Genic and Morphological Differentiation in Mexican (Caudata: ), with a Description of a New Species Author(s): James F. Lynch, David B. Wake and Suh Y. Yang Source: Copeia, Vol. 1983, No. 4 (Dec. 14, 1983), pp. 884-894 Published by: American Society of Ichthyologists and Herpetologists (ASIH) Stable URL: http://www.jstor.org/stable/1445090 Accessed: 28-01-2016 00:13 UTC

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Genic and Morphological Differentiation in Mexican Pseudoeurycea(Caudata: Plethodontidae), with a Description of a New Species

JAMES F. LYNCH, DAVID B. WAKE AND SUH Y. YANG

A new species of pseudoeurycea from the Transverse Volcanic Range of south- central is shown to be electrophoretically and morphologically distinct from P. leprosa, P. robertsi and P. altamontana. Pseudoeurycea robertsi and P. altamontana, although previously placed in different species groups, are shown to be closely similar genetically. , the most widely distrib- uted of the species that were studied, comprises a central "core" group of rela- tively undifferentiated populations, and a series of peripheral isolates that are genetically well-differentiated from one another and from the central group.

THE 23 recognized species of the pletho- METHODS dontid genus Pseudoeuryceaare widely distributed in humid montane areas of Electrophoresis.-Horizontal starch gel electro- Mexico and Guatemala (Wake and Lynch, 1976). phoresis was used to analyze variation in sam- Although these are the most abun- ples from 7 populations currently referred to dant vertebrates in many upland habitats, their P. leprosa, and one population each of P. alta- ecology is virtually unstudied. In addition, the montana, P. robertsi, and a previously unde- systematics of the genus is only imperfectly scribed species (Table 1). understood, for the dearth of taxonomically Salamanders were collected in 1975-1976, useful morphological characters has made it dif- and were shipped alive to Berkeley, where they ficult to identify stable groupings of closely re- were killed by immersion in dilute chloretone. lated species. In a previous paper (Lynch et al., Samples of liver, kidney, spleen, stomach, heart 1977) we employed multivariate morphometry and skeletal muscle were removed. Tissue ex- and starch gel electrophoresis to compare P. tracts that were not used at once were stored smithi, a well-known Oaxacan species, and P. at -70 C. Mixed tissue extracts were subjected unguidentis, an enigmatic sympatric form that to horizontal starch gel electrophoresis. We were earlier had been reduced to synonymy with P. able to consistently score 18 presumptive ge- smithi (Bogert, 1967). The results of our mor- netic loci. The enzymes, their abbreviations, and phological and genetic comparisons agreed in the appropriate gel/buffer systems (Selander et indicating that P. unguidentis is a valid, well- al., 1971) are given in Table 2. Nei's (1972) differentiated species. genetic distance (DN) and Rogers' (1972) ge- The present study applies similar methods to netic distance (DR) were calculated for each a group of populations of Pseudoeuryceathat in- pairwise combination of the seven populations habits the Transverse Volcanic Range (Cordil- of P. leprosa and the new species, and for one lera Volcanica) of south-central Mexico. The population of P. leprosa versus the new species, study began as a comparison of isolated popu- P. altamontana, and P. robertsi(Tables 3, 4). lations of P. leprosa; we later included two near- by species whose systematic status was poorly Morphometric methods.-Discriminant function understood, and the work received added im- analysis was employed to test the hypothesis that petus when our electrophoretic comparisons re- the four species of Pseudoeurycea, despite ob- vealed the presence of an undescribed species vious similarities in size and overall body form, of Pseudoeurycea.A continuing goal is to clarify can be reliably distinguished on the basis of con- the discrepancies between earlier attempts to ventional external measurements and tooth delimit species groups within the genus (Taylor, counts. A related question is the extent of in- 1944; Baird, 1951; Wake and Lynch, 1976) and traspecific, as opposed to interspecific, morpho- a recent immunological study (Maxson and logical differentiation in Pseudoeurycea.Because Wake, 1981) that indicated a radically different the fragmented geographic range of P. leprosa grouping of species. greatly exceeds the combined distributions of

? 1983 by the American Society of Ichthyologists and Herpetologists

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TABLE 1. LOCALITY INFORMATION FOR SAMPLES OF PseudoeuryceaUSED IN THE ELECTROPHORETIC ANALYSIS.

Sample Elevation number Species Locality (m) 1 P. leprosa 6 km W Rio Frio, Mexico 2 P. leprosa La , 10 km N Canoa, 3,000 3 P. leprosa 12 km N Tlaxco, Puebla 2,900 4 P. leprosa Xometla, 2,600 5 P. leprosa San Bernardino, Puebla 2,500 6 P. leprosa 12-14 km SW Las Vigas, Veracruz 2,950 7 P. leprosa ZempoalaDistrict, 10 km N Tres Cumbres,Morelos 2,770 8 P. longicauda 23 km W Villa Victoria, Mexico 2,840-2,970 9 P. robertsi , 4 km N Raices, Mexico 3,320 10 P. altamontana ZempoalaDistrict, 20 km N Tres Cumbres, Morelos 3,130

the other three species, it is of interest to com- Formalin, and stored in 70% ethanol. The fol- pare the level of morphological divergence lowing 8 characters were considered (see Lynch among isolated populations of P. leprosa to the et al., 1977, for detailed description of the mea- divergence between these populations and P. surements): head width, head length, fore limb altamontana, P. robertsi, and the undescribed length, hind limb length, body length, tail Pseudoeurycea.The SPSS computer program for length, combined number of maxillary and pre- stepwise discriminant function analysis was used maxillary teeth, and number of anterior vo- to generate discriminate functions, scores, and merine teeth. Because previous studies had in- an a posteriori classification matrix for 134 sal- dicated the existence of marked ontogenetic amanders assigned a priori to five populations changes in body proportions (Lynch et al., 1977) of P. leprosa, and one population each of P. analysis was restricted to post-juveniles (com- altamontana, P. robertsi, and the undescribed bined head and body length >40 mm). We con- Pseudoeurycea. sidered only individuals that possessed an intact All measurements were made on salamanders tail, and rejected specimens that were contorted that had been killed by immersion in dilute clor- or mutilated in such a way that accurate mea- etone solution, fixed in 10% neutral buffered surement was impaired. It would have been

TABLE 2. ENZYMES, THEIR ABBREVIATIONS AND BUFFER SYSTEMS USED IN THE ELECTROPHORETIC ANALYSIS.

Voltage and Enzyme Abbreviation(s) of loci Buffer time run phosphogluconatedehydrogenase Pgd Tris maleate with 100 V (3.5 h) NADP in gel peptidase (1-leucyl-alanine) Pept-1, Pept-2 Li 300 V (3 h) glutamate-oxaloacetate-transaminate Got-1, Got-2 mannose phosphate isomerase Mpi indophenol oxidase Ipo (from Mpi stain) \ Tris citrate II 100 V (3.5 h) isocitrate dehydrogenase Icd-1, Icd-2 sorbitol dehydrogenase Sordh leucine aminopeptidase Lap malate dehydrogenase Mdh-1, Mdh-2 Tris citrate IV 180 V (3 h) phosphate isomerase Gpi phosphoglucomutase Pgm-1, Pgm-2 lactate dehydrogenase Ldh-1, Ldh-2 Poulik 200 V (3 h)

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TABLE3. ALLOZYMEFREQUENCIES AT 18 Loci IN Pseudoeurycealeprosa (LOCALITIES1-7) ANDP. longicauda (LOCALITY 8). The most rapidly migrating band is indicated as "a." Numbered localities are identified in Table 1. Sample sizes are in parentheses.

Locality 1 2 3 4 5 6 7 8 Locus (21) (21) (5) (28) (3) (9) (20) (20)

Mpi c (.95) c c a (.05) c b (.78) c e (.78) d (.05) c (.91) c (.22) f(.22) d (.04) Ipo b b b b a (.83) b b b (.17) Icd-1 a a a a (.93) a (.33) a a (.63) b c (.07) c (.67) c (.37) Icd-2 a a c a a a (.78) a b c (.22) Sordh a (.88) a e a (.39) a (.67) a (.67) a (.75) e e (.12) b (.15) b (.17) b (.33) d (.25) c (.04) e (.16) e (.42) Pgd c (.95) b (.02) c c c b(.5) c c d (.05) c (.98) c (.5) Gpi b (.86) b b a (.12) a b b a (.37) c (.14) b (.88) b (.63) Pgm-l a a a a a a a a (.02) b (.98) Pgm-2 b b b b b b b a (.02) c (.98) Mdh-1 c c (.98) a c c e c b(.02) e (.02) d (.98) Mdh-2 b b b b b b b a

Lap a a a a (.98) a a a b c (.02) Pept-1 d d c b (.04) b (.67) c d c (.04) c (.17) d (.80) d (.16) e (.12) Pept-2 a a a a a a

Got-1 b a (.02) c a (.13) a (.50) b b d b (.98) b (.82) b (.50) c (.05) Got-2 c c c c (.98) c c(.25) c a (.02) d (.02) d (.75) b (.98) Ldh-i a a c a (.93) a a (.72) a b c (.07) c (.28) Ldh-2 c c c a (.20) .039 .005 0 .079 b (.80) Mean heterozygosity .039 .005 0 .079 .185 .056 .035 .064

Percent polymorphic loci 22 17 0 50 27 33 11 39

Alleles per locus (mean) 1.22 1.17 1.00 1.83 1.39 1.33 1.11 1.39

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TABLE 4. ALLOZYME FREQUENCIES FOR LOCI IN Pseu- preferable to analyze males and females sepa- doeuryceaaltamontana ANDP. robertsi.These loci have rately (Lynch et al., 1977), but this would have been standardizedrelative to populations 1 and 8 in unacceptably reduced our sample sizes. The fi- Table 2. Sample sizes are in parentheses. nal morphometric analysis was based on samples of from 6 to 30 (mean = 17) intact post-juvenile Species salamanders from each of 8 populations. P. altamontana P. robertsi Locus (7) (13) RESULTS Mpi e e In his analysis of the herpetofauna of Mi- c a Ipo (.72) (.65) choacan, Duellman (1961, 1965) reported high- d f (.28) (.35) elevation populations of Pseudoeuryceafrom the Icd-1 d d (.60) Sierra Temazcaltepec, near Michoacan's east- e (.40) ern border with the state of Mexico. Duellman Icd-2 a (.21) b referred these salamanders to P. robertsi,a species b (.79) that had been known previously only from the Sordh e e Nevado de Toluca, a high volcanic peak some 60 km SE of the Sierra Temazcaltepec. In 1976 Pgd c c (.40) we collected a large series of Pseudoeurycea in d (.60) the vicinity of the Michoacan-Mexico border, Gpi a (.14) d(.25) and were impressed by the differences in pro- b (.72) e (.75) portions and coloration between these f(.14) and topotypic P. robertsi.Subsequent analysis of variation and external Pgm-l d d allozyme morphology confirmed the distinctness of this new form, which we name in reference to its Pgm-2 c a (.30) unusually tail. c (.70) long Mdh-l f f Pseudoeurycealongicauda sp. nov. Mdh-2 a a Fig. 1 Holotype.-MVZ 137880. An adult female from b b Lap the forested slope just South Mex. hwy 15, 23.1 km (by rd) W. Villa Victoria, State of Mexico, Pept-l f(.21) g (.75) Mexico (elevation 2,850-2,970 m), collected 7 h g (.79) (.25) July 1976 by M. Feder, J. F. Lynch and D. B. Pept-2 b b Wake.

Got-i d(.57) c(.10) Paratypes.-MVZ 137879, 137881-921; Got-2 e e 138418-36; 138518-19 (63 specimens) same data as holotype; Kansas University (KU) 59397- Ldh-1 d d 409, 59969 (14 specimens) Puerto Lengua de Vaca, Michoacan, Mexico (elevation 2860 m); Ldh-2 e d (.10) University of Michigan Museum of Zoology e (.90) (UMMZ) 115130 (5 specimens) 8 km E Macho Mean heterozygosity .127 .167 de Agua, Michoacan, Mexico (elevation 2,880 m); UMMZ 115131 (4 specimens) 1.6 km E Macho de Agua, Michoacan, Mexico (elevation Percent polymorphicloci 27 44 2,800 m); UMMZ 115132 (3 specimens) Atzim- ba, Michoacan, Mexico (elevation 2,800 m). Alleles per locus (mean) 1.33 1.33 1.44 Diagnosis.-P. longicauda exhibits a mosaic of morphological similarities and differences with reference to three other similar highland Pseu- doeuryceathat occur in the Cordillera Volcanica

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j3 4

f

'4

' ,Y -'2.- , Po90

Fig. 1. Holotype of Pseudoeurycealongicauda.

east of the Mexico-Michoacan state border (Fig. species in relative tail length (mean tail length/ 2). Adult P. longicaudacan be distinguished from SL = .98 in longicauda vs .82 in altamontana, P. altamontana, P. leprosa and P. robertsiby the .80-.89 (mean .84) in 5 populations of leprosa, following combination of features, based on .80 in robertsi).P. longicauda also differs from P. comparisons of individuals with standard length altamontana in having a narrower head (mean (SL), the distance from the snout to the poste- head width/SL = .14 vs . 16), shorter legs (mean rior angle of the vent, greater than 40 mm: P. combined length of hind limbs plus fore limbs/ longicauda greatly exceeds all three of the other SL = .48 vs .54), fewer maxillary-premaxillary

100' 99? 98" Fig. 2. Map of the Transverse Volcanic region of central Mexico, showing distribution of (squares). P. robertsi(triangle), P. altamontana (open circle) and P. leprosa (solid circles). The latter species also has been reported from locality 9 and from Tianguistengo, Hidalgo, NE locality 3, but we have not examined material from these areas. The single known instance of sympatry involves the latter two species. Heavy Stippling: areas above 3,000 m elevation.

This content downloaded from 136.152.142.102 on Thu, 28 Jan 2016 00:13:21 UTC All use subject to JSTOR Terms and Conditions LYNCH ET AL.-MEXICAN PSEUDOEURYCEAVARIATION 889 teeth (mean = 41 vs 47), fewer vomerine teeth white iridophores are scattered across the chin, (mean = 19 vs 25), and lighter pigmentation (see and extend onto the chest in some specimens. below, Variation). P. longicauda differs from P. robertsiin having a narrower head (mean head Measurementsof the holotype(in mm).-Head width width/SL = .14 vs .16) and more maxillary and (HW) 9.1; snout to gular fold (HL) 14.2; head premaxillary teeth (mean = 41 vs 28). In P. rob- depth at posterior angle ofjaw 5.5; eyelid length ertsi the venter is uniform gray-black, and a 3.8, anterior of orbit to snout 3.0; horizontal striking tan to red-brown dorsal stripe is almost orbit diameter 2.0; interorbital distance 2.6; invariably present. P. longicauda is usually light- snout to insertion of fore limb 13.6; distance er than P. robertsi in overall color, the dorsal separating internal nares 1.7; distance separat- stripe is paler and more irregular, and the chin ing external nares 2.8; projection of snout past is distinctly lighter than the remainder of the mandible 1.0; snout to posterior angle of the venter. vent (SL) 65.3; posterior to anterior angle of Compared with P. leprosa, P. longicauda has the vent 4.2; axilla to groin (AG) 36.9; posterior longer legs (mean combined length of hind limb angle of vent to tip of tail (TL) 63.5; tail width plus fore limb/SL = .48 vs .42) larger feet (mean at base 4.6; tail depth at base 4.8; axilla to tip foot width = .09 vs .07), fewer maxillary-pre- of outstretched fore limb (FL) 14.5; groin to tip maxillary teeth (mean= 41 vs 67), and fewer of outstretched hind limb (HL) 15.1; width of vomerine teeth (mean= 19 vs 24). The two right hand 4.2; width of right foot (FW) 5.5. species are fairly similar in coloration, but P. The holotype has 43 maxillary teeth, 5 pre- longicauda tends to feature paler tones and a maxillary teeth, and 23 anterior vomerine teeth. lightening of the mid-dorsal pigmentation into an irregular stripe. In both species the chin re- Habitat.-P. longicauda is restricted to pine-fir gion is a lighter gray than is the belly, and there and pine-oak-fir forests at high elevations is a tendency toward dorsal lightening of the (2,850-3,000 m) in eastern Michoacan and tail. However, P. leprosa is usually a dark brown- westernmost Mexico. Specimens have been black salamander with obscure vermiculate found on the ground beneath logs, wood chips brown markings on the dorsum; P. longicauda and other debris, but most of the 64 individuals occasionally approach this coloration, but most collected at the type locality in July 1976 were individuals are a lighter gray-brown, and almost found off the ground, beneath the loose bark all show at least some development of an irreg- of large fir, pine, and cypress logs. Substrate ular light-colored mid-dorsal stripe. Whereas temperatures ranged from 10.5-16.0 C (mean = the venter is usually gray-black in P. leprosa, it 12.9 C; N = 14). is pale to medium gray in P. longicauda. Sympatric salamanders.-The only other sala- Variation.-The sexes broadly overlap in size, manders known to occur within the range of P. although females average slightly larger than longicauda are P. bellii and Ambystomaordina- males (mean SL for females >40 mm = 52.7, rium. All three species were collected by Duell- max. = 65.3 [N = 9]; mean SL for males = 51.0; man (1961) in pine-fir forest immediately to the max. = 61.6 [N = 16]). Adult males possess a west of the type locality, where we encountered prominent round mental gland. As noted by only P. longicauda. The three species also occur Duellman (1961), there is considerable varia- in close proximity in the Atzimba National Park, tion in color pattern. Some individuals have a some 50 km NW of the type locality (Duellman, featureless gray-black dorsum, but most are 1961). much paler, with dorsal coloration that ranges from dark brown through various shades of gray- Range.-Known from two clusters of localities brown, to an almost pink hue. The dorsum may in the Cordillera Volcanica of eastern Michoa- be mottled with obscure brown streaks and is can and adjacent Mexico (Fig. 2), an area termed frequently marked with a ragged mid-dorsal the "Sierra Temazcaltepec" by Duellman stripe of tan, yellow, orange, or brown. The (1961). P. longicauda may occur in other isolated light to medium gray lateral coloration is par- highland areas of pine-fir forest in the western tially obscured by varying amounts of silvery- Cordillera Volcanica (Duellman, 1961, 1965), white iridophore "frosting." The color of the but the species is absent from the Nevado de venter ranges from slaty to pale gray, but the Toluca and the Zempoala area, some 50-75 km chin is invariably lighter than the belly. Small east of the type locality.

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TABLE 5. COEFFICIENTS OF NEI'S DISTANCE (ABOVE DIAGONAL) AND ROGERS' SIMILARITY (BELOW DIAGONAL) FOR SEVEN POPULATIONS OFPseudoeurycea leprosa AND ONE POPULATIONOF Pseudoeurycea longicauda (SEE TABLE 1 FOR LOCALITIES).

I 2 3 4 5 6 7 8 1 - 0.002 0.397 0.015 0.191 0.244 0.012 1.867 2 0.978 - 0.404 0.020 0.197 0.240 0.011 1.883 3 0.660 0.667 0.325 0.539 0.379 0.413 1.558 4 0.935 0.929 0.693 - 0.167 0.242 0.022 1.672 5 0.778 0.774 0.561 0.791 0.437 0.176 1.847 6 0.727 0.731 0.644 0.720 0.585 0.261 2.016 7 0.954 0.962 0.652 0.923 0.798 0.711 -1.854 8 0.167 0.159 0.214 0.211 0.183 0.159 0.171

ELECTROPHORETIC COMPARISONS tances (DN) for conspecific samples ranged from .002-.539 (mean .22). Four populations (1, 2, Because we originally hypothesized that P. 4, 7) were closely similar genetically (maximum leprosa and P. longicauda were close genetic rel- DN = .022), and populations 1 and 2 were vir- atives, our initial electrophoretic survey com- tually identical (DN =.002). However, the pared the new species (Table 1, sample 8) with Xometla sample (population 4) was consider- several populations of P. leprosa. When these ably more variable than the other three, having two species proved to be strongly divergent ge- 33 alleles at 18 loci. The other three samples netically, we added two other local Pseudoeury- (Rio Frio, La Malinche, Zempoala) had 20 to cea (P. altamontana and P. robertsi)to our com- 22 alleles at 18 loci, and were well differentiated parison. Thus, the second part of the survey from the first four samples, as well as from one compared one population each of P. robertsi another. The smallest genetic distance separat- (sample 9) and P. altamontana (sample 10) with ing any of these three divergent populations P. longicauda and one population of P. leprosa from other P. leprosapopulations was DN = .176 (sample 1). Data are summarized in Tables 3, (between samples 4 and 5, the Xometla and San 4, and localities are indicated in Fig. 2. Readers Bernardino populations); the greatest genetic are cautioned not to make any assumptions con- distance separated populations 3 and 5 (DN = cerning homologies between allozymes that oc- .539). The Tlaxco population (sample 3) ap- curred in P. robertsi and P. altamontana as op- peared to be the most distinctive. The range of posed to P. leprosapopulations 2-7, unless those DN values separating this population from the same allozymes were found in P. leprosa popu- other P. leprosapopulations was .325-.539 (mean lation 1. .41). The sample size was sufficiently small (N = Most of the populations we studied displayed 5) that we may have failed to encounter infre- substantial allozyme variation (Tables 3, 4). In quent alleles whose presence might have in- P. leprosa heterozygosity ranged from 0 (pop- creased the resemblance of population 3 to oth- ulation 5) to .079 (population 4) and .185 (pop- er populations, but the Tlaxco population did ulation 5, a small sample). The other three Pseu- show one unique, apparently fixed allele (Mdh- doeuryceaspecies exhibited high heterozygosity 1, allele a). Sample 6 (Las Vigas population) was (maximum = .167 in P. robertsi). Polymorphism the next most distinctive (DN = .240-.437, mean reached a maximum of 50% (population 4 of P. .30). For peripheral populations of P. leprosa leprosa), and ranged from 27%-44% in the oth- genetic distance appeared to be a function of er species and populations. The average num- geographic separation. Thus, for population 5 ber of alleles per locus was also high, reaching (San Bernardino), DN increased from .167 (vs a maximum of 1.83 in P. leprosa (population 4), population 4) to .437 (vs population 6) to .539 and 1.33-1.44 in the remaining three species. (vs population 3). Population 3 of P. leprosa displayed the lowest The Xometla population (sample 4) con- variability (fixed at all loci in five specimens). tained almost all of the alleles that occurred in There was substantial interpopulational vari- any of the 7 P. leprosa populations. Even rela- ation within P. leprosa (Table 5), and Nei dis- tively well-differentiated populations (e.g., the

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.015 003 i I Rio Frio TABLE 6. STANDARDIZED COEFFICIENTS FOR FIRST .020 0007 2 Malinche THREE MULTIPLE DISCIMINANT FUNCTIONS BASED ON Best fit 004 028 7 Zempoala 8 MORPHOMETRIC CHARACTERS (% SD- 6,5) .032032 .081 4 Xometla Variable* Function 1 Function 2 Function 3 ~.~171~ 5 San Bernardino > .148 . 148 6 Las Vigas SL -.471 -.765 -.984 .862 208 3 TlOxc TL -.551 -1.178 -.972 8 Villa Victoria HW .385 .090 2.058 Fig. 3. "Best" Fitch-Margoliash trees constructed CL 1.537 1.763 -.685 from electrophoretic data for seven populations of P. AG -1.071 -.304 .633 leprosa (1-7) and one population of P. longicauda (8). FW .474 -.036 -.798 MP -.556 .785 -.175 VT .134 .199 .235

* See text for definition of variables. Tlaxco population) differed from the Xometla population mainly in allozyme frequencies. The Tlaxco and Las Vigas populations (samples 3 in grouping population 6 with populations 1, 2, and 6) shared a fixed allele for Pept-1, but showed 4, 5 and 7, rather than with population 3. Both no other clear-cut similarities in their patterns trees have a very small negative branch, an in- of allele distribution. dication of some irregularity in the rate of mo- In view of the great genetic distances sepa- lecular evolution in this group. rating most species of Pseudoeurycea (Lynch et and the substantial differ- al., 1977), genetic MORPHOMETRIC ANALYSIS entiation we found in P. leprosa, we were sur- prised at the relatively small genetic difference The results of the multiple discriminant func- between P. altamontana and P. robertsi (DN = tion analysis (Fig. 4) were consistent with the .149). This value, which is well within the range electrophoretic analysis. The first discriminant of values for isolated populations of P. leprosa, axis (Table 6) accounted for 74% of the varia- is the smallest genetic distance yet recorded be- tion in morphology among populations. This tween any pair of species in the genus. To put dimension segregated P. leprosa from the other this value into context, we can note that it is 3 species, and also separated P. robertsifrom P. about the minimum genetic distance one en- longicauda. The characters with the highest counters between sympatric species of the tem- loadings on this axis were combined limb length perate plethodontid genus Plethodon (Highton and axilla-groin length. The second discrimi- and Larson, 1979). nant axis accounted for an additional 12% of Although P. longicauda previously had been referred to P. robertsi(Duellman, 1961, 1965), the two species are very distinct genetically I I I . . . I I 6 (DN = 1.41). P. longicauda is also very different 5 from P. altamontanla(DN = 1.01). Based on gross we had ex- morphological similarities, initially z that P. would to be a 0 pected longicauda prove z close relative of P. leprosa, but the mean DN 4 between P. and the seven K longicauda populations I of P. leprosa is 1.81 (range 1.56-2.02). These distances match or exceed those which separate g, C v robertsi P. robertsi = and P. (DN 1.83) altamontanza (1.57) - z ( from population 1 of P. leprosa. \_ P longicauda We used the EVOLVE program (provided by Walter Fitch) to generate Fitch-Margoliash trees -4 -3 -2 -I FIRST DISCRIMINANT2 3 4 FUNCTION5 6 7 9 from DR values. These trees were rooted using FIRST DISCRIMINANT FUNCTION P. as an The "best" tree longicauda outgroup. Fig. 4. Plot of first two discriminant functions for (i.e., the one with the lowest percent standard morphometric data from 6 populations of P. leprosa deviation [see Prager and Wilson, 1978]) is il- and one population each of P. altamontana, P. lon- lustrated in Fig. 3. The second best tree differs gicaudaand P. robertsi.

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TABLE7. CLASSIFICATIONMATRIX FOR EIGHT POPULATIONS OF Pseudoeurycea, BASED ON STEPWISEMULTIPLE DISCRIMINANT FUNCTION ANALYSIS OF 8 MORPHOMETRICCHARACTERS. The Orizaba population (sample a) was collected on the NW flank of the , near Sierra Negra, Puebla. Localities for other samples are given in Table 1.

Predicted population Actual population Locality a b c d e f g h a. P. leprosa Orizaba 14 4 3 2 4 0 0 0 b. P. leprosa Xometla 6 16 0 3 5 0 0 0 c. P. leprosa Las Vigas 0 1 4 1 0 0 0 0 d. P. leprosa Malinche 0 0 4 21 2 0 0 0 e. P. leprosa Zempoala 1 0 0 1 8 0 0 0 f. P. altamontana Zempoala 0 0 0 0 0 9 0 0 g. P. robertsi N. de Toluca 0 0 0 0 0 0 8 1 h. P. longicauda Villa Victoria 0 0 0 0 0 0 0 16

the total among-population variation, and sep- arating P. longicauda from P. robertsi or P. al- arated P. altamontana from P. longicauda and P. tamontana are somewhat smaller, but are never- robertsi,mainly on the basis of tail length, com- theless substantial (DN= 1.41 and 1.00, bined limb length and tooth counts. Successive respectively). Recently obtained electropho- axes accounted for 14% of the total among- retic data for 16 of the 24 known species of population variation. As indicated by the clas- Pseudoeuryceaindicate that P. robertsiand P. al- sification matrix (Table 7), the canonical anal- tamontanaare more similar genetically to a num- ysis resulted in only one "mistaken" assignment ber of Guatemalan and Oaxacan species of Pseu- of a specimen to the wrong species. Thus, more doeurycea (e.g., P. rex, P. goebeli, P. smithi, P. than 99% of the specimens could be assigned unguidentis) than either is to P. longicauda. to the correct species solely on the basis of a The status of species groups within Pseudoeu- few external characters. A number of individual rycea remains in a state of flux. The situation P. leprosa were assigned to conspecific popula- has been reviewed recently by Maxson and Wake tions other than their own, but in no case was (1981), who found that immunological data the assignment to a heterospecific population. conflicted with a number of previous clusterings In all, 56% of 107 P. leprosa could be assigned of species into groups. For example, although correctly not only to species, but also to local Maxson and Wake prepared five antisera and population. tested a total of 18 of the 23 Pseudoeuryceaspecies that were then known, they discovered no close DISCUSSION relatives of P. leprosa. Wake and Lynch (1976) had provisionally assigned P. altamontana to the Interspecific diferentiation.-The results of the cephalica group and P. robertsi to the leprosa electrophoretic and morphological analyses groups, but Maxson and Wake (1981) found agree in indicating that P. longicauda is well dif- that both species were much more similar im- ferentiated, both genetically and phenetically, munologically to P. smithi (gadovii group) than relative to other plethodontid species that have either was to P. leprosaor P. cephalica. Curiously, been studied. Contrary to our original expec- P. altamontana gave consistently lower immu- tations, neither P. leprosa nor P. robertsi(nor P. nological distances than P. robertsito all antisera altamontana) is a clear sister species to P. longi- that were tested. cauda. The magnitude of the genetic distance P. altamontana and P. robertsiare very similar between P. longicauda and P. leprosa is similar genetically, and certainly warrant placement in to that which separates many "large" and the same species group. Based on the immu- "small" eastern North American Plethodon nological comparisons of Maxson and Wake species (Highton and Larson, 1979), and ex- (1981), and on unpublished electrophoretic data, ceeds the genetic distance between most Eu- the gadovii species group seems the most rea- ropean and Californian species of Hydromnantes sonable provenance for these two species, al- (Wake et al., 1978). The genetic distances sep- though this group is admittedly a very hetero-

This content downloaded from 136.152.142.102 on Thu, 28 Jan 2016 00:13:21 UTC All use subject to JSTOR Terms and Conditions LYNCH ET AL.-MEXICAN PSEUDOEURYCEAVARIATION 893 geneous assemblage. Because P. altamontana and lations (DN = .20), although there is no indica- P. robertsiare so similar genetically and are allo- tion that the Las Vigas sample is particularly patrically distributed, a case could be made for distinctive in morphology (Table 6). Indeed, in synonymizing them. However, because the two view of the absence of a major physiographic species are distinct in color and body propor- or climatic barrier between Pico de Orizaba and tions we continue to recognize them as separate (Fig. 2), the genetic distinc- taxa for the present. tiveness of this population is somewhat surpris- ing. In contrast, the San Bernardino and Tlaxco Intraspecificvariation in P. leprosa.-P. leprosa, populations are presently well isolated from the which has by far the most extensive geograph- main Transverse Volcanics axis by climatic-to- ical range of the four Pseudoeuryceaspecies we pographic barriers. studied, presently comprises numerous isolated Taylor (1938) believed that "P. leprosa" com- populations (Fig. 2). Accordingly, we were not prised two allopatric species. He restricted P. surprised to discover considerable morpholog- leprosa to salamanders from the vicinity of Vol- ical and genetic variation among populations of can Orizaba (our Xometla and Sierra Negra P. leprosa, although the extent and geographical samples fall within this geographic area), and pattern of this variation could not have been referred all remaining populations of P. leprosa- predicted. A core group of populations along like salamanders (including those from Rio Frio, the main E-W axis of the Cordillera Volcanica Las Vigas, and the Zempoala lakes region) to (Fig. 2) shows only slight genetic differentiation P. orizabensis.Taylor and Smith (1945) later syn- (Table 3, Fig. 3). From west to east these pop- onymized P. orizabensisand P. leprosa, a decision ulations inhabit the Ajusco Range (Zempoala which is supported by the results of our study. sample), the Popocatepetl-Iztaccihuatl volcanic If any populations currently referred to P. le- massif (Rio Frio sample), La Malinche Volcano prosa were to be recognized taxonomically, the (Malinche sample), and the Pico de Orizaba area northern and northeastern outliers (Tlaxco and (Xometla sample). These populations are re- Las Vigas) would be the most obvious candi- stricted to humid, high elevation (2,500-3,000 dates. However, in view of the considerable ge- m) forested areas that today are separated by netic and morphological overlap that exists lower, somewhat drier country which is not in- among leprosa populations (Table 6, Fig. 4) any habited by P. leprosa. However, during Pleis- taxonomic change at this time would be un- tocene glacial epochs, elevational zones in this warranted. region were depressed by 800 m or more (Simp- The results of the present study are in ac- son, 1979; White, 1960). During pluvial periods cordance with Wake's (1981) observation that continuous forest undoubtedly connected pres- both concordance and discordance between ently isolated areas of montane forest. Indeed, morphological and electrophoretic variation coniferous forest persisted in much of the valley characterize salamander lineages. Populations of Mexico until historical times, when signifi- of the widespread P. leprosa show moderate dif- cant deforestation by Amerindian populations ferentiation with respect to both external mor- began. Deforestation of the foothills around the phology and allozyme frequencies. In contrast, Valley of Mexico was completed in the 16th P. robertsiand P. altamontana, species which are century when Cortes denuded the slopes of tim- sufficiently dissimilar in morphology to have ber in order to construct present-day Mexico been placed in different species groups by ear- City. Clearly, the isolation of populations of P. lier workers, show only trivial genetic diver- leprosa along the main volcanic axis was a rel- gence. The frequent decoupling of morpholog- atively recent event. ical and genic variation in salamanders means Along the Caribbean escarpment, an isolated that both morphometric and biochemical ap- population of P. leprosa approximately 40 km S proaches will be indispensable in future at- Volcan Orizaba (San Bernardino) is moderately tempts at systematic revision of this group. divergent from the "core" group (DN = .18), but it is the Tlaxco population, some 60 km N Malinche, that shows the greatest degree of ge- ACKNOWLEDGMENTS netic differentiation (DN = .41). The popula- tion on the flanks of Cofre de Perote (Las Vi- We wish to thank Martin Feder, James Han- gas), at the NE periphery of the species range, ken, Theodore Papenfuss, Samuel Sweet and is also well-differentiated from the core popu- Thomas Wake for their help in collecting the

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Pseudoeuryceafor this study. Monica Frelow per- SELANDER, R. K., M. H. SMITH, S. Y. YANG, W. E. formed most of the electrophoretic work. Wal- JOHNSON AND J. B. GENTRY. 1971. Biochemical and in the Pero- ter Fitch generously provided a copy of the polymorphism systematics genus EVOLVE used in the myscus. I: Variation in the old-field mouse (Pero- program phylogenetic Ibid. McKim and Vicki Dedrick- myscuspolionotus). 7103:49-90. analysis. Margaret B. B. 1979. of son the final The illustrations SIMPSON, Quaternary biogeography typed manuscript. the mountain of South America, p. are the work of Susan Strok. Financial high regions support 157-188. In: The South American herpetofauna: for the research came from the Museum of Ver- its origin, evolution and dispersal. W. E. Duellman tebrate Zoology, the Smithsonian Institution and (ed.). Univ. Kans. Mus. Nat. Hist. Monogr. NSF Grant DEB78-03008 (D. B. Wake, prin- TAYLOR, E. H. 1938. Concerning Mexican salaman- cipal investigator). ders. Bull. Univ. Kans. 39:259-313. 1944. The genera of plethodont salamanders in Mexico, Part 1. Univ. Kans. Sci. Bull. 30:189- LITERATURE CITED 232. ,AND H. M. SMITH. 1945. of the I. L. 1951. An anatomical of certain Summary BAIRD, study collections of made in Mexico under salamandersof the Univ. Kans. genusPseudoeurycea. the Walter Rathbone Bacon Sci. Bull. traveling scholarship. 34:221-265. Proc. U.S. Nat. Mus. 95:521-613. C. M. 1967. New salamandersof BOGERT, the pleth- D. B. 1981. The of evi- odontid from the Sierra Madre WAKE, application allozyme genus Pseudoeurycea dence to in the evolution of del Sur of Mexico. Amer. Mus. Novit. 2314. problems morphology, 257-270. In: Evolution G. G. E. Scudder E. p. today. DUELLMAN, W. 1961. The amphibians and reptiles L. Reveal Proc. 2nd Int. of Mexico. Univ. of Kans. andJ. (eds.). Congr. Syst. Michoacan, Publ. Mus. Evol. Biol. Nat. Hist. 15:1-148. AND F. LYNCH. 1976. The . 1965. A , J. distribution, biogeographic account of the her- and of of Mexico.Ibid. ecology, evolutionary history plethodontid petofauna Michoacan, 15:627-709. salamanders in America. Contrib. Sci. Nat. AND A. LARSON. 1979. The tropical HIGHTON,R., genetic Hist. Mus. Los Co. 25. of the salamandersof the Pleth- Angeles relationships genus L. R. MAXSONAND G. Z. WURST. 1978. Ge- odon. Zool. 28:579-599. , Syst. netic differentiation, albumin evolution, and their Y. AND LYNCH,J. F., S. YANG T.J. PAPENFUSS. 1977. in sala- Studies of salamandersof the biogeographic implications plethodontid neotropical genus manders of California and I: status of (genus Hydromantes) Pseudoeurycea, systematic Pseudoeurycea southern Evolution 32:529-539. 33:46-52. Europe. unguidentis.Herpetologica WHITE,S. E. 1960. Late Pleistocene L. AND D. B. WAKE. 1981. Albumin glacialsequence MAXSON, R., for west side of Mexico. Geol. Soc. evolution and its in the Iztaccihautl, phylogenetic implications Amer. Bull. 71:2001. plethodontid salamandergenera Pseudoeuryceaand Chiropterotriton.Herpetologica 37:109-117. NEI,M. 1972. Genetic distancebetween populations. CHESAPEAKE BAY CENTER FOR ENVIRONMENTAL Amer. Nat. 106:283-292. STUDIES, SMITHSONIAN INSTITUTION, EDGE- PRAGER, E. M., ANDA. C. WILSON. 1978. Construc- WATER, MARYLAND 21037, MUSEUM OF VER- tion of phylogenetic trees for proteins and nucleic TEBRATE ZOOLOGY AND DEPARTMENT OF acids: empirical evolution of alternative matrix methods. J. Mol. Evol. 11:129-142. ZOOLOGY, UNIVERSITY OF CALIFORNIA, ROGERS, J. S. 1972. Measures of genetic similarity BERKELEY,CALIFORNIA 94720, AND DEPART- and genetic distance.Studies in Genetics VII. Univ. MENT OF BIOLOGY, INHA UNIVERSITY, INCHON, Texas Publ. 7213:145-153. REPUBLIC OF KOREA. Accepted 18 Oct. 1982.

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