BREVIORA Museum of Comparative Zoology US ISSN 0006-9698 CAMBRIDGE, Mass. 17 OcrosBerR 2011 NUMBER 526 A NEW CRYPTIC SPECIES OF SALAMANDER, GENUS OEDIPINA (CAUDATA: PLETHODONTIDAE), FROM PREMONTANE ELEVATIONS IN NORTHERN NICARAGUA, WITH COMMENTS ON THE SYSTEMATIC STATUS OF THE NICARAGUAN PARATYPES OF O. PSEUDOUNIFORMIS BRAME, 1968 JAVIER SUNYER,! JosIAH H. TOwNSEND,? DAvip B. WAKE, Scott L. TRAVERS,*-6 SERGIO C. GONZALEZ,4 LENIN A. OBANDO,! AND ARDIEL Z. QUINTANA® ABSTRACT. We describe a new species of Oedipina (subgenus Oedipina) from premontane elevations of three isolated mountains in northern Nicaragua. The new cryptic species differs in molecular characters from its closest relatives: Oedipina cyclocauda (an Atlantic lowland species with a distributional range from central Panama to extreme southeastern Nicaragua) and Oedipina pseudouniformis. We regard all Nicaraguan specimens previously referred to as O. pseudouniformis to be conspecific with the new species herein described and restrict O. pseudouniformis as a Costa Rican endemic species. We also record the fourth known locality (and southernmost) of the Nicaraguan endemic O. nica and discuss additional species of Oedipina that are likely to be found in Nicaragua as field research continues in the country. RESUMEN. Describimos una nueva especie de Oedipina (subgénero Oedipina) de alturas premontanas de tres montamas aisladas del norte de Nicaragua. La nueva especie criptica difiere en caracteres moleculares de sus parientes mas cercanos: Oedipina cyclocauda (una especie de las tierras bajas del Atlantico entre el centro de Panama y el ‘Museo Herpetologico de la UNAN-Leén (MHUL), Departamento de Biologia, Facultad de Ciencias y Tecnologia, Universidad Nacional Autonoma de Nicaragua-Leon, Leon, Nicaragua; e-mail: javier_sunyer@yahoo.com; lenin_obando@yahoo.com *School of Natural Resources and Environment, University of Florida, Gainesville, Florida 32611, U.S.A., and Centro Zamorano de Biodiversidad, Escuela Agricola Panamericana Zamorano, Departamento de Francisco Morazan, Honduras; e-mail: jtwnsnd@ufl.edu Museum of Vertebrate Zoology and Department of Integrative Biology, 3101 VLSB, University of California Berkeley, California 94720-3160, U.S.A.; e-mail: davidbwake@gmail.com 4 Department of Wildlife Ecology and Conservation, and Florida Museum of Natural History, University of Florida, Gainesville, Florida 32611, U.S.A.; e-mail: stravers@ufl.edu; sergeman@ufl.edu >Hebron 3, 35018 Las Palmas de Gran Canarias, Islas Canarias, Spain; e-mail: aquintan@gmx.de © Present ApprESss: Department of Biology, Villanova University, 800 Lancaster Avenue, Villanova, Pennsylvania 19085, U.S.A. © The President and Fellows of Harvard College 2011. 2 BREVIORA No. 526 extremo sureste de Nicaragua) y O. pseudouniformis. Consideramos a todos los especimenes nicaragiienses previamente referidos como Oedipina pseudouniformis representantes de la nueva especie aqui descrita y a O. pseudouniformis una especie endéemica de Costa Rica. También registramos la cuarta localidad conocida (y mas al sur) de la salamandra endémica O. nica y discutimos acerca de especies adicionales de Oedipina candidatas a ser encontradas en Nicaragua a medida que se continue investigando en el pais. KEY WORDs: Despite their remarkably conserved external morphology, Neotropical worm salamanders (Caudata: Plethodontidae: Oedipina) have been shown in _ recent phylogenetic studies to exhibit a surprising degree of evolutionary diversity (Garcia- Paris and Wake, 2000; McCranie ef al, 2008). Most recently, Sunyer et al. (2010) described a new species of Oedipina of the cryptic subgenus Oéeditriton (O. nica) from three isolated highlands in north-central Nicaragua. The description of O. nica marked the first in a series of papers dealing with the systematics of various unresolved populations of Oedipina from around the country. The present contribution is the second of these papers and addresses the systematic status of three additional isolated populations of Oedipina (two of which had previously been assigned to Oedipina pseu- douniformis) from northern Nicaragua. The taxon O. pseudouniformis has been reported in Nicaragua from two isolated premontane localities (Brame, 1968; Kohler et al., 2004). In his seminal revision of the genus, Brame (1968) included eight para- types of O. pseudouniformis based on a series collected in July 1957 from “Hacienda La Cumplida, 1.5 km north of Matagalpa, 731 m elevation,’ Dept. Matagalpa, Nicaragua. Premontane elevations in this area have undergone severe human alteration in the last five decades, and cattle and agriculture (mostly coffee plantations) have isolated the few remaining forest patches to extreme upper portions of the surrounding mountain peaks. Kohler et al. (2004) additionally 16S; cyt b; mtDNA; environmental modeling; Oedipina koehleri sp. nov.; Oedipina nica; Parque Nacional Cerro Saslaya; Reserva Natural Cerro Musun reported two specimens referred to O. pseudouniformis from pristine forest between approximately 600 and 945 m elevation in Parque Nacional Cerro Saslaya, Region Autonoma Atlantico Norte, Nicaragua. Although we have so far failed to secure fresh samples of Oedipina from premontane elevations in the surroundings of Hacienda La Cumplida in Matagalpa, we recently collected an additional specimen from pre- montane elevations at Parque Nacional Cerro Saslaya, as well as two specimens of Oedipina from Reserva Natural Cerro Mu- sun, Dept. Matagalpa, Nicaragua. Although external morphology has been unhelpful in elucidating taxonomic assignments for these populations, phylogenetic analysis of data from two mitochondrial genes (cytochrome b [cyt b] and 16S) demonstrates that the Saslaya and Musun populations represent a single undescribed species in the subgenus Oedipina, a sister of a clade of Costa Rican taxa Oedipina cyclocauda and O. pseudouni- formis. We herein provide a taxonomic description of this new species, evaluate its phylogenetic relationships within the genus Oedipina, and further comment on our understanding of Nicaraguan populations of Oedipina. MATERIAL AND METHODS Taxon Sampling. We formulated a new phylogenetic hypothesis for the genus Oedi- pina, and included representatives of both the Saslaya and Mustn populations of the new species, as well as all congeners available 2011 A NEW CRYPTIC SPECIES OF OEDIPINA FROM NICARAGUA 3 TABLE 1. LOCALITY, VOUCHER, AND GENBANK ACCESSION NUMBERS FOR TAXA AND SAMPLES USED IN PHYLOGENETIC ANALYSES. Taxon Locality Voucher 16S Cyt b Nototriton barbouri Honduras: Atlantida UF 156538 GU971733 GU971734 Oedipina alleni Costa Rica: Puntarenas MVZ 190857 AF199207 AF199149 Oedipina carablanca Costa Rica: Limon No voucher FJ196862 FJ196869 Oedipina collaris Panama: Coclé SIUC H 8896 FJ196863 FJ196870 Oedipina complex Panama: Colon MVZ 236255 AF199213 AF199157 Oedipina cyclocauda Costa Rica: Heredia MVZ 138916 AF199214 AF199158 MVZ 203747 AF199215 AF199159 Oedipina elongata Guatemala: Izabal UTA A-51906 AF199216 AF199160 Oedipina gephyra Honduras: Yoro UF [JHT2443] JN190930 JN190936 Oedipina gracilis Costa Rica: Heredia MVZ 210398 AF 199219 — Oedipina grandis Costa Rica: Puntarenas MVZ 225904 FJ196864 AF199164 Oedipina ignea Honduras: Ocotepeque USNM 530586 AF199231 AF199192 Oedipina kasios Honduras: Olancho MVZ 232825 FJ196866 FJ196872 Honduras: Francisco Morazan UF 156500 HM113477 HM113484 Oedipina koehleri Nicaragua: Atlantico Norte UF 156456 JN190926 JN190933 sp. nov. Nicaragua: Matagalpa SMF 90079 JN190927 JN190934 Oedipina leptopoda Honduras: Yoro MVZ 167772 — AF199193 Oedipina maritima Panama: Bocas del Toro MVZ 219997 AF199221 AF199166 Oedipina nica Nicaragua: Matagalpa MHUL 003 JN190928 _ Nicaragua: Jinotega MVZ 263774 HM068306 HM068303 UF 156447 HM113474 HM113482 UF 156453 HM113475 HM113483 Oedipina pacificensis Costa Rica: Puntarenas UCR 12063 AF199222 AF199169 Oedipina petiola Honduras: Atlantida USNM 343462 AF199217 AF199161 Oedipina poelzi Costa Rica: Alajuela MVZ 181235 -— AF199172 MVZ 206398 AF199223 AF199171 Oedipina Costa Rica: Cartago MVZ 203749 AF199227 AF199178 pseudouniformis MVZ 181229 — AF199179 MVZ 190852 -- AF199177 Oedipina quadra Honduras: Gracias a Dios MCZ 232824 FJ196865 FJ196871 Oedipina savagei Costa Rica: Puntarenas UCR 14587 AF199209 AF199152 Oedipina sp. 1 Nicaragua: Jinotega SMF 78738 FJ196868 ~— Oedipina stenopodia Guatemala: San Marcos MVZ 163649 AF199228 AF199181 Oedipina taylori Guatemala: Zacapa USAC 1134 HM068304 HM068302 Oedipina tomasi Honduras: Cortés UF 150066 JN190929 JN190935 Oedipina uniformis Costa Rica: Cartago MVZ 203751 AF199230 AF199190 on GenBank (http://www.ncbi.nlm.nih.gov); species, locality, and voucher information for these taxa are summarized in Table 1. Acronyms for museum collections follow those of Leviton et al. (1985), except MHUL (Museo Herpetologico de la UNAN-Leon, Universidad Nacional Autonoma de Nicara- gua—Leon, Leon, Nicaragua), N field num- bers, which correspond to specimens col- lected in Nicaragua between 2007 and 2008 by University of Florida field teams in collaboration with the UNAN-—Leon, and JHT (field series of the second author), which is used for a specimen of Oedipina gephyra donated to the University of Florida (UF) in May 2009 that remains uncataloged. Nototriton barbouri, a member of the sister genus to Oedipina (Garcia-Paris and Wake, 2000; Wiens et al, 2007), was used as an outgroup. 4 BREVIORA Morphological Examinations. Measure- ments follow those used in McCranie et al. (2008). All measurements are in millimeters (mm) and made to the nearest 0.1 mm with dial calipers and a dissecting microscope with ocular micrometer. SL is the distance from the tip of the snout to the posterior angle of the vent. Males were determined by the presence of mental glands behind the tip of the mandible and small papillae in the anterior part of the vent and females by the presence of folded vent margins and absence of mental glands and vent papillae. Maxil- lary and vomerine tooth counts are totals of the paired bones. Limb interval equals the number of costal interspaces between ad- pressed limbs. We provide a list of the Nicaraguan comparative specimens exam- ined in the Appendix. DNA Extraction and Sequencing. Template DNA was extracted from tissue with a Qiagen PureGene DNA Isolation Kit (Qia- gen, Valencia, California) following manu- facturer’s instructions. Fragments of two mitochondrial genes were targeted for ampli- fication: a 516-bp fragment of the 16S large subunit rRNA (16S) was amplified using primers 16Sar-L and 16Sbr-H (Palumbi et al., 1991), and a 692-bp fragment of cyt b was amplified using primers MVZ15 and MVZ16 (Moritz et al., 1992). Conditions for PCR did not differ between genes, with initial dena- turation at 94°C for 3 min, 35 amplification cycles of 45 s denaturation at 94°C, 45 s annealing at 50°C, 45 s extension at 72°C, and a final extension of 5 min at 72°C. Samples were cleansed of unincorporated dNTPs through application of USB Exo- SAP-IT before following standard sequenc- ing protocol on an ABI 3130xl automated sequencer (Applied Biosystems, Foster City, California) at the University of Florida WEC/SFRC Molecular Ecology Lab. Phylogenetic Analyses. Cytochrome b se- quences were trimmed to 385 bp to match No. 526 available sequences in GenBank from other studies (Garcia-Paris et al., 2000; McCranie et al., 2008; Sunyer et al, 2010), and sequence alignment was estimated using MAFFT (Katoh et al., 2002). Bayesian phylogenetic analysis was performed in MrBayes v3.1.2 (Huelsenbeck and Ronquist, 2001), with sequence data partitioned by gene for 16S and by codon position for cyt b. Using the Akaike Information Criterion in MrModeltest2.2 (Nylander, 2004), we se- lected the nucleotide substitution model GTRG+I+I for 16S, and GTR+I+I, GTR+T, and HKY+I for first, second, and third codon position of cyt b, respectively. Bayes- ian analysis consisted of two parallel runs of four chains, run for 20 X 10° generations and sampled every 1,000 generations, with the first 4,000 samples discarded as burn-in. The remaining 16,001 post—burn-in trees from both runs were used to generate a 50% majority rule consensus tree. Cumulative and sliding window plots of split frequencies and the correlation of split frequencies in first versus second runs were visualized in Gen- eious v4.8 (Drummond et al., 2009) to assess convergence around posterior quantities. Maximum likelihood (ML) phylogenetic analysis was conducted in RAxML v/7.2.6 (Stamatakis, 2006) using the same data partitions as the Bayesian analysis and the GTR+I model for each partition; 1,000 bootstrap replicates were performed using the rapid bootstrapping algorithm. Macroecological Modeling. We created an environmental niche model based on the localities of Oedipina koehleri using the maximum entropy method (Phillips et al., 2006) as implemented in the program Max- Ent, to determine whether a resulting distri- bution model would predict occurrence of O. koehleri at Hacienda La Cumplida, Dept. Matagalpa, the locality given for eight paratypes of O. pseudouniformis (Brame, 1968). Using presence-only data (i.e., local- 2011 ities of occurrence), MaxEnt uses the princi- ple of maximum entropy density estimation to generate a probability distribution (Phil- lips et al., 2006; Phillips and Dudik, 2008). It has been shown to produce more accurate models with lower sample sizes than other niche-modeling methods (Elith et al., 2006; Hernandez et al, 2006). We used the 19 WorldClim Current BioClim climate layers, which have a resolution of 30 arc-seconds (about 1 km2). These layers are based on data from 1950 to 2000 and include variables reflecting annual, as well as seasonal, climat- ic trends and extremes of precipitation and temperature (Hymans et al., 2005). We did not make any assumptions of correlation among these variables and thus chose to use the entire set of environmental layers. We clipped the WorldClim layers in ESRI ArcGIS 9.3 to our working extent before using them in MaxEnt. A model representing the probability of occurrence of O. koehleri was produced in MaxEnt using a cross- validation approach for our specimen local- ities. The cross-validation function splits the data set into nm samples of one case that are individually tested against all remaining samples, which become the training set of localities during each run (Araujo ef al., 2005). This eliminated the need to partition a data set into large training and testing sets. In this case, this approach was necessary because splitting the data would have result- ed in a training set of insufficient size. The final mean logistic output of the model runs was used to assess our results. The area under the curve (AUC) of the receiver operating characteristic plot was used to evaluate model performance. SYSTEMATIC ACCOUNT Oedipina koehleri sp. nov. Figure | Oedipina pseudouniformis (Brame, 1968) in part: Brame (1968:25—28, fig. 20). Paratypes A NEW CRYPTIC SPECIES OF OEDIPINA FROM NICARAGUA 5 UMMZ 119523(1-8) from “Hacienda La Cumplida, 1.5 km N of Matagalpa, elevation 731 m (2,400 ft), Dept. Matagalpa, Nicara- gua’; Villa (1972:51—54); Villa (1983:21); Villa et al. (1988:7-8); Kohler (1999:11); Kohler (2001:23); Ruiz and _ Buitrago (2003:36-37); Kohler et al. (2004:18); Sunyer and Kohler (2010:500); Kohler (2011:86). Holotype. UF 156456, an adult male, from Parque Nacional Cerro Saslaya (along the trail from “Las Guardiolas” to “El Reve- nido’’), 13°42.8’N, 85°01.9'W, 724 m above sea level (a.s.l.), Region Autonoma Atlantico Norte, Nicaragua, collected 1 August 2008 by Scott L. Travers, Stephen Doucette-Rtise, Sergio C. Gonzalez, Atanasio Baldonado, and Ignacio Cruz (original field num- ber N614). Paratypes. Four; SMF 82225, an adult male, from the southern slope of Parque Nacional Cerro Saslaya (along the trail from Campamento “Las Pavas” [13°44.5’N, 85°01.5'W] to Campamento “Los Monos” [13°45.1’N, 85°02.2’W)), 945 ma.s.l., Region Autonoma Atlantico Norte, Nicaragua; SMF 82874, an unsexed juvenile, from the southern slope of Parque Nacional Cerro Saslaya (along the trail from Campamento “El Carao” [13°42.8'N, 84°58.7’W] to Campamento “Las Pavas’ [13°44.5’N, 85°01.5’W]), 400-600 m as.l., Region Autonoma Atlantico Norte, Nicaragua; SMF 90078-79, both adult females, from the southern slope of Reserva Natural Cerro Mustin (FUNDENIC Cabins [12°57.3’'N, 85°13.9'W]), 628 m a.s.l., Dept. Matagalpa, Nicaragua. Referred Specimens. Eight; UMMZ 119523, four adult males and four adult females, all from Hacienda La Cumplida, 1.5 km N of Matagalpa, 731 m a.s.l., Dept. Matagalpa, Nicaragua. Diagnosis. A slender species of moderate size (largest referred specimen 50.4 mm SL) and robustness assigned to the genus Oedi- 6 BREVIORA Figure 1. No. 526 Dy ak Bae a Adult female paratypes of Oedipina koehleri (a) SMF 90078 and (b) SMF 90079 from Reserva Natural Cerro Mustn; and (c) juvenile paratype of O. koehleri (SMF 82874) from Parque Nacional Cerro Saslaya. pina based on the presence of more than 13 costal grooves between the short limbs, and tail much longer than head plus body. This species is a member of the subgenus Oedipina (based on molecular data; Fig. 2) and is distinguished from its closest relatives, O. pseudouniformis, in having fewer maxillary teeth (mean 38 vs. 49.8 in males; 39.8 vs. 45 in females) and vomerine teeth (mean 20.5 vs. 25.9 in males; 22.2 vs. 25.7 in females), and O. cyclocauda in having a more rounded snout, a broader head, and somewhat longer 2011 A NEW CRYPTIC SPECIES OF OEDIPINA FROM NICARAGUA 7 100/1.0 O. pseudouniformis (MVz 181229) BEA O. pseudouniformis (MVz 190852) 92/1.0 O. pseudouniformis (mvz 203749) ee/to 100/1.0 4 O. cyclocauda (MVZ 203747) O. cyclocauda (Mvz 138916) 100/1.0 O. koehleri sp. nov. (Cerro Saslaya; Holotype, UF 156456) O. koehleri sp. nov. (Cerro Mustn; Paratype, SMF 90079) O. leptopoda (mvz 167772) S 0.69 O. collaris (S\UC H-8896) a 52/0.75 O. grandis (mMvz 219593) my O. poelzi (MVzZ 206398) 1) 61/0.88 O. uniformis (MVz 203751) 74/0.95 O. pacificensis (UCR 12063) O. gracilis (MVZ 203753) 202 Oedipina sp. 1 (SMF 78738) eee O. stenopodia (Mvz 163649) 62/0.97 O. ignea (USNM 530586) O. taylori(USCG 1134) Bea] 100/1.0 96/1.0 O. gephyra (UF JHT2443]) (aa sy YAO O. petiola (USNM 343462) 0.71 O. tomasi (UF 150066)