumof VertebrateZoology, the Smithsonian 1972. Genetic variability in the Anolis lizards Institution,and especiallyby grantsGB 17112and of Bimini. Evolution 26:523-535. BMS74-20922 from the NationalScience Founda- YANG, S. Y., M. SOULE, AND G. C. GORMAN. tion. 1974. Anolis lizards of the eastern Caribbean: a case study in evolution. I. Genetic relation- LITERATURECITED ships, phylogeny, and colonization sequence of the roquet group. Syst. Zool. 23:387-399. AvISE, J. S. 1974. Systematic value of electro- phoretic data. Syst. Zool. 23:465-481. Received: 16 June 1976 BLACKITH,E. E., AND R. A. REYMENT. 1971. Accepted: 22 November 1976 Multivariate morphometrics. Academic Press, New York. BOGERT,C. M. 1967. New salamanders of the JFL: Chesapeake Bay Center for En- plethodontid genus Pseudoeurycea from the vironmental Studies, Smithsonian Institu- Sierra Madre del Sur of Mexico. Am. Mus. tion, Edgewater, Maryland 21037, USA; Novit. (2314): 1-27. SYY and TJP: Museum of Vertebrate HIEDGECOCK,D., AND F. J. AYALA. 1974. Evo- lutionary divergence in the genus Taricha. Zoology, Universityof California,Berkeley, Copeia 1974:738-747. Calfornia94720, USA