A NEW SPECIES OF PRISTIMANTIS (ANURA: STRABOMANTIDAE) …

A NEW SPECIES OF PRISTIMANTIS (ANURA: STRABOMANTIDAE)FROM THE PACIFIC COAST OF THE DARIEN PROVINCE, PANAMA,WITH A MOLECULAR ANALYSIS OF ITS PHYLOGENETIC POSITION

ANDREW J. CRAWFORD1,2,3,6, MASON J. RYAN

4, AND CESAR A. JARAMILLO1,2,5

1Smithsonian Tropical Research Institute, Apartado 0843-03092, Balboa, Ancon, Republic of Panama2Cırculo Herpetologico de Panama, Apartado 0824-00122, Panama, Republic of Panama

3Departamento de Ciencias Biologicas, Universidad de los Andes, AA 4976, Bogota, Colombia4University of New Mexico, Albuquerque, NM, USA

5Universidad de Panama, Facultad de Medicina, Departamento de Histologıa y Neuroanatomıa Humana,Panama, Republic of Panama

ABSTRACT: We describe a new species of Pristimantis (Anura: Strabomantidae) from the Pacific coast ofthe Darien Province, Panama. The type locality is on Cerro Pina, in the Serranıa de Sapo, at 700–800 melevation. This new species is readily distinguished from all other known congeners from the region based onexternal morphology. Despite the small size of the two type specimens, histological analysis of the gonadsshows these individuals were potentially reproductive males. Molecular phylogenetic analyses based on theND2-WANCY mitochondrial gene region reveal that this new species is genetically distinct. Molecular andmorphological data place the new species in the Pristimantis (Hypodictyon) ridens species series, and suggestthat P. cerasinus be moved to this group as well. Molecular analyses also reveal a potential synapomorphy ofthe genus Pristimantis relative to the other five genera sampled here: the loss of the D-stem of the tRNACYS

gene. Our finding of another endemic frog from eastern Panama lends further support to the concept of theDarien as a center of endemism and not just a conduit between continents.

RESUMEN: Describimos una nueva especie del genero Pristimantis (Anura: Strabomantidae) de la costadel Pacıfico de la Provincia de Darien en Panama. La localidad tipo es Cerro Pina a 700–800 metros sobre elnivel del mar. Con base en la morfologıa externa esta nueva especie es facilmente distinguible de todas lasotras especies conocidas en la region. A pesar del pequeno tamano de los dos especımenes tipo, analisishistologicos de sus gonadas muestran que estos individuos eran machos potencialmente reproductores. Losanalisis de filogenia molecular basados en la region de ADN mitocondrial ND2-WANCY revelan que laespecie nueva es geneticamente distinta. Los datos moleculares y morfologicos sugieren asignartentativamente a la nueva especie dentro de la serie de especies ‘‘Pristimantis (Hypodictyon) ridens’’ yque la especie, P. cerasinus, debe ser incluida en este grupo tambien. Los analisis moleculares tambienrevelan una posible sinapomorfıa del genero Pristimantis en comparacion con los otros cinco generosincluidos en este analisis: la perdida de la ‘‘rama D’’ del gen ARNtCYS. El descubrimiento y descripcion deotra especie endemica del oriente de Panama da mas apoyo a nuestro concepto del Darien como un centro deendemismo y no solo como una vıa entre dos continentes.

Key words: Gonads; Histology; Molecular phylogenetics; Pristimantis; Species description; Spermdevelopment; tRNA secondary structure

THE DARIEN region of eastern Panama ispart of the Tumbes–Choco–Magdalena globalbiodiversity hotspot (Mittermeier et al., 1999;updated information available at http://www.biodiversityhotspots.org), and a particularlyendangered portion of this diversity comprisesamphibians (Stuart et al., 2004). Panama hosts197 species of amphibian as of 2008 (Amphib-iaWeb, 2008). Across the border in theColombian Choco region of the Pacific coastthere are 139 named species of amphibians in

the area below 800 m elevation (Lynch andSuarez-Mayorga, 2004).

Eastern Panama is home to roughly 20named species of direct-developing anurans inthe clade Terrarana sensu Hedges et al.(2008), also known as brachycephalids (Frostet al., 2006). Terraranan frogs have been acontinual challenge for taxonomists because ofsubstantial homoplasy and concomitant dearthof autapomorphies to identify species andunambiguous synapomorphies to identifyclades (e.g., Campbell and Savage, 2000;Lynch and Duellman, 1997). Molecular phy-logenetic analyses have revealed that tradi-6 CORRESPONDENCE: e-mail, [email protected]

Herpetologica, 66(2), 2010, 192–206

E 2010 by The Herpetologists’ League, Inc.

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tional taxonomy often does not reflect rela-tionships within or among the major lineagesof Terrarana (Crawford and Smith, 2005;Heinicke et al., 2007). Herein we describe anew species in the genus Pristimantis basedon morphological characters, and provide aDNA sequence–based analysis of its distinct-ness and phylogenetic position. We alsouncover an unusual feature of the inferredsecondary structure of a mitochondrial tRNAgene, which may help in resolving higher-levelrelationships.

MATERIAL AND METHODS

Morphological AnalysesFrogs were collected in the field, photo-

graphed by A. J. Crawford in the field on theday following collection, and euthanized withdilute chloretone. Specimens were fixed in10% formalin and stored in 70% ethanol(Pisani, 1973). We examined comparativematerial from the Cırculo Herpetologico dePanama (CH), the Instituto de CienciasNaturales of the Universidad Nacional, Bo-gota, Colombia, and recent field collections byA. J. Crawford (Appendix 1). The terminologyfor morphological descriptions follows mainlyLynch and Duellman (1997), Savage (2002),and Savage and Villa (1986). Morphometricabbreviations used throughout the text are asfollows: snout–vent length (SVL), length oftibia (tibia), length of head (measured fromthe tip of snout to posterior of mandible; HL),greatest head width (HW), interorbital dis-tance (IOD), distance from eye to nostril (E–N), distance from the corners on eyes (eyelength), and distance between nostrils (ND).All measurements were taken using dialcalipers accurate to the nearest 0.1 mm.Observations on the color of the frogs in lifewere based on field notes and color slides ofspecimens. The holotype (MVUP 2255, AJC0922) resides in the Museo de Vertebrados dela Universidad de Panama and the paratype(CH 8132) resides in the CH.

Histological MethodsGonads were extracted and processed using

standard paraffin-embedding techniques(Luna, 1968). Four-micrometer sections werecut with a rotary microtome and stained using

hematoxylin and eosin (Luna, 1968). Identifi-cation of histological features was confirmedby comparison with published accounts(Rugh, 1951; Uribe A., 2002).

Molecular Phylogenetic AnalysesFresh liver samples were preserved in a

NaCl-saturated buffer containing 0.25 m eth-ylenediaminetetraacetic acid and 20% dimeth-yl sulphoxide (Seutin et al., 1991), and storedin the natural history collection of the CH. Weconducted molecular phylogenetic analyses of39 frog specimens comprised of 36 terraranidsand three outgroup taxa (Appendix 2). Theseoutgroups have been shown to be outside ofTerrarana but within the larger Nobleobatra-chia (a.k.a. Hyloidea) clade (Darst and Can-natella, 2004; Roelants et al., 2007). Analysesincluded molecular data from the holotypeand paratype of the new species describedherein. A segment of 1450 bp of contiguousmitochondrial DNA sequence containing thecomplete ND2 gene, five complete tRNAgenes, small portions of the COI gene, and asixth tRNA gene were used in molecularanalysis of each sample. However, no third-position sites from ND2 or COI were used inthe analyses (see below). Twenty-two sampleswere taken from Crawford and Smith (2005)and 14 were taken from Wang et al. (2008)and detailed molecular laboratory techniquesare provided in these two publications. Thenew species described here was referred to as‘‘P. sp. nov. A’’ in Wang et al. (2008). Twoterraranid sequences (Oreobates quixensisand Pristimantis altamazonicus) and one out-group sequence (Ceratophrys cornuta) arepublished for the first time here. In additionto polymerase chain reaction and sequencingprimers listed in Table 2 of Crawford andSmith (2005), we used three previouslyunpublished internal primers for sequencing:TRPf.J (L5551) 59-AGACCAARARCCTT-CAAAGC-39, ALAf.A (L5603) 59-AAGAC-TTGCAGGACATTAACC-39, and ND2r.F1(H4980) 59-ATCTTCCGGATTTGTGTTT-GATT-39.

Resulting DNA sequences were alignedusing the inferred amino acid translations forthe ND2 and COI gene regions, whereas thetRNA genes were aligned using the inferredsecondary structure based on the work of J.

June 2010] HERPETOLOGICA 193

Robert Macey and colleagues (e.g., Macey etal., 1997a–c; Weisrock et al., 2001). We ranthree molecular phylogenetic analysis of ouraligned dataset: a partitioned metropolis-coupled Monte Carlo Markov Chain (MCMC)Bayesian phylogenetic analysis (Rannala andYang, 1996; Yang and Rannala, 1997), amaximum likelihood analysis (ML; Felsen-stein, 1981) and a maximum parsimony (MP;Camin and Sokal, 1965) nonparametric boot-strap evaluation of nodal support (Felsenstein,1985).

For the Bayesian model–based analysis, wepartitioned the data into four groups: onepartition for each codon position (of the ND2gene plus the small fragment of the COI gene)and the combined tRNA genes. We tested thetotal data and each data partition for signifi-cant departure from the assumption ofstationarity using a x2 test implemented inPAUP* 4.0b10 (Swofford, 2000). For each ofour four data partitions, we used a Bayesianinformation criterion as implemented in theperl script, DT-ModSel (version: 13-Aug-02)by Minin et al. (2003) to select the mostappropriate model among the 56 commonlyused models of molecular sequence evolution.Model selection procedures included only in-group taxa.

We performed Bayesian phylogenetic anal-yses using the software, MrBayes v. 3.1.2(Ronquist and Huelsenbeck, 2003). We rantwo simultaneous analyses, each using fourmetropolis-coupled MCMC chains with theheating parameter set at 0.08, and all otherparameters and prior distributions left at theirdefault values. Runs lasted five million gener-ations, with trees sampled every 1000 gener-ations. For comparison, we ran an ML analysison the unpartitioned data set using PAUP*,starting from a neighbor-joining tree andusing a heuristic search with subtree prun-ing–regrafting branch-swapping.

For comparison with Bayesian marginalposterior probability (mpp) support valuesfor each clade, we also conducted a MPnonparametric bootstrap analysis usingPAUP*. MP bootstrap support is regarded asa more conservative estimate of clade supportthan mpp, but under certain conditions it maybe less accurate (e.g., Alfaro et al., 2003). Weemployed heuristic searches with tree bisec-

tion–reconnection branch-swapping and 50random-addition sequence replicates on eachof 2000 bootstrapped data sets. Nodal supportwas estimated from the proportion of pseu-doreplicates that contained a given node.

RESULTS

Morphological characters and molecularphylogenetic analyses (see below) suggest thatpopulations from Serranıa del Sapo represent anew species that belongs to the Pristimantis(Hypodictyon) ridens species series. Thisgroup is characterized by expanded digitaldisks, toe V longer than toe III, absence ofsupernumary tubercles under fingers and toes,absence of toe webs, a coarsely areolate venter,and tympanic membrane and annulus usuallydistinct (Hedges et al., 2008; Lynch, 1976;Savage, 2002). We name this species below.

SPECIES ACCOUNT

Pristimantis adnus, sp. nov.Fig. 1

Holotype.—An adult male (see histologyresults below; MVUP 2255; collector numberAJC 0922) collected by A. J. Crawford,accompanied by Carolina Polanıa, Dr. ChrisJiggins, and other members of the laboratory ofDr. Eldredge Bermingham of the SmithsonianTropical Research Institute. This frog wasfound along a trail approximately 700 m inelevation at the foot of Cerro Pina in theSerranıa del Sapo, above the Rıo Pina on thePacific coast of the Darien Province, Republicof Panama (Fig. 2). Global positioning system(GPS) coordinates were not taken at this point,but elevation was estimated using a wristwatchaltimeter. GPS points were taken both belowthe collecting site (07.67314, 2078.20639,datum WGS 84, 546 m elevation) and aboveit (07.69080, 2078.19803, datum WGS 84,1150 m elevation). The collecting site wasreached by following a ridge up from our basecamp located at the coordinates 07.682,2078.2022 (elevation by GPS: 85 m) next tothe Rıo Pina, 6.2 km upstream from BahıaPina. The holotype was collected in the leaflitter on the ground on 13 May 2003, at 1310 h.Liver and testes were removed from holotypethrough a ventrolateral incision for genetic andhistological analyses, respectively.

194 HERPETOLOGICA [Vol. 66, No. 2

Paratopotype.—CH 8132 (collector num-ber AJC 0924). Collection data as above by A.J. Crawford, but at approximately 800 melevation and at 1220 h. Liver and testes wereremoved from paratopotype through a ven-trolateral incision for genetic and histologicalanalyses, respectively.

Diagnosis.—A member of the Pristimantis(Hypodictyon) ridens species series with the

following qualities: (1) skin on dorsum isshagreen with scattered enlarged granules;small supratympanic ridge present; venterareolate; dorsolateral fold absent; (2) tympanicmembrane not concealed; tympanic annulusfaintly visible; (3) canthus rostralis distinct andconcave; nostrils protuberant; area betweennostrils concave; (4) enlarged supraoculartubercle absent; series of superciliary tuber-

FIG. 1.—Photographs A–D are of holotype specimen MVUP 2255 (collector number AJC 0922) taken during thedaytime. (A) profile, with white arrow indicating the enlarged heel tubercle on the right leg. (B) Dorsum and posteriorthighs (note spots on posterior thighs). (C) Venter (note silver-colored musculature posterior to pectoral girdle). (D)Lateral view including groin (note slight red coloration in groin). Images (E) and (F) are histological sections of (E)MVUP 2255 and (F) CH 8132 (collector number AJC 0924), demonstrating the sex and reproductive status of thesemales. Letters indicate the following structures: spermatogonia (g), spermatids (t), and spermatozoa (z).


cles on edge of upper eyelid weakly devel-oped; cranial crests absent; (5) vomerineodontophores not observed; (6) vocal slitsand vocal sac absent; (7) finger II longer thanfinger I; disks expanded; (8) fingers lackinglateral fringes; (9) ulnar tubercles absent; (10)heel bearing single enlarged but modestlysized tubercle; a small elongate inner tarsal,tuberculate fold present; (11) inner metatarsaltubercle elongate, ovoid and elevated; outermetatarsal tubercle small, conical and round; asmall elongate inner tarsal, tuberculate foldpresent; (12) lateral fringes on toes absent;webbing absent; subarticular tubercles on toesprojecting; supernumary tubercles absent;plantar tubercles absent; third toe longer thanfifth; (13) posterior thigh dark brown to red,with lighter colored red or orange spots; palered or orange spots in groin and anteriorsurface of thighs; (14) SVL in males 19–20 mm; females unknown.

Among Panamanian species, P. adnus issuperficially similar to P. ridens, P. caryophyl-laceus, and P. cruentus of the Pristimantis(Hypodictyon) ridens species series; P. cer-asinus of the Pristimantis (Hypodictyon)rubicundus species series; and P. taeniatus ofthe Pristimantis (Pristimantis) frater speciesgroup (Hedges et al., 2008; Savage, 1981).However, none of these aforementionedspecies has spotting on the posterior thigh,whereas P. adnus shows obvious spotting(Fig. 1B,D). In P. adnus and P. cerasinusthe tip of toe V does not reach the distalsubarticular tubercle of toe IV. Pristimantiscruentus and P. caryophyllaceus have apointed or enlarged supraocular tuberclewhereas P. adnus and P. cerasinus lack apointed or enlarged supraocular tubercle. InP. adnus the series of superciliary tuberclesalong the edge of the upper eyelid is weaklydeveloped (strongly developed in P. cerasi-nus). The canthus rostralis of P. adnus issharper (more angular) than that of P.cerasinus and is longer than that of P.cruentus. The tip of the rostrum of P. adnusis rounded (distinctly pointed in P. caryophyl-laceus). The supertympanic stripe in P. adnusis faint or lacking (dark and distinctive in P.taeniatus of Panama). Although P. adnus andP. ridens can be readily distinguished usingthe above characteristics, these two species do

resemble one another superficially in terms ofdorsal coloration, small size, and silverymusculature visible through the skin on theventral side of the body (posterior to thepectoral girdle in males). Pristimantis adnuspossesses an enlarged pointed heel tubercle,but in P. adnus the heel tubercle is lessobvious than that found in P. cruentus, P.caryophyllaceus, and P. cerasinus. Our reviewof specimens of P. ridens and P. taeniatusshowed that these frogs sometimes have asmall pointed heel tubercle as well, suggestingthat this character may be of limited value inidentifying P. adnus in Panama.

Among Colombian species, P. adnus is mostsimilar to P. thectopternus from the CordilleraOccidental of Colombia (Lynch, 1975), amember of the Pristimantis (Pristimantis)conspicillatus species group (Hedges et al.,2008). However, these two species can bedistinguished as follows: In P. thectopternusthe spots on the posterior thighs are distinctlywhite (dark red-orange in P. adnus), thetympanum is half as wide as the eye (one-third as wide in P. adnus), limb bars on thedorsal surface of the legs are one-fourth thewidth of the gray interspaces (bars andinterspaces have the same width in P. adnus),and a supratympanic stripe is dark and distinct(faint to lacking in P. adnus). Pristimantisroseus from the Pacific lowlands of Colombiaresembles P. ridens but has ‘‘posterior thighcream peppered with brown and marbledwith brown’’ (Lynch, 1980). Both P. ridensand P. roseus have concealed tympana (dis-tinct in P. adnus). Pristimantis adnus may bedistinguished from western Colombian mem-bers of the Pristimantis (Hypodictyon) rubi-cundus species series as follows: Pristimantislanthanites has vocal sac and slits (absent in P.adnus), and a uniform posterior thigh (Lynch,1975; spotted in P. adnus). Pristimantislabiosus from the Pacific lowlands of southernColombia and Ecuador has vocal slits (absentin P. adnus), a conical tubercle on uppereyelid (absent in P. adnus), and very broadfinger and toe disks (Lynch, 1994; less broadin P. adnus). Pristimantis orpacobates fromthe northern end of the Cordillera Occidentalof Colombia (1140–2000 m elevation) has oneto three conical tubercles on upper eyelid(absent in P. adnus) and large finger and toe


disks (smaller in P. adnus), and tiny whiteflecks on posterior thighs (Lynch, 1994;posterior thighs have red-orange spots in P.adnus). Pristimantis ocellatus from southernPacific Colombia lacks a heel tubercle (Lynchand Duellman, 1997; present in P. adnus).Pristimantis w-nigrum has prominent blackspots on flanks, groin, and concealed surfaces(no prominent black spots in P. adnus); theposterior thigh color is black with white spots(brown with red-orange spots in P. adnus, andmales have vocal slits (absent in P. adnus).

Description of the holotype.—A small frog,19.0 mm SVL; head wider than body; headlonger than wide; canthus rostralis distinctand concave; nostrils protuberant; area be-tween nostrils concave; nostrils directed lat-erally and slightly posteriorly; eyes directedlaterally; eye length greater than ND; vocal

slits and vocal sac absent; enlarged supraocu-lar tubercle absent; series of superciliarytubercles on edge of upper eyelid weaklydeveloped, with faint white spots; upper eyelidrugose but lacking an enlarged super-ocular tubercle; eyes do not extend beyondjaw in dorsal view; head round in dorsal viewand truncate in profile; tympanum distinct,ovoid, not concealed, with annulus faintlyvisible; small supratympanic ridge present;one round postrictal tubercle; interobital dis-tance greater than width of upper eyelid;distance of eye to tympanum almost equal todistance from eye to lip; finger length formula I, II , IV , III; tip of finger IV reachesbeyond distal subarticular tubercle on fingerIII; tip of finger II barely reaches distalsubarticular tubercle of finger III; disks onfingers: I and II expanded and even; III and IV

FIG. 2.—Map of Panama and northwestern Colombia, with arrow indicating the type locality of Pristimantis adnus.Elevation in meters indicated by shading. On the western edge of the map lie the Talamanca Mountains of Costa Rica,and the Colombian Andes lie on the eastern edge.


fan shaped; disk pads on fingers: I rounded; II,III, IV broadened; a raised lateral keelextending from base of finger disks to digit;subarticular tubercles distinct and projecting,pungent, and round; supernumary tuberclesabsent; accessory palmar tubercles absent;thenar tubercle elongate and wide, (0.5 mm)one-third length of finger I; palmar tubercledeeply lobed U; toe length formula I, II, III, V , IV; tip of toe V reaches distalsubarticular tubercle on toe IV; tip of toe IIIreaches medial subarticular tubercle on toe IV;disks on toes: I and II expanded and even, IIIand IV fan-shaped, V truncate; pads on toes: I,II, and III broadened, IV and V truncate;subarticular tubercles on toes distinct andprojecting, ovoid and pungent; supernumarytubercles absent; plantar tubercles absent;inner metatarsal tubercle elongate, ovoid andelevated, almost half the length of toe I; lateralfringes absent; outer metatarsal tubercle small,conical and round; a small elongate inner tarsal,tuberculate fold present; a pointed heel calcar;a small lateral skin fold anterior to cloacalopening, much smaller than opening.

Dorsum is shagreen with some scatteredenlarged granules; interocular fold absent; apair of postocular folds extend posteriorlyfrom eye to back; throat texture granular withfaint black pigmentation under scatteredwhite spots; venter coarsely areolate withscattered white spots and sparse dark pigmen-tation.

Histology.—Histological analysis of thegonads confirmed that both type specimensare males. Both individuals were potentiallyreproductive adults at the time they werecollected, as evidenced by the spermatozoa(Fig. 1E,F).

Color in preservative.—Holotype: The orig-inally reddish-brown dorsum (Fig. 1) turned alight ashen gray in preservative; extremitieslighter than trunk dorsally; posterior thighsreddish-brown with pale, slightly orangishspots; dorsal surface of legs with barred withbrown and gray; venter pale with fine dustingof brown pigmentation on anterolateral ofgular area.

Color in life.—The following are taken fromthe field notes of A. J. Crawford written within0–3 days of capture. For holotype (AJC 0922):Ventral abdomen coated in fine white dots,

ventral skin see-through (transparent); anteri-or 40% of abdomen shows silvery-white chestmusculature. Gular ashen grey with mildwhite flecking. Blotchy red-orange on groinand anterior thighs, plus some on ventralthighs and ventral calves. Posterior thighs aredark brown with a few light dots showing red-orange color. Suprascapular ‘‘W’’ pattern oftubercular ridging. No supraocular tubercles.Calcars tiny but clearly visible on heels.Protuberant nares and big head are reminis-cent of P. cerasinus. Iris more maroon thangold-colored. Tissued 16 May 2003.

A. J. Crawford field notes for paratype (CH8132): Ventral abdomen covered in fine whiteflecks but still see-through (transparent);anterior one-third of abdomen shows silverychest muscles. Reddish orange coloration (oris it orangish-pink?) found in groin, anteriorthighs, ventral calves, and even a tiny spot inarmpits. Posterior thighs have same color, too,but localized to lots of dots amid a dark brownbackground color; dark brown color localizedto posterior thighs. Tiny calcar tubercles andtiny supercilliary tubercles. Sharp canthusrostralis forming a concave area between itand upper lip. Nares prominent. Iris is a rosygold color. Pickled 16 May 2003.

Measurements of holotype (and paratype)in millimeters.—SVL: 19.1 (20.0); tibia: 10.8(12.3); HL: 7.5 (7.8); HW: 6.9 (7.2); IOD: 2.0(2.0); E–N: 2.0 (2.4); eye length: 2.3 (2.5);IND: 1.7 (1.6); width of finger IV disk: 0.7(0.8); width of finger III: 0.6 (0.8). ratios(3100%) of holotype: SVL/tibia 5 56; SVL/HL 5 39 IOD/HW 5 28; HL/HW 5 92.

Etymology.—The name of this small spe-cies refers to the Spanish acryonym, ADN, foracido deoxyribonucleico, meaning deoxyribo-nucleic acid (DNA) in English. This epony-mous reference to DNA in Spanish refers tothe great potential for these data-rich mole-cules to accelerate phylogenetic inference,species discovery, and species identification,especially in species-rich clades of morpho-logically homoplastic Neotropical amphibians,such as Terrarana.

Remarks.—The molecular and morpholog-ical analyses reported here suggest that thisnew species be assigned to the Pristimantis(Hypodictyon) ridens species series (Hedgeset al., 2008). This species is currently known


only from the type locality, located in WorldWildlife Fund ecoregion NT0122, the easternPanama montane forest (Olson et al., 2001).Photographic evidence hints that this speciesmight also occur on the Pacific coast ofColombia or on the Serranıa Tacarcuna tothe northeast, but these reports cannot beverified at this time. Both examples of thisspecies were found in the leaf litter during theday, but we do not yet know whether thisspecies should be considered diurnal. Search-es at this elevation were limited to a singleafternoon, so we cannot comment on therelative abundance of this species. Limitedtaxonomic sampling of molecular characterssuggests that the closest relative of P. adnus isP. cerasinus (Fig. 4).

Molecular Phylogenetic ResultsAlthough we found some length variation

among DNA sequences for the ND2-WANCYregion, we inferred that all data came frommitochondrial DNA rather from than pseudo-genes transferred to the nucleus because (1) wedid not observe nonsense mutations in the ND2or COI genes; (2) the light strand showsstrongly biased nucleotide frequencies as ob-served previously in animal mtDNA (Macey etal., 1998; Sperling and Hickey, 1994), especiallyin third position sites (see below); and (3) alltRNA genes appeared to code for tRNAmolecules with functional secondary structures.However, an unusual secondary structurewas observed in all samples of Pristimantis.These samples appeared to be missing 4, 7, or9 bp from the region of the D-stem of thetRNACYS gene, relative to all other samples,including Craugastor, which showed the stan-dard secondary structure (Fig. 3). The standardD-stem of the tRNACYS gene is 9 bp long. TwoPristimantis sequences (GenBank accessionnumbers EU443188 and EU443184) showeda distinct 5-bp sequence in this gene region thatcould potentially form a tiny stem structure.However, these two 5-bp sequences show nosimilarity with each other and no apparenthomology with the other Terrarana sequences(Fig. 3), and may represent novelties.

The null hypothesis of stationarity ofnucleotide frequencies among taxa was reject-ed for the combined data set (P 5 0.0012) andfor third position sites alone (P 5 0.0000), but

not for first (P 5 0.9849) or second positionsites (P 5 1.0) or for the combined tRNAgenes (P 5 1.0). Therefore, we removed thirdposition sites from all phylogenetic analyses(Bayesian, ML, and MP bootstrap). We alsoremoved all gapped sites as well as thehypervariable 39 end of the ND2 gene, whichwas difficult to align even at the amino acidlevel because of the high level of sequenceand length variation (Crawford and Smith,2005). All analyses, therefore, included 968characters, of which 521 were parsimonyinformative and 124 were ‘‘singletons.’’

Based on our model selection results weapplied a six-parameter general time-rever-sible (GTR) model (Tavare, 1986) + C (Yang,1994) to the three partitions included in ouranalysis (positions 1 and 2, plus tRNA genes).In the ML analysis we adopted a single GTR +C model for all sites, as chosen by DT-ModSel. Likelihood topology and MP boot-strap consensus tree topology were identicalto the Bayesian consensus tree topology(Fig. 4), suggesting that the inferred relation-ships are robust to method of inference. Inaddition, most nodes on the tree wereresolved with very high statistical support asmeasured by both MP bootstrap and mpp(Fig. 4). Key to this study, P. adnus came outsister to P. cerasinus with high nodal supportin both analyses. The placement of Oreobatesrelative to other genera was not resolved inthis analysis.

DISCUSSION

The high level of species diversity of theDarien and northern Choco may be explainedin part by the mixing of Central and SouthAmerican faunas during the Great AmericanBiotic Interchange (Marshall, 1988; Savage,1982, 2002; Stehli and Webb, 1985; Vanzoliniand Heyer, 1985). Pristimantis most likelyoriginated in South America, where it is mostdiverse (Duellman, 1999; Heinicke et al.,2007; Lynch, 1971), and spread into CentralAmerica as far north as Honduras (Duellman,2001; Savage, 1982) in the Pliocene with thecompletion of the Isthmus of Panama, orperhaps earlier (Wang et al., 2008).

In addition to its role as a conduit, theIsthmus of Panama has also fostered its ownendemic amphibian fauna, perhaps fostered in


part by the relatively low capacity for migra-tion or dispersal and relatively high populationstructure among amphibians (Beebee, 2005;Crawford, 2003; Vences and Wake, 2007). Inaddition to P. adnus, other endemics includemontane frogs such as Strabomantis laticor-pus, Atelopus glyphus, and Rhinella acrolopha(Frost, 2009). Although the isthmian landbridge was not completed until approximately3 million yr ago (Ma; Coates and Obando,1996), the inchoate Darien began to appeararound 12 Ma or earlier (Kirby et al., 2008),forming long, narrow islands by 6 Ma (Coateset al., 2004). These islands may have allowedcolonization of the Darien from the north orsouth in advance of the completion of the landbridge in the Pliocene (Wang et al., 2008;

Weigt et al., 2005). The increasing number ofknown Darien endemic species of amphibians(e.g., Ibanez and Crawford, 2004) and reptiles(e.g., Myers, 2003) supports the hypothesizedold age of the fauna of eastern Panama.

Comparison of our phylogeny with Hedgeset al. (2008) is not straightforward because onthe one hand the tree presented here hasrelatively scant sampling, whereas on theother hand our tree includes some speciesnot sampled by Hedges et al. (2008). Hedgeset al. (2008) recovered a clade containing P.ridens, P. cruentus, and other members of theP. ridens series as sister to a clade containingmembers of the Pristimantis (Hypodictyon)rubicundus species series. We sampled onepresumed member of the P. rubicundus

FIG. 3.—DNA sequence alignment of the tRNACYS gene based on inferred secondary structure, illustrating theinferred absence of the D-stem structure in Pristimantis, relative to other samples used in this study. Secondarystructure follows Macey et al. (1997a–c). Structural annotations are indicated on the bottom line: amino acid stem (AA),amino acid acceptor base (*), D-stem (D), T-stem (T), and anti-codon (Cys). The length of a given stem structure isindicated by the less-than symbol (,) to the left of the corresponding annotation. (This gene is encoded on the lightstrand, and would be transcribed from right to left.) To aid in visualization, the T-stem and D-stem positions areunderlined, and the anti-codon positions are highlighted in bold. The nucleotides in the D-stem region of twoPristimantis samples could potentially form a diminutive 2-bp stem structure. Note that our phylogenetic analysesexcluded all gapped sites, the D-stem, and positions annotated here with the # symbol. The tRNACYS gene sequencewas unavailable for two individuals: Craugastor fitzingeri (FMNH 257745) and P. cerasinus (FMNH 257713).


series, P. cerasinus, which was not sampled byHedges et al. (2008). In our analysis, P.cruentus is more closely related to P. cerasinusthan to P. ridens, a result that would appear toconflict with that of Hedges et al. (2008),assuming that the samples are correctly

allocated to species series. To restore mono-phyly to these taxonomic groups, we recom-mend that P. cerasinus be moved to the P.ridens series, along with P. adnus. Moving P.cerasinus to the P. ridens series makes P.achatinus the only member of the P. rubi-

FIG. 4.—Bayesian consensus phylogram of four genera of terraranid frogs and two outgroup genera based on 968 bpof mtDNA comprised of first and second position sites of ND2, five tRNA genes, and flanking sites. Bayesian marginalposterior probabilities (mpp) for a given node are indicated above each branch, with maximum parsimony bootstrapsupport (bss) indicated below the branch. A star indicates 1.0 mpp or .94% bss. A circle indicates 0.95–0.99 mpp or84%–94% bss. Note that Pristimantis cruentus is paraphyletic with respect to P. museosus.


cundus series that enters Central America andrestricts the P. rubicundus series to theeastern side of the Panama Canal.

Possible Synapomorphy of Pristimantis

The loss of the D-stem of the tRNACYS

gene was observed previously in acrodontlizards and was suggested to be a synapomor-phy of the clade (Macey et al., 1997b), but hasalso been observed to occur independently inother squamate clades (Macey et al., 1997c).We can readily infer that the absence of theD-stem of the tRNACYS gene also representsthe derived condition within the phylogenypresented here (Fig. 4). Although the taxo-nomic sampling in the present study islimited, this structural feature of the mito-chondrial genome is a potential synapomor-phy of the genus Pristimantis, as well. Wesuggest that changes in secondary structureand other higher-level features of mitochon-drial DNA may serve as a source of importantcharacters useful for resolving deeper-levelrelationships among Terrarana and otheranurans.

Acknowledgments.—We thank the owners and manag-ers of Tropic Star Lodge (http://www.tropicstar.com/) forinviting us to survey their property, and B. and A. Coatesfor organizing the expedition. We thank R. Ibanez for helpin identifying the gonads in these very small frogs, I. Wangfor sequencing the Louisiana State University (LSU)samples, and D. Medina for help reviewing specimens.We thank D. Dittmann and R. Brumfield of LSU and J. P.Caldwell of Oklahoma University for loaning tissuesamples. Many thanks go to the Autoridad Nacional delAmbiente of Panama for their gracious and continuedapproval of our research activities, and to O. Arosemenafor administrative help in acquiring permits. B. Moon, C.J. Raxworthy, J. Padial, one anonymous reviewer, and ElBrujo S. Poe provided much appreciated comments on anearlier draft of this manuscript. Research on live animalsconformed to the Smithsonian Tropical Research Insti-tute’s Animal Care and Use Protocol. AJC was supportedby an National Science Foundation (NSF) InternationalPrograms postdoctoral fellowship during fieldwork, and aSmithsonian Molecular Evolution postdoctoral fellowshipduring laboratory work. MJR was supported by NSFDEB-0844624 to S. Poe and by the Panama Frog RescueProject 2004.

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.

Accepted: 31 January 2010.Associate Editor: Christopher Raxworthy

APPENDIX I

Specimens Examined

Pristimantis adnus (holotype and paratype).—PANA-MA: Darien Province: Serranıa del Sapo, above the RıoPina, 6.2 km upstream from Bahıa Pina, 700 m, MVUP2255 (AJC 0922); 800 m, CH 8132.Pristimantis cruentus (five specimens).—PANAMA:

Cocle Province: Cano Sucio, Santa Marıa, 95 m, CH5369; Parque Nacional General de Division Omar TorrijosHerrera, 700 m, AJC 0568, CH 0387. Comarca Kuna Yala:Nusagandi, 420 m, CH 3452. Darien Province: Cana,Pirre high camp, 1550 m, AJC 0597.Pristimantis cerasinus (nine specimens).—PANAMA:

Bocas del Toro Province: Culubre, 170 m, CH 6519; RıoChanguinola, Guayacan, 245 m, CH 6571. Cocle Province:San Miguel Arriba, 400 m, CH 5098; Parque NacionalGeneral de Division Omar Torrijos Herrera, 800 m, CH6588, 6591. Colon Province: Parque Nacional Sober-anıa, Quebrada Juan Grande, 30 m, CH 7108-9. ComarcaKuna Yala: Nusagandi, 420 m, CH 7102. PanamaProvince: Chilibre District, Altos de Cerro Azul, 600 m,AJC 0971.Pristimantis ridens (six specimens).—PANAMA: Cocle

Province: Tres Hermanas, Rıo Indio, 25 m, CH 4968.Colon Province: Parque Nacional Soberanıa, Rıo Limbo,50 m, CH 4418; Rıo Cocle del Norte, Los Almendros,10 m, CH 5224; Sherman, 70 m, CH 3850; Sierra Llorona,200 m, CH 4836. Panama Province: Cerro Trinidad,875 m, CH 4278.Pristimantis taeniatus (five specimens).—PANAMA:

Darien Province: Trail to Serranıa de Pirre, 700 m, CH5529. Panama Province: Altos de Cerro Azul, 800 m, CH3663, 6796; Lago Alajuela, Quebrada Tranquilla, 80 m,CH 3859; Rıo Chagres, Estacion de la ACP en Rıo Chico,200 m, CH 6854.


APPENDIX

IISp

eciesnames,institutionalvouche

rnu

mbe

rs,fieldtagnu

mbe

rs,localityinform

ation,

andGen

Bankaccessionnu

mbe

rsforallfrog

samples

used

inthisstud

y.MostDNA

sequ

enceswerepreviouslypu

blishe

din

Crawford

andSm

ith(200

5)or

Wanget

al.(20

08).Three

DNAsequ

encesarene

wlypu

blishe

dhe

re:tho

sefrom

Ceratop

hrys

cornuta,

Pristiman

tisaltamazon

icus,andOreobates

quixensis.

Species

Institutionalvouche

rnu

mbe

raField

collectionnu

mbe

rbCollectionlocalityc

Lon

gitude

Latitud

eGen

Bank

accessionno

.

Ceratop

hrys

cornuta

LSU

MZH-174

16JPC

14732

PE

Guajara-M

irim

,Ron

donia,

BR,?m

264

.55

210

.316

GU1687

81Craug

astorau

gusti

MVZ22

6839

RCS12

112-A

BitterLakeNWR,R

oswell,New

Mexico,

US,

1085

m210

4.41

0433

.599

09AY27

3109

Craug

astorbocourti

UTAA-552

35GAR

181

Purulha,BajaVerapaz,GT,?m

290

.164

415

.247

5AY27

3110

Craug

astorbran

sfordii

FMNH

2577

00AJC

0213

Nusagandi,Kun

aYala,

PA,42

0m

278

.983

309

.316

7AY27

3140

Craug

astorda

ryi

UTAA-552

51MEA22

48Sierra

deXucaneb

,AltaVerapaz,GT,?m

289

.78

15.65

AY27

3107

Craug

astorfitzing

eri

FMNH

2577

45AJC

0102

EBLas

Cruces,Pun

tarenas,CR,11

00m

282

.975

08.783

AY27

3117

Craug

astorgollm

eri

FMNH

2575

61AJC

0230

EBFortuna,Chiriqu

ı,PA,11

00m

282

.217

08.75

AY27

3124

Craug

astormegacepha

lus

FMNH

2577

14AJC

0072

EBLaSe

lva,

Hered

ia,CR,10

0m

284

.025

10.417

AY27

3111

Craug

astorno

blei

FMNH

2576

16AJC

0333

PN

Omar

Torrijos,Cocle,PA,80

0m

280

.592

08.667

AY27

3127

Craug

astorpo

diciferus

FMNH

2576

53SJA23

703

EBLas

Cruces,Pun

tarenas,CR,11

00m

282

.975

08.783

AY27

3135

Craug

astorpy

gmaeus

UTAA-552

46ENS95

95Sierra

Madre

delSu

r,Oaxaca,

MX,12

45m

297

.097

516

.194

2AY27

3119

Craug

astorrano

ides

MVZ20

7277

DAG

3071

VulcanCacao,Guanacaste,

CR,11

00m

285

.45

10.933

3AY27

3112

Craug

astorrhod

opis

UTAA-552

31ENS10

024

Jacaltep

ec,Oaxaca,

MX,15

0m

296

.238

817

.863

8AY27

3131

Craug

astorsartori

UTAA-511

05ENS83

28Aldea

LaFraternidad,SanMarcos,GU,?m

291

.881

514

.929

7AY27

3121

Craug

astortabasarae

SIUC

H-069

64KRL89

18PN

Omar

TorrijosH.,Cocle,PA,80

0m

280

.592

08.667

AY27

3115

Craug

astortrachy

derm

usUTAA-485

00ENS67

51Livingston,

Izabal,GU,93

5m

289

.24

15.71

AY27

3106

Pristiman

tisad

nus

MVUP22

55AJC

0922

Rıo

Pina,

Pue

rtoPina,

Darien,

PA,80

0m

278

.202

207

.682

EU4431

91Pristiman

tisad

nus

CH

8132

AJC

0924

Rıo

Pina,

Pue

rtoPina,

Darien,

PA,70

0m

278

.202

207

.682

EU4431

92Pristiman

tisaltae

UCR

16472

AJC

0398

MNH

LaPaz,SanRam

on,Alajuela,

CR,12

30m

284

.558

5510

.182

23EU4431

85Pristiman

tisaltamazon

icus

LSU

MZH-154

67JPC

14628

Rio

Ituxi,Amazon

as,BR,?m

??

GU1687

82Pristiman

tiscerasinu

sFMNH

2577

13AJC

0071

EBLaSe

lva,

Sarapiqu

ı,Hered

ia,CR,75

m284

.007

0010

.430

33EU4431

94Pristiman

tiscruentus

none

yet

AJC

0603

Cana,

PN

Darien,

Darien,

PA,16

00m

277

.716

677.7711

1EU4431

88Pristiman

tiscruentus

UCR

16448

AJC

0458

SanRam

on,Alajuela,

CR,96

0m

284

.596

9810

.218

77EU4431

76Pristiman

tiscruentus

UCR

16443

AJC

0463

SanRam

on,Alajuela,

CR,96

0m

284

.596

9810

.221

00EU4431

86Pristiman

tismuseosus

SIUC

H-069

70KRL88

81PN

Omar


0m

280

.591

678.6666

7AY27

3103

Pristiman

tispa

rdalis

FMNH

2576

75AJC

0188

Fortuna,Chiriqu

ı,PA,10

00m

282

.216

678.7500

0AY27

3102

Pristiman

tispirrensis

CH

5641

AJC

0594

Cana,

PN

Darien,

PA,50

0m

277

.684

057.7560

7EU4431

90Pristiman

tisridens

FMNH

2578

33AJC

0336

Cerro

Cam

pana,Panam

a,PA,90

0m

279

.927

388.6856

4EU4431

59Pristiman

tisridens

FMNH

2576

97AJC

0211

Nusagandi,Panam

a,PA,40

0m

278

.983

309.3167

0EU4431

64Pristiman

tisridens

MVUP17

87KRL06

92PN

Omar


0m

280

.591

678.6666

7EU4431

65Pristiman

tisridens

UTAA-570

17ENS10

727

Agalta,

Olancho

,HN,10

80m

286

.148

0014

.959

00EU4431

54Pristiman

tisridens

FMNH

2577

46AJC

0103

Las

Cruces,Pun

tarenas,CR,60

m282

.975

008.7833

3AY27

3101

Pristiman

tissp.no

v.B

none

yet

AJC

0580

Cana,

PN

Darien,

PA,13

00m

277

.722

257.7635

8EU4431

93Pristiman

tissp.C

none

yet

AJC

0601

Cana,

PN

Darien,

PA,50

0m

277

.684

057.7560

7EU4431

84Euh

yaspa

nton

iUSN

M32

7872

SBH

103516

Hardw

arGap,St.And

rew

Parish,

JM,

276

.72

18.08

AY27

3104

Leptoda

ctylus

fragilis

UTAA-486

66ENS71

04Pue

rtoBarrios,Izabal,GT,50

m288

.71

15.6

AY27

3100


Species

Institutionalvouche

rnu

mbe

raField

collectionnu

mbe

rbCollectionlocalityc

Lon

gitude

Latitud

eGen

Bank

accessionno

.

Leptoda

ctylus

melan

onotus

UTAA-538

17ENS93

68SanRafael,SanMarcos,GT,60

0m

291

.914

.87

AY27

3099

Oreobates

quixensis

LSU

MZH-127

84JPC

1026

4Cuyaben

o,Su

cumbıos,EC,?m

??

GU1687

83Sy

rrho

phus

pipilans

UTAA-510

50JA

C19

141

Nen

ton,

Hue

hueten

ango,GT,?m

291

.842

15.761

AY27

3105

aCH

5Cırculo

Herpe

tologico

dePanam

a,Panam

aCity,Panam

a;FMNH

5Field

Museu

mof

Natural

History,C

hicago,Illino

is,U

SA;L

SUMZ5

Lou

sianaStateUniversityMuseu

mof

Zoology

(NaturalSciences),Baton

Rou

ge,

Lou

isiana,U

SA;M

VUP5

Museo

deVertebrados

delaUniversidad

dePanam

a,Panam

aCity,Panam

a;MVZ5

Museu

mof

VertebrateZoology,B

erkeley,California,USA

;SIU

C5

Southe

rnIllin

oisUniversityat

Carbo

ndale,Illin

ois,

USA

;UCR

5Museo

deZoologıa,

Universidad

deCosta

Rica,

SanPed

ro,Costa

Rica;

USN

M5

NationalMuseu

mof

Natural

History,Washing

ton,

DC,USA

;UTA5

Universityof

Texas

atArlington

,Texas,USA

.bAJC

5And

rewJ.Crawford;DAG

5David

AGood;

ENS5

EricN.Sm

ith;

GAR5

Ron

yGarciaAnleu

;JA

C5

Jonathan

A.C

ampb

ell;JPC

5JanaleeP.Caldw

ell;KRL5

Karen

R.Lips;LDW

5Larry

David

Wilson

(Randy

McC

ranie,

collector);MEA5

Manue

lE.Acevedo

;RCS5

Rob

ertC.Steb

bins;SB

H5

S.BlairHed

ges;SJA5

Stevan

J.Arnold(AJC

,collector).

cMHN

5Mon

umen

toHistorico

Natural;P

E5

Parqu

eEstadual;PN

5Parqu

eNacional;NWR5

NationalW

ildlifeRefug

e.Cou

ntries

areindicatedby

theirISO

3166

two-letter

code

s.Finalnu

mbe

rsindicate

elevationin

meters.

APPENDIX

IICon

tinu

ed.


A NEW SPECIES OF PRISTIMANTIS (ANURA: STRABOMANTIDAE) …

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