Zootaxa 3666 (3): 358–368 www.mapress.com / zootaxa / Copyright © 2013 Magnolia Press
ISSN 1175-5326 (print edition)
Article
ZOOTAXA ISSN 1175-5334 (online edition)
http://dx.doi.org/10.11646/zootaxa.3666.3.6 http://zoobank.org/urn:lsid:zoobank.org:pub:3CC662F2-2EFF-421B-A665-9E4DEA621F38
A new Nototriton (Caudata: Plethodontidae) from Parque Nacional Montaña de Botaderos in northeastern Honduras JOSIAH H. TOWNSEND1,5, MELISSA MEDINA-FLORES1,2, ONÁN REYES-CALDERÓN3 & JAMES D. AUSTIN4 1
Department of Biology, Indiana University of Pennsylvania, Indiana, Pennsylvania 15705–1081, USA Escuela de Biología, Universidad Nacional Autónoma de Honduras, Tegucigalpa, Francisco Morazán, Honduras 3 Departamento Académico de Estudios Generales, Universidad Nacional de Agricultura, Catacamas, Olancho, Honduras 4 Department of Wildlife Ecology and Conservation, University of Florida, Gainesville, Florida 32611, USA Corresponding author. E-mail:
[email protected] 2
Abstract The highlands of northeastern Honduras remain under-characterized in terms of biological diversity, as exemplified by the regularity of new amphibian and reptile taxa discoveries. Following the recent description of a new species of Nototriton from the Sierra de Agalta in northeastern Honduras, we report the discovery of a second new species of Nototriton from the nearby Parque Nacional Montaña de Botaderos. This new taxon, Nototriton mime sp. nov., is distinguished from other Nototriton by its distinctive pale brown dorsal coloration in adult males, relatively large nares, a relatively broad head, mitochondrial sequence divergence, and phylogenetic relationships, and is geographically isolated from other populations of Nototriton. Key words: Nototriton mime sp. nov., mtDNA, 16S, cytochrome b, Nototriton picucha, sexual dichromatism
Resumen Las tierras altas del noreste de Honduras continúan insuficientemente caracterizadas en términos de diversidad biológica, ejemplificado por la regularidad con la que nuevas especies de anfibios y reptiles son descubiertos. Siguiendo la reciente descripción de una nueva especie de Nototriton de la Sierra de Agalta en el noreste de Honduras, reportamos el descubrimiento de una segunda nueva especie de Nototriton, procedente del vecino Parque Nacional Montaña de Botaderos. Este nuevo taxón, Nototriton mime sp. nov., se distingue de otras Nototriton por su distintiva coloración dorsal marrón pálido en machos adultos, orificios nasales relativamente grandes, cabeza relativamente ancha, divergencia en la secuencia mitocondrial y relación filogenética, y se encuentra aislada geográficamente de otras poblaciones de Nototriton.
Introduction Despite the presence of over a dozen isolated tropical montane cloud forests, the highlands of northeastern Honduras remain largely uncharacterized in terms of regional endemic biodiversity (Townsend et al. 2011). Only two cloud forest areas above 1,000 m elevation in northeastern Honduras have been studied in any detail: Parque Nacional Sierra de Agalta (Castañeda 2006) and Parque Nacional La Muralla (Espinal et al. 2001), both in Departamento de Olancho. Despite being one of the herpetologically best-known of cloud forests in northeastern Honduras, a new species of moss salamander (Nototriton picucha, Townsend et al. 2011) was recently described from Parque Nacional Sierra de Agalta. The Sierra de Agalta forms the westernmost portion of a narrow cordillera extending into the lowland plain of La Mosquitia and containing the easternmost 2,000+ m elevation cloud forests in Nuclear Central America (Fig. 1). To the north of the Sierra de Agalta, separated by an arid intermontane basin known as the Valle de Agalta and
358 Accepted by Miguel Vences: 24 Apr. 2013; published: 31 May 2013
its bordering xeric pine-oak forest foothills, lies another mountain range, the Sierra de Botaderos (Fig. 1). A relatively large area of intact broadleaf rainforests between 700 and 1,724 m elevation that straddles the border delimiting the departments of Olancho, Colón, and Yoro was recently declared a new protected area, Parque Nacional Montaña de Botaderos (ICF 2011). Previous biological inventory work in the Sierra de Botaderos was limited to two expeditions, one in 2002 and another in 2005, which led to the descriptions of two new pitviper taxa: Atropoides indomitus Smith & Ferrari-Castro (2008) and Cerrophidion wilsoni Jadin, Townsend, Castoe, & Campbell (2012). In April 2011, the authors participated in the first biological inventory of the highest ridge (1,700–1,724 m elevation) in the Sierra de Botaderos. Our team documented herpetofaunal diversity along the targeted ridge on 16– 17 April 2011, and collected a series of four Nototriton, the adult males of which had a distinctive yellow-brown dorsal coloration. Analyses of molecular and morphological characteristics confirm that these samples represent a previously unknown species-level taxon that apparently exhibits sexual dimorphism with respect to coloration. We present a new phylogenetic hypothesis for Nototriton based on mitochondrial sequence data and describe this new taxon below.
FIGURE 1. Map showing localities mentioned in the text and the distribution of Nototriton in northeastern Honduras. 1) Type locality of N. mime sp. nov., Cerro Ulloa, 1,735 m elevation. 2) Type locality of N. picucha, northwestern slope of Cerro La Picucha, 1,890 m. 3) Type locality of N. lignicola, Cerro de Enmedio, 1,780 m.
Method and materials Taxon sampling. Taxa used in this study and their associated metadata are presented in Table 1. Nototriton taxa known to occur in the Chortís Highlands (the region east and south of the tectonic boundary between the Maya and Chortís blocks and north of the Nicaraguan Depression; Townsend 2011) were included in this study, with N. saslaya, the only northern representative of the N. picadoi group (Townsend et al. 2011; Boza-Oviedo et al. 2012), used as an outgroup. Institutional abbreviations follow those standardized by the American Society of Ichthyologists and Herpetologists (Sabaj-Pérez 2012). Specimens were deposited in the Museum of Vertebrate Zoology, University of California Berkeley (MVZ) and the National Museum of Natural History, Smithsonian Institution (USNM). Forest formations follow Holdridge (1967) as applied by McCranie & Wilson (2002).
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TABLE 1. Samples used in sequence divergence and phylogenetic analyses, with GenBank accession and museum voucher numbers; GT = Guatemala, HN = Honduras, NI = Nicaragua. Taxon
Locality
GenBank voucher GenBank ID 16S
cyt b
N. barbouri
HN: Yoro: Montaña Macuzal
UF 156538
GU971733
GU971734
N. brodiei
GT: Izabal: Sierra de Caral
UTA A-51490
AF199202
AF199139
N. lignicola
HN: Olancho: Sierra de La Muralla
USNM 497540
AF199204
AF199141
HN: Francisco Morazán: Montaña de Yoro
UF 156543
GU971735
GU971736
HN: Santa Bárbara: San Luís Planes
MVZ 263852
JN377383
JQ899197
HN: Comayagua: Azul Meámbar
UF 156539
GU971737
GU971738
HN: Comayagua: Azul Meámbar
UF 156541
JN377386
JN377396
HN: Olancho: Sierra de Botaderos
USNM 579870
KC905090
KC905094
HN: Olancho: Sierra de Botaderos
USNM 579871
KC905091
KC905095
HN: Olancho: Sierra de Botaderos
USNM 579872
KC905088
KC905093
N. limnospectator
N. mime sp. nov.
HN: Olancho: Sierra de Botaderos
MVZ 269306
KC905089
KC905092
HN: Olancho: Sierra de Agalta
USNM 578299
JN377388
JN377392
HN: Olancho: Sierra de Agalta
USNM 578298
JN377389
JN377393
N. sp. A
HN: Atlántida: Quebrada de Oro
USNM 339712
AF199201
AF199136
N. sp. B
HN: Atlántida: Texíguat
USNM 578300
JN377387
JN377391
N. stuarti
GT: Izabal: Montañas del Mico
USAC 3357
JQ899167
JQ899196
N. tomamorum
HN: Yoro: Texíguat
UF 155377
GU971731
GU971732
N. saslaya
NI: Atlantíco Norte: Cerro Saslaya
UF 156352
JN377390
JN377394
N. picucha
DNA extraction, PCR amplification, and sequencing. Whole genomic DNA was extracted from tail tips preserved in SED buffer (20% DMSO, 0.25 M EDTA, pH 7.5, NaCl saturated; Seutin et al. 1991, Williams 2007) using the Qiagen PureGene DNA Isolation Kit (Qiagen, Valencia, CA), following manufacturer’s instructions. Fragments of the mitochondrial genes 16S large subunit RNA (16S) and cytochrome b (cyt b) were amplified using the primers 16Sar-L and 16Sbr-H for 16S (Palumbi et al. 1991) and MVZ15L and MVZ16H for cyt b (Moritz et al. 1992). PCR reactions were 20mL in total volume, containing ~25ng (~1.8 mL) of DNA template, 4 mL 5X PCR buffer, 1.2 mL MgCl2 (25mM), 0.09 mL dNTPs (10 mM), 0.8 mL of each primer (10mM), 0.2 mL GoTaq Flexi polymerase (Promega, Madison, WI, USA), and 11.91 mL H2O. Amplification profiles were as follows: 16S, initial denaturation for 3 minutes at 94°C, 35 cycles of denaturation at 94°C for 45 seconds, annealing at 50°C for 45 seconds, and extension at 72°C for 45 seconds, with a final elongation at 72°C for 5 minutes; and cyt b, denaturation for 3 minutes at 94°C, followed by 38 cycles of 94°C for 30 seconds, 48°C for 1 minute, and 1 minute for 45 seconds, and final elongation at 72°C for 5 minutes. Unincorporated nucleotides were eliminated using 1 uL of ExoSAP-IT (USB, Santa Clara, CA, USA) per 10uL of PCR product. We cycle sequenced complimentary strands using the BigDye Terminator 3.1 Cycle Sequencing kit, followed by spin column filtration through Sephadex before electrophoresing on an ABI 3130xl (Applied Biosystems, Inc). Sequence alignment and analyses. Newly generated sequence data for Nototriton from northern Central America were combined with published data available from NCBI (http://www.ncbi.nlm.nih.gov/) for phylogenetic evaluation of Nototriton samples from Montaña de Botaderos, and included trimmed sequences of 490 bp of 16S and 385 bp of cyt b. Sequences were individually aligned for each gene using ClustalW (Thompson et al. 1994) in the program package MEGAv5.05 (Tamura et al. 2011) using the default parameters, adjusted manually, and concatenated, with a final total aligned length of 875 bp. We partitioned the dataset by gene (16S) and by codon position (1st, 2nd, 3rd) for the protein-coding gene cyt b. Best-fit models of nucleotide evolution were selected for each gene and each partition using jModeltest v2.1 (Posada 2008), which uses PhyML 3.0 (Guindon & Gascuel 2003) for model estimation under a likelihood framework. The number of substitution schemes was set to three to limit the number of models tested to 24, corresponding to the number of different models that can be implemented in MrBayes 3.1.2 (Huelsenbeck & Ronquist 2001).
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For both phylogenetic analyses, the dataset was partitioned by gene for 16S and by codon position for cyt b. Bayesian inference (BI) was performed using MrBayes 3.1.2 (Huelsenbeck & Ronquist 2001), and consisted of two parallel runs of four Markov chains (three heated, one cold) run for 20,000,000 generations and sampled every 10,000 generations, with a random starting tree and the first 4,000,000 generations discarded as burn-in, as determined by graphically examining the log likelihood trace plots for stationarity within the program package Geneious Pro R6.1 (Biomatters 2012) The following models were used for BI partitions: 16S – GTR+G, cyt b (1st and 2nd codon position)—HKY+G, cyt b (3rd codon position) – GTR+G. Maximum likelihood (ML) analysis was carried out on the partitioned dataset using RAxML v7.2.8 (Stamatakis 2006; Stamatakis et al. 2008), with 10,000 bootstrap replicates under the default GTR-GAMMA substitution model. Comparative morphology. Measurements were taken with digital calipers, with finer scale measurements taken with a stereo microscope with an optical micrometer; all measurements rounded to the nearest 0.1 mm. Abbreviations used for morphological measurements are as follows: snout to posterior edge of vent, SL; axilla– groin length, AG; trunk width at mid-body, TW; head length from tip of snout to gular fold, HL; head width taken at maximum, HW; tail length, TL; hind limb length, HLL; forelimb length, FLL; combined forelimb and hind limb lengths, CLL; hind foot width, HFW; nares length, NL; eye length, EL; eye width, EW; interorbital distance, IOD. To negate the effects of allometric differences and allow for comparative studies across taxa, most measurements are standardized by SL. Specimens examined are listed in the Appendix. Comparative data for other taxa not examined by the authors are taken from Good & Wake (1993), Campbell & Smith (1998), Lynch & Wake (1978), McCranie et al. (1998), Ehmcke & Clemen (2000), Köhler (2002), McCranie & Wilson (2002), Savage (2002), García-Paris & Wake (2000) and Wake & Campbell (2000). Color names and numbers used in the description follow Köhler (2012); descriptions of coloration in life were taken from field notes and a series of photographs of the type specimens.
Results Phylogenetic analyses were performed on the concatenated and partitioned 16S/cyt b datasets (Fig. 2), and both BI and ML analyses recovered topologies generally congruent to those in previous studies (Townsend et al. 2010, 2011). Nototriton in the Chortís Highlands are members of six geographically discrete clades (Fig. 2): a northeastern clade (bootstrap score [bs]=98; posterior probability [pp]=1.0), delimited here for the first time, containing N. picucha and four samples from Parque Nacional Montaña de Botaderos; a northern clade (bs=100; pp=1.0), consisting of N. brodiei, N. stuarti, and two unnamed candidate species (N. sp. A and B) presently referred to N. barbouri (sensu lato); a central clade (bs=86; pp=0.99) consisting of N. barbouri (sensu stricto) and N. limnospectator; a N. lignicola clade (bs=100; pp=1.0) and a N. tomamorum clade (single sample), both of whose relationships remain unresolved with respect to the remaining taxa; and N. saslaya (single sample), a member of the otherwise lower Central American N. picadoi clade. Monophyly of the four samples from Parque Nacional Montaña de Botaderos (bs=100, pp=1.0), as well as their sister lineage N. picucha (bs=98, pp=1.0), is unambiguous; one Botaderos sample, MVZ 269306, was excluded from both phylogenetic analyses because the 16S and cyt b sequence fragments were identical to that of USNM 579872. Based on the phylogenetic results, supplemented by morphological data, we herein describe the Parque Nacional Montaña de Botaderos population as a new species.
Systematics Nototriton mime sp. nov. Figures 3A–D Holotype. USNM 579870 (Fig. 3A), an adult male from Cerro Ulloa (15.3833°N, 86.0399°W), Parque Nacional Montaña de Botaderos, 1,705 m, Departamento de Olancho, Honduras, collected 16 April 2011 by M. MedinaFlores, O. A. Reyes-Calderón, and J. H. Townsend; original field number JHT 3400; GenBank accession numbers KC905090 (16S) and KC905094 (cyt b).
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FIGURE 2. Bayesian phylogram showing phylogenetic relationships of Nototriton mime sp. nov. and other northern Central American species of Nototriton. Maximum likelihood bootstrap scores and Bayesian posterior probability values are shown below branches; bootstrap scores below 50 and posterior probabilities below 0.5 are not shown.
Paratopotypes. Three (same collectors as holotype); a female (Fig. 3B; MVZ 269306) and a juvenile (Fig. 3D; USNM 579872), collected 16 April 2011, 1,710 m, 15.3843°N, 86.0396°W; and a male (Fig. 3C; USNM 579871), 17 April 2011, 1,720 m elevation, 15.3874°N, 86.0467°W. Diagnosis. A member of the genus Nototriton diagnosed by possessing 13 costal grooves (>16 costal grooves in Oedipina), hands and feet longer than broad (hands and feet broader than long in Bolitoglossa), and small nares (0.007–0.010 NL/SL; 0.017–0.029 NL/SL in Cryptotriton and Dendrotriton). Distinctiveness of the new species and its assignment to the Nototriton barbouri species group is supported by analysis of sequence fragments from the mitochondrial genes 16S and cyt b (Fig. 2). Nototriton mime is unique among described congeners in the Chortís Highlands by having a yellow-brown dorsal coloration in adult males, as well as relatively longer front and hind limbs (FLL/SL 0.195–0.246, HLL/SL 0.224–0.254; versus 0.142–0.174 and 0.153–0.200 in N. barbouri, 0.148–0.151 and 0.166–0.180 in N. brodiei, 0.137–0.160 and 0.158–0.181 in N. lignicola, 0.156–0.183 and 0.164– 0.211 in N. limnospectator, 0.179–0.191 and 0.204–0.218 in N. picucha, 0.172 and 0.178 in N. stuarti, and 0.160 and 0.197 in N. tomamorum). The new species may be further differentiated from the other species of Nototriton found in the Chortís Highlands as follows: from N. brodiei by having a broader head (HW/SL 0.121–0.138, versus 0.120), larger nares (NL/SL 0.007–0.010, versus 0.004–0.005), a shorter tail (TL/SL 0.698 –1.117, versus 1.420– 1.440), and fewer maxillary teeth (27–46, versus 60–62); from N. lignicola by having a broader head (HW/SL 0.121–0.138, versus 0.103–0.118); from N. limnospectator by having a broader head (HW/SL 0.121–0.138, versus 0.095–0.118) and larger nares (NL/SL 0.007–0.010, versus 0.003); from N. picucha by having a narrower head (HW/SL 0.121–0.138, versus 0.140–0.148); from N. saslaya by having narrower hind feet (HFW/SL 0.044 –
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0.050, versus 0.075–0.091), larger nares (NL/SL 0.007–0.010, versus 0.002–0.003), and a higher number of maxillary (27–46, versus 17–22) and vomerine (12–24, versus 3–11) teeth; from N. stuarti by having a slightly narrower head HW/SL 0.121–0.138, versus 0.138), a shorter tail (TL/SL 0.698 –1.117, versus 1.264), and smaller nares (NL/SL 0.007–0.010, versus 0.012); and from N. tomamorum by having well-developed digits with subdigital pads (versus hands and feet syndactylous and lacking subdigital pads), a narrower head (HW/SL 0.121– 0.138, versus 0.145), and smaller nares (NL/SL 0.007–0.010, versus 0.018).
FIGURE 3. Type series of Nototriton mime sp. nov. A) Male holotype (USNM 579870). Female paratype (MVZ 269306). Male paratype (USNM 579871). Juvenile paratype (USNM 579872). Photographs by JHT.
Description of holotype. An adult male (SL = 31.8 mm, total length = 67.3 mm) with a slender body and reduced limbs. The head is rounded, slightly broader than the body; nostrils are relatively small (NL/SL=0.010),
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and the snout is acutely rounded and of moderate length. Nasolabial grooves are visible but slight, and nasolabial protuberances are not apparent. The eyes are relatively large and protuberant, and the elongate parotoid glands are relatively flat and not well-defined. There are 46 maxillary teeth, 3 slightly enlarged premaxillary teeth in line with the maxillaries, and 24 vomerine teeth. The limbs are short (CLL/SL=0.40), with the adpressed limb interval approximately 4.5 costal grooves. The hands and feet are narrow with well-developed digits that bear poorly developed subdigital pads. The relative length of the digits is I
