HERPETOLOGICAL JOURNAL, Vol. 13, pp. 21-26 (2003)

A NEW SPECIES OF PSEUDOEUR YCEA (CAUDATA: PLETHODONTIDAE) FROM NORTHERN OAXACA, MEXICO

LUIS CANSECO-MARQUEZ' AND GABRIELA PARRA-0LEA2

'Museo de Zoologia, Departamento de Biologia Evolutiva, Fa cultad de Ciencias, UNA M, AP 70-399 Mexico, DF 04510, Mexico

2/nstituto de Biologia, Departamento de Zoologia, UNA M, AP 70- 153 Mexico, DF 04510, Mexico

We describe a new species of Pseudoeurycea fromthe northern-most high peak of the Sierra de Juarez, Oaxaca, Mexico. This species belongs to the P. juarezi group, a monophyletic assemblage restricted to northern Oaxaca and comprising three species: P. juarezi, P. saltator, and P. aurantia sp. nov. Pseudoeurycea aurantia is the sister taxon to the clade formed by P. juarezi and P. saltator. The new species is diagnosed by a distinctive coloration and by di vergent mitochondrial DNA sequences.

Keywords: mitochondrial DNA, new species, phylogeny, salamander, systematics

INTRODUCTION The Sierra de Juarez, where all known species of the juarezi group occur, is a moderately high mountain Pseudoeurycea is a large clade of neotropical sala­ P. chain (2900 m maximum altitude) which from the manders that displays extensive morphological diversity runs highlands of Cuicatlan Valley (Cerro Pefia Verde) to the and genetic differentiation (Parra-Olea & Wake, 2001; east of Ciudad de Oaxaca, with southern limits in the Parra-Olea, 2002). It ranges from northern Mexico area of Cerro San Felipe and Cuajimoloyas. At Cerro across the Isthmus ofTehuantepec into Guatemala and - San with few exceptions - its species occur at elevations Felipe, the Sierra Aloapaneca intersects the Sierra de Juarez, and runs west and north of Cerro San Felipe. above 1200 m. The vegetation changes according to altitudinal gradi­ Tlie systematics of Pseudoeurycea have changed re­ ents from the nearly treeless summits of Cerro Pel6n cently, with several species newly described or in the covered by low shrubs to the mesic cloud forest. The process of description (Parra-Olea et al., 2001; Wake & herpetofauna of the region, one of the richest in montane Campbell, 2001) and several more identified as new Mexico (Casas-Andreu 1996) keeps providing (Parra-Olea, 1999, 2002). A recent phylogenetic analy­ et al., many new species despite past intensive search. During sis (Parra-Olea, 2002) based on mtDNA showed the the last eight years, six new salamanders (Hanken & paraphyly of Ps eudoeuryceawith respect to Lineatriton Wake, 1994, 2001) and 10 anurans (Campbell & and lxalotriton. This paraphyly forcedthe transferof P. Duellman, 2000; Mendelson, 1997; Mendelson & parva to Jxalotriton, but thetaxonomic problem involv­ Campbell, 1999; Toal, 1994; Toal & Mendelson, 1995; ing Lineatriton is still unsolved (Parra-Olea & Wake, Ustach 2000) have been described for the Sierra 200 1). et al., de Juarez. The analyses of Parra-Olea (2002) found phylogenetic support forthe recognition ofth ree species A visit to the Sierra Pefia Verde - the northern-most high peak ofthe Sierra de Juarez in the highlands above groups within Pseudoeurycea (P. be/Iii, P. gadovii and the Cuicatlan Valley- uncovered a morphologically dis­ P. leprosa species groups). Additionally, three taxa (P. tinct new salamander that resembles species of the P. juarezi, P. saltator and P. unguidentis) are not grouped group. The analysis of mitochondrial DNA with any of these and formpart of the basal polytomy. P. juarezi (mtDNA) partial sequences of the Cytochrome juarezi and P. saltator are sister taxa, and their relation­ b (Cytb) and 16S genes confirmed its close relationship with ships to the rest of Pseudoeurycea are not resolved. P. members of the P. juarezi group, but it is not the sister unguidentis forms part ofthe basal polytomy on its own. taxon of any of the known species. We present here a The group f rmed by P. juarezi and P. saltator (here � new phylogenetic hypothesis for thegroup together with termed the P. juarezi group), includes species character­ the description of this new tax on. We provide some eco­ ized by morphology associated with semiarborial life, logical data and describe the eggs of the new species. with long limbs and toes and slender bodies. The species inhabit terrestrial habits, under rocks, logs and under the MATERIALS AND METHODS bark oflogs on the ground, or bromeliads. Their range is fromhigh altitude, unforested habitats dominated by MORPHOLOGICAL DESCRIPTION grasses to mid-elevation cloud forestin the Sierra de The descriptions follow the format used by Lynch & Juarez of northern Oaxaca, Mexico. Wake (1989) for other species in the genus Pseudoeurycea, and include the same basic characters Correspondence: Gabriela Parra-Olea, Jnstituto de Biologia, · and measurements. Larger measurements were taken us­ UNAM, AP 70-153 Mexico, DF 045 10, Mexico. E-mail: ing dial calipers (to the nearest 0. 1 mm), but [email protected] measurements of feet, toes and some head dimensions 22 L. CANSECO-MARQUEZAND G. PARRA-OLEA

(e.g. additional measurements of the holotype), as well 2-parameter; Kimura, 1980), were obtained using the as tooth counts were taken using a stereoscopic micro-_ computer program PAUP*4.0b8a (Swofford, 2002). scope equipped with an eyepiece graticule. All Phylogenetic inference was based primarily on maxi­ measurements are in mm. The distance from the tip of mum parsimony analyses (MP: Swofford, 2002). MP the snout to the posterior end of the vent is treated as phylogenies were estimated using the heuristic search standard length (SL). Colour notes are based on field algorithm for each tree-building methodology. We used notes taken fromliving specimens, and on preserved l 0 repeated randomized input orders for taxa in all MP specimens. Osteological descriptions are based on two analysis to minimize the effect of entry sequence on the cleared and bone-cartilage differentially stained speci­ topology of the resulting cladograrns. MP analyses were mens (IBH 13797, 13798). conducted without the steepest descent option, and with Ma terial examined: Nine specimens from4 km W accelerated character transformation (ACCTRAN) opti­ Pefia Verde, Oaxaca, Mexico, 2805 m elevation, 17° mization, tree bisection-reconnection (TBR) branch 50.20' N, 96° 47.05' W, (EBUAP2052-2058; swapping, and zero-length branches collapsed to yield IBH13793-13794). Material for coloration and mtDNA polytomies. We used nonparametric bootstrapping comparisons consisted of specimens of P. juarezi, and ( l OOO pseudoreplicates) and decay indices to assess the P. saltator recently collected (Parra-Olea et al., l 999, stability of internal branches in the resulting topologies Parra-Olea, unpubl. data). Comparative measurements (Felsenstein, 1985; Felsenstein & Kishino, 1993). Each between P. juarezi and P. saltator were taken from base position was treatedas an unordered character with Lynch & Wake (1989). Institutional abbreviations: four alternative states. Gaps were treated as missing EBUAP: Escuela de Biologia, Universidad Autonoma data. de Puebla, Mexico; IBH: Coleccion Nacional de Anfibios y Reptiles, Instituto de Biologia, UNAM, DESCRIPTION OF NEW SPECIES Mexico. PSE UD OEUR YCEA AURANTIA, NEW SPECIES PENA VERDE SALAMANDER MTDNA SEQUENCES SALAMANDRA DE PENA VERDE We obtained partial sequences of l 6S and Cyt b for Holotype. EBUAP205 l, an adult male collected 4 km the single specimen from which tissue was available W PefiaVerde, Oaxaca, Mexico; 2805 m elevation; 17° (IBH13793). Whole genomic DNA was extracted from 50.20' N, 96° 47.05' W, on 7 May 2000 by Luis Canseco small amounts of ethanol-preserved tissues, using the Marquez (Fig. la). Quiagen DNA extraction kit. We sequenced 554 base Paratypes. All from Pefia Verde, Oaxaca, Mexico: pairs of the large 16S subunit ribosomal mtDNA gene IBH13793-13794 (two specimens), and EBUAP2052- corresponding to positions 251 0-3059 in the human mi­ 2058 (seven specimens), same data as the holotype. tochondrial genome (Anderson et al., 1981 ), and 570 Diagnosis. We include this species in the genus base pairs of the Cytochrome b gene, expanding from Pseudoeurycea based on the following osteological codon 7 of the Xenopus Cyt b gene (Roe et al., 1985). characters: distal tarsal five separated from tarsal four, Amplification was done via the polymerase chain reac­ smaller than four, and not articulated with central; pre­ tion (PCR) (Saiki et al., 1988), using the primers maxillary single; middle digits markedly larger than the "MVZ15", "MVZ18" (Moritz et al., 1992) for Cyt b, outer; and presence of sublingual fold. It is distin­ and the primers "16Sar" and "l6Sbr" (Palumbi et al., guished from all other Pseudoeurycea by its orange 1991) for 16S. PCR reactions consisted of 38 cycles coloration. This species is closely related to members of with a denaturing temperature of 92°C ( 1 min), anneal­ the P. juarezi group based on morphology and mtDNA ing at 48-50°C ( l min), and extension at 72°C ( l min) in (Figs. l, 4 ). It is distinguished fromP. juarezi in having a Techne PHC-1 thermocycler. PCR reactions were run fe wer maxillary and premaxillary teeth (63-81 vs. 74- in a total volume of25 µl, using 0.5 pmol of each primer. 100 for P. juarezi), and a shorter tail (relative tail Double strandtemplates were cleaned using a QIAquick length=0.88 vs. 0.96 in P. juarezi); and from P. saltator PCR purification kit (QIAgen). We used 1 µl of PCR in having a larger size (maximum SL = 51 mm vs. 48 in product as the template forcycle sequencing reactions in P. saltator), and a shorter tail (relative tail length =0 .88 a l 0 µl total volume with the Perkin-Elmer Ready Reac­ vs. 1.05 in P. saltator). tion Kit to incorporate dye-labeled dideoxy terminators. Description. A medium-sized salamander; SL in two Thermal cycling was performed using standard condi­ adult males 43-43.4 (mean=43.2) and in eight females tions. Cycle sequencing products were purified using 38.5-5 1.7 (mean=44.6), with relatively robust habitus; ethanol precipitation and run in an ABI 310 capillary head relatively broad, ( 15-16% SL in both males, and sequencer. fe males); snout broadly rounded, more truncate in males The sequences were compiled using Sequence Navi­ than in females; neck region ill-defined, only slightly gator™ version 1.0. 1 (Applied Biosystems), and narrower than head; eyes moderate in size, only slightly aligned to thepreviously published data set for species protuberant. Adult males present mental gland well de­ of Pseudoeurycea (Parra-Olea, 2002). Pairwise com­ veloped. Parotoid glands not evident. Costal folds 13, parisons of corrected sequence divergence (Kimura counting one each in axilla and groin. Limbs are rela- NEW SPECIES OF MEXICAN SALAMANDER 23

much shorter than the fourth. Digits in order of decreas­ ing length: fingers 3-2-4- 1; toes 3-4-2-5-1. Tail relatively stout, and short - 72-1 00% SL; Maxillary teeth 66 (mean=66) in males, 55-67 (mean=62.5) in fe ­ males; premaxillary teeth 2-4 (mean=3) and enlarged in adult males, 8-19 (mean=13.5) and smailer in fe males; vomerine teeth in long rows, 17-20 (mean=18.5) in males, 17-29 (mean=2 l.5) in females. Co/oration in life. The ground colour is reddish­ brown dorsally grading to pale yellow ventrally. A broad and conspicuous orange mid-dorsal stripe extends from the scapular region to the tip of the tail. In the head region the dorsal stripe is broken into darker flecks. Bright yellow spots are present all over the dorsum and are more concentrated on the tail. A dark line runs from the nostril, posteriorly to the neck region. Some speci· mens present a few dark spots along the sides of the body and tail. The limbs generally are the same reddish­ brown as the dorsum. The underside of the body and tail is uniform pale yellow with no spots. There is a bright yellow stTipe beneath the lip area. The iris is coppery gold. Co/oration in alcohol. The col oration in alcohol con­ trasts with that of the living animals, only in the degree

of brightness. The dorsum is dark brown, with a dark orange stripe nnming from the scapular region to the tip of the tail. The venter is pale yellow. Measurements of holotype (in mm). Head width 6.8; FIG. l. Live specimens of members of the P.juarezi group ­ head depth 3.6; eyelid length 3.0; eyelid width 2.1; ante­ a, f'. aurantia, EBUAP 2052 (paratype); b, P. juarezi (Cerro rior rim of orbit to snout 2.4; intcrorbital distance 2.7; Pel6n, Oaxaca, Mexico); c, (La Esperanza, P saltator distance between comers of eyes 5 .6; snout to forelimb Oaxaca, Mexico). l .'.i . l; nostril diameter 0.2; distance between external lively Jong; digits typically meet when limbs are pressed nares 2.4; projection of snout beyond mandible 0.7; to the side of trunk in males, or separated by no more snout to gular fold 11.2; width across shoulders 6.3; than one costal interspace; in females, appressed limbs snout to posterior angle of vent 45.0; snout to anterior fail to meet by one to one and one half costal angle of vent 41.6; axilla to groin 23 .2; tail length 47.0; interspaces. Hands and feet relatively well developed, tail depth at base 4.7; tail width at base 5.3; forelimb digits relatively long and slender for Pseudoewycea, length l 0.0; width of hand 3 .O; hind limb length 12.7; without subtenninal pads; fi ft h toe well developed but width of foot 4.7; length oflongest (third) toe 1 .8; length of fifth toe 1.0. Numbers of teeth: premaxillary 4; max­ illary 29/37; vomerine 9/8. -- . Gulf of Me.xlco Va riation. About 75% of fe males reach a larger size

\ than males and have a more robust body. The basic col­ .\ .'"" our pattern is the same, but some individuals display L. _ _ _ black spots along the sides ofthe body and tail. The dark line that runs from the nostril to the neck area across the t; _____ ------/ .l eye is Jess evident in adult females. . ,-, . � Distribution. Pseudoeu1ycea aurantia has been col­ I ( . · lected only at the type locality - Pena Verde, Oaxaca, OA XAC. A _ ,, , ! "·;" ) \ " ! Mexico, at 2805 m ( J 7° 50.20' N, 96° 47.05' W) (Fig. 2). ,; · Na tural histmy. This species occurs in cloud forest ;.·· Pacific Ocean where it is found under rocks, the bark of logs, and in or • PseudoewyCl.'a aur 2000 Ill. 2000, 49.3 SL, was guarding a clutch of 22 eggs under 24 L. CANSECO-MARQUEZAND G. PARRA-OLEA

of the embryos. The lens is white. Rudiments of all five digits are visible on the hind limbs and rudiments of all four digits are visible in the forelimbs. Gills are tribranchiate. Etymology. The specific epithet is derived from the Latinaurantium, and is used as an adjective in reference to the distinctive orange coloration of this species. Molecular characters. Parra-Olea (2002) recently re­ ported the results of an extensive molecular analysis of phylogenetic relationships of members of the genus Pseudoeurycea (sensu lato). Three species groups were recognized (P. bellii, P. gadovii and P. /eprosa). The relationships of three additional species (P. juarezi, P. saltator and P. unguidentis) were uncertain. The sam­ ples used by Parra-Olea (2002) and in the present study FIG. 3. Egg clutch of P. aurantia, at stage 3 I of Marks and under the designation of P. unguiden tis were collected Collazo (I 998). Egg diameter, including surrounding inan area relatively distant fromthe type locality of the capsules ranges from 4.2 to 5.2 mm (mean=4.89) after species (Cerro San Felipe, Oaxaca, Mexico) and based preservation. on preliminary morphological data they might represent a different species (D. B. Wake, pers. comm.). the bark of a log. The eggs were attached in a bead like We have now added sequences of l 6S and Cyt b for fashion. The eggs were preserved 10% neutral buff­ in the new species, P. aurantia, to the data set. The small­ ered formalin. Egg diameter, including surrounding est divergences of P. aurantia from any other species capsules ranges from 4.2 to 5.2 (mean=4.89) after mm are for P. juarezi (3.9% Cyt b, 0.3% 16S) and P. preservation. All embryos were preserved at approxi­ saltator (3.8% Cyt b, 0.3% 16S). Divergences between mately the same stage of development {Fig. 3), which P. aurantia and sequences from all other corresponds to stage 31 of Marks & Collazo (1998). Ps eudoeurycea species groups are large, including P. Dorsal pigmentation is extensive and extends onto aurantia to P. gadovii group (10.7-14.6% Cyt b, 3.8- proximal portions of each limb. The retina is black in all .------l�:":= 4.8% 16S); P. aurantia to P. bellii group (15.0- 16.6% ParWriage Cyt b, 5.4-6.4% 16S); P. aurantia to P. leprosa group 1aeh11«1 nl{lel (11.7- 17.0% Cyt 3.5-4.9%16S). l:xa/"rilorr p/111/US b, i------P.unguidtntis A maximum parsimony analysis produced nine P}U8!eZi equally parsimonious trees (L=l467 steps, CI=0.406, P. s81t4ta RI=0.564, 318 characters were parsimony informative). P ;varezigoup P.11Ur1nn LineallitQrl The strict consensus topology (Fig. 4) does not differ LJ{)ealJ//(IJ considerably from the published topology for the genus P. wet/a l > P. my&ax Pseudoeurycea (Parra-Olea, 2002). There is a high level Une ttrfl.on of support forrecognition of the P. gadovii (decay 9, bs Uneatr;ion 99%) and P. be/Iii (decay 5, bs 86%) species groups, P. Jeposa and low support for the group (bs <50%). P. ll!fXOS8 P. leprosa 100 Pseudoeurycea aurantia is a sister taxon to the clade 14 P. lynch/ p sp. 3 formed by P. juarezi and P. saltator (P. juarezi group, P. $mithi decay 16, bs 100%). The clade forms part of P. smith! P. juarezi P. loflgicaudll. the basal polytomy. P. rabert6i P gaO{migoup P. 81tamcn1ana DISCUSSION P. sp. 2 P. tencha/11 The salamander fauna of the Sierra de Juarez in J------P.melanomolg.a Oaxaca, Mexico i� highly diversified, including 22 de­ P coch1anae scribed species of the genera Pseudoeurycea, Thorius, P. coct111Y1ae P. ga/eanae Lineatriton, Chiropterotriton and Cryptotriton. System­ P Cefilsl1C8 atic studies of the salamander fauna ofthe region started P. ceph alica during the 1930's and 40's with descriptions of P. P btM'1lgoup P. bel11i p bellil smithi, P. cochranae and P. unguidentis (Taylor, 1939, Thorius 1941, 1943) and species of Thorius (Taylor, 1940). BatractJoseps Several more species of salamander have been de­ scribed recently (Hanken & Wake, 1994, 2001 ; FIG. 4. Maximum Parsimony strict consensus tree of the I 6S Papenfuss & Wake, 1987; Wake & Campbell, 200 1), and Cyt b combined data set (I 092 bp, TL= I 467 steps, and there are still a few others yet undescribed (Wake, CJ=0.406, Rl=0.564). Numbers above branches are pers. corn.). This is one of the regions of Mexico most nonparametric bootstrap values ( 1 OOO replicates), numbers below branches are decay index values. frequented by herpetologists, but P. aurantia occurs in a NEW SPECIES OF MEXICAN SALAMANDER 25

remote area of Sierra de Juarez (Fig. 2). Pena Verde is provided for gene coalescence and the possibility of the northern-most peak, and is of difficultaccess even introgression along the ecotones between these geo­ now, probably the reason this species remained un­ graphically close taxa. The recency of the lineage split known despite field work in the general area. This among species ofthe P. juarezi group is an unusual case species seems to be another montane specialist with re­ within tropical salamanders, which generally display stricted distribution, as is true of closely related species. large mtDNA divergences among species (Parra-Olea, Based on morphology and mtDNA P. aurantia is re­ 2002). Salamanders of the P.juarezi species group rep­ lated to the other two members of the P. juarezi group: resent an ideal study case for species formation in P. juarezi and P. saltator, both of which also occur in tropical regions since different stages of the speciation the Sierra de Juarez. These three species share a general process are represented by different taxa within the common morphology (Fig. I) and their differences in­ group in a reduced geographic area. clude size (P. saltator is the smallest of the three) ACKNOWLEDGMENTS number of teeth and tail length. Pseudoeuryceajuarezi and P. saltator live in the same general area and differ in We thank G. Gutierrez-Mayen, S. Larios-Guzman, the altitudinal segregation of their habitats. L.-E. Chong-Alcaraz and I. Fentanes-Patifio for their Pseudoeurycea saltator is an arboreal dweller of tropi­ collaboration during field work; D. B. Wake and M. cal mesic cloud forest found at mid-elevations Garcia-Paris forcomments and discussion on this paper; (1500-1800 m). P. aurantia is the most distant geo­ L. Marquez (Laboratorio de Biologia Molecular, graphically, and occurs at elevations similar to P. Instituto de Biologia, UNAM)for assistance obtaining juarezi (2200-2900 m); both occupy terrestrial habitats DNA sequences and M. Valladolid (Museo Nacional de in the pine forestand barren alpine lands at higher eleva­ Ciencias Naturales, Madrid, Spain) forthe skeletal clear tions. and stain procedure. Partial support forthis project was The addition of mtDNA sequences of P. aurantia to provided by CONABIO (Mexico) to G. Gutierrez­ the previously published data set forPseudoeurycea, Mayen (FB615/R067/98), CONACyt to G. Parra-Olea did not alter the resulting topology (Parra-Olea, 2002). ( 139332-V) and by the National Science Foundation There is strong support for the P. gadovii, and P. be/Iii (DEB-0 102383 to J. A. Campbell). SEMARNA T pro­ groups, and lower support for .the P. /eprosa group. vided the necessary collecting permits. There is strong bootstrap support for the new P. juarezi group that now includes P. aurantia. The basal relation­ REFERENCES ships are umesolved, so the affinities of the P. juarezi Anderson, S., Bunkier, A. T., Barrell, B. G., Debruijn, M. group to the other clades of Pseudoeurycea and allied H. L., Coulson, A. R., Drouin, J., Eperon, I. C., genera are undefined. The geographic and genetic dis­ Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H. , tances between P. juarezi and P. sa/tator are smaller Smith, A. J. H., Staden, R. & Young. I. G. (1981). than those fromP. aurantia to either, but morphological Sequence and organization of the human differentiation is larger between P. saltator and P. mitochondrial genome. Na ture 290, 457-465. juarezi. Comparedwith P.juarezi, P. saltator is smaller Campbell, J. A. & Duellman, W. (2000). 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