This article was downloaded by: [University of Oxford] On: 24 February 2009 Access details: Access Details: [subscription number 789072836] Publisher Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Studies on Neotropical Fauna and Environment Publication details, including instructions for authors and subscription information: http://www.informaworld.com/smpp/title~content=t713817190 The Golden Tree Frog of Trinidad, Phyllodytes auratus (Anura: Hylidae): systematic and conservation status Michael J. Jowers ab; J. R. Downie b; B. L. Cohen a Institute of Biomedical and Life Sciences, Division of Molecular Genetics, University of Glasgow, Glasgow, UK b Institute of Biomedical and Life Sciences, Division of Environmental and Evolutionary Biology, University of Glasgow, Glasgow, UK a First Published:December2008 To cite this Article Jowers, Michael J., Downie, J. R. and Cohen, B. L.(2008)'The Golden Tree Frog of Trinidad, Phyllodytes auratus (Anura: Hylidae): systematic and conservation status',Studies on Neotropical Fauna and Environment,43:3,181 — 188 To link to this Article: DOI: 10.1080/01650520801965490 URL: http://dx.doi.org/10.1080/01650520801965490 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material. Studies on Neotropical Fauna and Environment Vol. 43, No. 3, December 2008, 181–188 ORIGINAL ARTICLE The Golden Tree Frog of Trinidad, Phyllodytes auratus (Anura: Hylidae): systematic and conservation status Michael J. Jowersa,b*, J. R. Downieb and B. L. Cohena a Institute of Biomedical and Life Sciences, Division of Molecular Genetics, University of Glasgow, Glasgow, UK; bInstitute of Biomedical and Life Sciences, Division of Environmental and Evolutionary Biology, University of Glasgow, Glasgow, UK Downloaded By: [University of Oxford] At: 16:10 24 February 2009 (Received 6 December 2006; accepted 4 February 2008) Analyses of mitochondrial 12S and 16S rDNA sequences lead us to propose that the Neotropical hylid genus Phyllodytes is paraphyletic. The level of divergence between the Trinidadian endemic P. auratus and the two Brazilian Phyllodytes included in the analyses (Phyllodytes sp. and P. luteolus) is greater than that of inter-generic distances within the Lophiohylini. The molecular evidence here reported, behavioural differences and restriction to a single host plant in a geographically limited area differentiates Phyllodytes auratus from other supposed Phyllodytes evidencing its unique taxonomic status. We therefore propose a new genus, Phytotriades gen. nov., for Phyllodytes auratus. P. auratus mitochondrial cytochrome b sequences obtained from one locality (seven individuals) contained two haplotypes, one individual differing by a single transition. The other locality (one individual) had the commoner haplotype. The low genetic divergence between the two populations suggests recent isolation at these two localities. Keywords: DNA; Golden Tree Frog; Phyllodytes auratus; Phytotriades gen. nov.; Trinidad Introduction Phyllodytes auratus (Boulenger, 1917), the Golden Tree Frog of Trinidad, has been reported from the two highest peaks of the island’s Northern Range, El Tucuche and Cerro del Aripo. Kenny (1969) found this species only on El Tucuche at elevations higher than 600 m above sea level and put forward evidence, from his own observations, suggesting that it was absent from four other adjacent peaks with adequate altitude. However, Read (1982) was first to find the frog on a second mountain, Cerro del Aripo. Murphy’s (1997) review of the amphibians of Trinidad reported Phyllodytes auratus from El Tucuche, Cerro del Aripo ‘‘and probably Morne Bleu Ridge’’. Clarke et al. (1995), in a paper not known to Murphy (J. Murphy, personal communication), recorded the Golden Tree Frog only on El Tucuche and Cerro del Aripo. They surveyed two other mountains, Morne Bleu Ridge and Chaguaramal, but both lacked the specific vegetation apparently required by the frog, though Kenny (1969) had found suitable vegetation on these peaks 30 or so years earlier. The vegetation required by Phyllodytes auratus is the epiphytic giant tank bromeliad Glomeropitcairnia erectiflora. All reported sightings of the frog have been from on or within this plant, which can hold considerable quantities of water. Clarke et al. (1995) opened 27 G. erectiflora on El Tucuche and found a mean water content of nearly 700 ml. Six of these contained Golden Tree Frog adults and/or tadpoles. At Cerro del Aripo, of 13 G. erectiflora opened over 2 years, six contained Golden Tree Frog adults and/or tadpoles (Clarke et al. 1995). The Golden Tree Frog’s requirement for bromeliads containing large volumes of water presumably relates to the frog’s reproductive mode. Clutch size is unknown but there is evidence that it is small. Kenny (1969) reported a maximum of five tadpoles per bromeliad tank and Clarke et al. (1995) reported a maximum of six. S. Rudd (personal communication) found a clump of four eggs stuck to a leaf by their jelly coats. On hatching, tadpoles are 14 mm long (J.R. Downie, unpublished data). They grow to a maximum length of 40 mm (Kenny 1969), which may take some time given the limited resources available in a bromeliad tank. The Global Amphibian Assessment (GAA) (Hardy 2004) rated Phyllodytes auratus as Critically Endangered on the basis of its highly restricted range, but also because of ‘‘continuing decline in extent and quality of its habitat’’. Although the GAA was unaware of any conservation measures, the frog’s main location, El Tucuche, is both part of a game *Corresponding author. Michael J. Jowers, Loc. Pischinazza 10, Trinitá D’Agultu, O-T, Sardegna, Italy. Email: michaeljowers@ hotmail.com ISSN 0165-0521 print/ISSN 1744-5140 online # 2008 Taylor & Francis DOI: 10.1080/01650520801965490 http://www.informaworld.com Downloaded By: [University of Oxford] At: 16:10 24 February 2009 182 M. J. Jowers et al. sanctuary and a prohibited area where access requires government permission (N. N-Gyan, personal communication): however, it would be fair to say that policing access to the mountain is not easy. Given that the Golden Tree Frog now exists as two small nearby isolated populations (El Tucuche and Cerro del Aripo are about 15 km apart), we expected that these are remnants of a recently larger population and thus to find low genetic divergence between the two populations. Another aim of the work reported here was an assessment of the phylogenetic status of the Golden Tree Frog. The species is placed in the genus Phyllodytes. The genus currently contains 12 species, divided into four species groups, on the basis of color patterns (Peixoto et al. 2003; Caramaschi et al. 2004; Caramaschi & Peixoto 2004). Apart from P. auratus, all occur in Brazil (Caramaschi & Peixoto 2004; Caramaschi et al. 2004; Cruz et al. 2006). Based on the unusual generic biogeography (to the best of our knowledge there is no other genus within the family Hylidae with a similar distribution) and P. auratus’s morphological and behavioral differences from all other Phyllodytes species, we hypothesized that current classification of P. auratus is likely to be erroneous. In this paper we assessed the relationships of P. auratus using a molecular phylogenetic approach which has proved to be a useful tool in anuran taxonomy (Faivovich et al. 2005; Manzanilla et al. 2007). Hitherto, the assemblage of these taxa into a single genus has not been based upon either wellestablished morphological synapomorphies or molecular divergence, but upon variable characters that could prove to be unreliable, such as bromeliad use and the presence of odontoids (‘‘fangs’’) on the mandible, nowadays known as a homoplasious character subject to sexual selection (Fabrezi & Emerson 2003). Phyllodytes auratus is distinguished from other Phyllodytes by its restriction to cloud forest woodland sites above 600 m elevation, by the unique presence of longitudinal colored dorsal stripes (Bokermann 1968) and by its apparent lack of vocalization. Clarke et al. (1995) and Read (1982) reported spending several nights in total in the Golden Tree Frog’s habitat without hearing it call. Faivovich et al.’s (2005) review of the Hylidae followed Caramaschi et al. (2004) in recognizing Phyllodytes as a single taxonomic unit, but they were able to include in their molecular analysis only two Brazilian species, P. luteolus and Phyllodytes sp. Since their sample did not include P. auratus they were unable to provide a molecular test of monophyly. In this paper, in addition to molecular analyses using mitochondrial rRNA markers of samples from the two Trinidad locations of P. auratus, we provide a comparison with 10 genera within the Lophiohylini (Faivovich et al. 2005) including the two Brazilian Phyllodytes species used in Faivovich et al. (2005). Materials and methods Study sites and specimen collection Phyllodytes auratus was collected in August 2003 from leaf axils of tree-growing bromeliads (Glomeropitcairnia erectiflora) approximately 1–4 m above the ground, near the summits of El Tucuche (937 m above sea level) and Cerro del Aripo (940 m above sea level) (Figure 1). Sampling was done around the summit of El Tucuche (within 30 m from the 150 m2 cleared area), for 2 h. Because we did not have authorization to cut down G. erectiflora at any of the sites, sampling had to be carried out by gently pulling back the bromeliad leaves. When disturbed, P. auratus adults leaped out of the plants and were then hand caught. Seven adults were caught from ,20 G. erectiflora at El Tucuche and we took toeclips from them. Cerro del Aripo was accessed through the Morne Bleu Ridge, but no G. erectiflora or P. auratus were seen there. Most bromeliads growing within 4 m from the ground were sampled (,20) at the summit of Cerro del Aripo or in the surrounding area but only one P. auratus tadpole was found after a 2 h search and a clip of its tail was taken. Tissue samples were preserved immediately Figure 1. Map of Trinidad with the localities where Phytotriades auratus comb. nov. (circles) and Glomeropitcairnia erectiflora are found. A, El Tucuche; B, Cerro del Aripo; C, Chaguaramal. Gray areas represent hill ranges. Studies on Neotropical Fauna and Environment in 95% ethanol. All animals were released after sampling. Downloaded By: [University of Oxford] At: 16:10 24 February 2009 DNA studies Total DNA was extracted and purified using standard phenol/chloroform protocols (Sambrook et al. 1989). Excised bands and PCR product were recovered TM using a QIAquick Gel Extraction Kit. The oligonucleotide primers used for amplification and sequencing are shown in Table 1. Polymerase chain reactions (PCR) were performed with commercial reagents and recommended reaction mix concentrations (Promega, Glasgow, UK). Sequencing of both strands was performed using fluorescent dideoxy chain terminators (BigDyes, PE Biosystems) and analysed in an ABI 377 gel electrophoresis apparatus (Applied Biosystems, Perkin Elmer). Sequences were aligned and edited in Seqapp 1.9a (Gilbert 1993). Terminal amplification primers were excluded from the alignment and two alignments were prepared. 12S and 16S rDNA fragments from 10 genera within the Lophiohylini (see Table 2) were obtained from GenBank (www.ncbi.nlm.nih.gov), concatenated and aligned, using Clustal-X 1.81 (Thompson et al. 1997) with 10/10 open/extend gap penalties and minor modifications by eye. The alignment of sequences (including specimen field note number T521) followed Xenopus laevis secondary structure models (www.rna.icmb.utexas.edu) to make decisions about ambiguous regions. After exclusion of 104 sites whose alignment was potentially ambiguous this alignment of 1170 nucleotide sites (728 nt 12S rDNA; 442 nt 16S rDNA) was used to examine the relationships of Phyllodytes within Lophiohylini. Because the 12S and 16S amplified segments from different P. auratus individuals were almost invariant, within-species variation was assessed using a 390 nt fragment of the faster-evolving cytochrome b gene (cyt b). The sequences obtained could be aligned without indels and showed an open reading frame 183 when translated with the vertebrate mitochondrial genetic code. All systematic analyses were performed with PAUP*4.b.10 (Swofford 2002) or MrBayes 3.0 (Huelsenbeck & Ronquist 1999). Heuristic search of 1000 pseudoreplicates was used to test for the presence of non-random structure in the data (PTP Test; Faith & Cranston 1991) and for incongruence of rDNA data partitions (Partition Homogeneity Test; Farris et al. 1995). Trees were constructed by maximum likelihood (ML) and Bayesian maximum likelihood (BML) optimality criteria. Clade support in BML was obtained by the frequency with which a clade appeared in the saved trees. The best-fit ML model under the Akaike information criterion (Posada & Buckley 2004) was identified using MrModelTest 2.0 (Nylander 2004) and Modeltest (Posada & Crandall 1998) for the ML tree construction. MrBayes was used with default priors and Markov chain settings, and with random starting trees. The gamma shape parameter and the proportion of invariant sites were estimated from the data. Trees were sampled every 100 generations for 1,500,000 generations. The log-likelihood scores plateau was reached at 30,000 generations. A consensus tree was constructed from the last 5000 trees (500,000 generations). Relative rate tests were performed with RRtree 1.1.13 (Robinson et al. 1998). Saturation of substitutions was evaluated by plotting (in Excel) transition against transversion ‘p’ distances. The linear or power regression with the highest r2 value was identified as the best-fitting one. Results and discussion The concatenated rDNA alignment base composition was not heterogeneous (x2 tests, P51) and there was strong non-random structure (PTP tests, P50.01). Inclusion of Trachycephalus jordani (used in Faivovich et al. 2005) in the analyses significantly increased the heterogeneity between rDNA partitions. Exclusion of Table 1. Primers used to amplify and sequence cyt b, 12S and 16S rDNA. Primers L15172 H15557 L617 H1066 L1091 H1478a Gene cyt b cyt b 12S rDNA 12S rDNA 12S rDNA 12S rDNA (I, II domain) (I, II domain) (III domain) (III domain) Sequence 59-TGAGGACAAATATCATTCTGAGG-39 59-GGCGAATAGGAARTATCATTC-39 59-CAAAGCAYAGCACTGAAGATG-39 59-GCATAGTGGGGTATCTAATCCCAGYYYG-39 59-AAAAAGCTTCAAACTGGGATTAGATACCCCACTAT-39 59-CYCTGACGGGCGRTDTGT-39 Reference Hillis et al. (1996) Hillis et al. (1996) Feller and Hedges (1998) Feller and Hedges (1998) Kocher et al. (1989) Kocher et al. (1989) Note: Phyllodytes auratus 12S rDNA was amplified with two pairs of primers resulting in two non-overlapping fragments while all other 12S rDNA sequences included in the alignment did not lack this region. Thus, other 12S rDNA sequences had ca. 30 extra sites that corresponded to the non-sequenced region between the two P. auratus 12S rDNA non-overlapping fragments. This region was excluded from the alignments. 184 M. J. Jowers et al. Table 2. List of specimens examined, localities, genes sequenced, voucher numbers, Phytotriades auratus comb. nov. specimen field note numbers and GenBank accession numbers. Downloaded By: [University of Oxford] At: 16:10 24 February 2009 Taxa Hylini Smilisca phaeota Anotheca spinosa Triprion petasatus Hyla versicolor Isthmohyla rivularis Lophiohylini Trachycephalus venulosus T. mesophaeus T. resinifictrix T. hadroceps T. nigromaculatus Osteopilus septentrionalis O. vastus O. crucialis O. dominicensis Osteocephalus cabrerai O. oophagus O. leprieurii O. taurinus Itapotihyla langsdorffii Nyctimantis rugiceps Aparasphenodon brunoi Corythomantis greeningi Argenteohyla siemersi Tepuihyla edelcae Phyllodytes luteolus Phyllodytes sp. Phytotriades auratus comb. nov. P. auratus comb. nov. P. auratus comb. nov. P. auratus comb. nov. P. auratus comb. nov. P. auratus comb. nov. P. auratus comb. nov. P. auratus comb. nov. P. auratus comb. nov. P. auratus comb. nov. P. auratus comb. nov. P. auratus comb. nov. P. auratus comb. nov. Vouchers or field numbers Locality GenBank accession Genes Captive raised Mexico: Oaxaca, Ixtlan de Juarez Belize: Hummingway Hwy Venezuela: Bolivar: Cerro Guanay Costa Rica: Heredia Rds 786 ENS 10034 Rds 749 UMFS5545 MVZ 149750 12S 12S 12S 12S 12S and and and and and 16S 16S 16S 16S 16S rDNA rDNA rDNA rDNA rDNA AY843764 AY843566 AY843774 AY843682 AY843659 Guyana: Dubulay Ranch Brazil: Rio de Janeiro Venezuela: Amazonas French Guyana: Kaw Road Brazil: Espirito Santo Cuba: Guantanamo Pet trade Jamaica: Manchester Parish Pet trade Brazil: Acre French Guyana: Kaw Road Venezuela: Amazonas Venezuela: Amazonas Argentina: Misiones Ecuador: Napo Brazil: Espirito Santo Brazil: Alagoas Argentina: Corrientes Venezuela: Estado Bolivar Brazil: Espirito Santo Brazil: Bahia Trinidad, Cerro del Aripo Trinidad, Cerro del Aripo Trinidad, Cerro del Aripo Trinidad, El Tucuche Trinidad, El Tucuche Trinidad, El Tucuche Trinidad, El Tucuche Trinidad, El Tucuche Trinidad, El Tucuche Trinidad, El Tucuche Trinidad, El Tucuche Trinidad, El Tucuche Trinidad, El Tucuche AMNH-A 141142 CFBH 5780 AMNH-A 131201 MNHNP 2001.0814 N/A VSNM 317830 AMNH A-168415 N/A AMNH A-168210 LSUMZ H-13720 MNHNP 2001.0828 AMNH A-131254 AMNH A-131245 MACN 38643 N/A CFBH 2715 CFBH 2968 MACN 38644 MNHNP 1998-311 N/A MRT 6144 T511 (N/A) T511 (N/A) T511 (N/A) T521 (N/A) T521 (N/A) T521 (N/A) T521 (N/A) T522 (N/A) T523 (N/A) T524 (N/A) T525 (N/A) T550 (N/A) T551 (N/A) 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S and 16S rDNA 12S rDNA (III) 16S rDNA cyt b 12S rDNA (I, II) 12S rDNA (III) 16S rDNA cyt b cyt b cyt b cyt b cyt b cyt b cyt b AY549362 AY843718 AY843719 AY843717 AY843772 AY843712 AY843713 AY843710 AY843711 AY843705 AY843708 AY549361 AY843709 AY843706 AY843780 AY843567 AY843578 AY843570 AY843770 AY843721 AY843722 DQ403728 DQ403731 DQ403733 DQ403726 DQ403727 DQ403730 DQ403734 DQ403735 DQ403736 DQ403737 DQ403738 DQ403739 DQ403740 Note: Taxonomy follows Faivovich et al. (2005). AMNH-A, American Museum of Natural History (New York); ENS, Collection Eric N. Smith (Texas); CFBH, Collection Célio F. B. Haddad, Universidad Estadual Paulista (São Paulo); MVZ, Museum of Vertebrate Zoology, University of California (California); MNHNP, Museum National d’Histoire Naturelle (Paris); VSNM, National Museum of Natural History, Smithsonian Institution (Washington, DC); MACN, Museo Argentino de Ciencias Naturales (Buenos Aires); LSUMZH, Tissue Collection, Louisiana State University, Museum of Zoology (Louisiana); MRT, field numbers of Miguel Trefaut Rodrigues (São Paulo); Rds, field numbers Rafael de Sá (Virginia); UMF, University of Miami (Miami); N/A, non-available voucher. this taxon resulted in no significant heterogeneity between partitions (P50.28). The scatter-plot of uncorrected transition and transversion distances (Figure 2) was best fitted by a linear regression that shows no saturation. Reconstruction of the relationships of Phyllodytes spp. to other genera within the Hylinae requires outgroup polarization. Five genera from the subfamily Hylini, sister clade to the Lophiohylini (Faivovich et al. 2005), were chosen for this purpose, and a ML tree reconstruction is shown in Figure 3. In this tree, Phyllodytes is paraphyletic. As in the study of Faivovich et al. (2005), P. luteolus+P. sp. are a sister clade (posterior probability PB: 1.00) to the remaining Lophiohylini, and Osteopilus (PB: 0.99), Osteocephalus (PB: 0.99) and Trachycephalus (PB: 1.00) form three well-supported monophyletic groups. Thus, although the phylogenetic signal may Downloaded By: [University of Oxford] At: 16:10 24 February 2009 Studies on Neotropical Fauna and Environment Figure 2. Scatter-plot of pairwise uncorrected transition and transversion distances estimates of the 12S and 16S rDNA genes (r250.71). Outgroup taxa are excluded. not be strong enough to resolve inter-generic relationships, it is adequate to resolve intra-generic relationships. Figure 3 shows noticeable branch-length differences between lineages and this is confirmed 185 by relative rate tests in which most lineages showed significant differences in evolutionary rates (P,0.05). Maximum likelihood pairwise distances within genera of the Lophiohylini indicate that the highest genetic distance within the ingroup is between Phyllodytes auratus and P. luteolus (18.3%) and Phyllodytes sp. (22.6%). Thus, divergence within Phyllodytes is higher than other inter-generic distances within the Lophiohylini. Intra-population analysis reveals that with the exception of one P. auratus individual (T524, from El Tucuche), with one purine (A « G) transition occurring in the first codon position, all remaining individuals shared the same cyt b haplotype. The individual from Cerro del Aripo (T511) was identical to the other six specimens from El Tucuche. Our data show that the genus Phyllodytes is paraphyletic, showing approximately as much divergence as exists between other genera of the Lophiohylini. Separate generic status for P. auratus can therefore be justified by molecular divergence and on differences in ecology, morphology and lifehistory. For example, the possible lack of voice or ultrasonic communication in P. auratus suggests Figure 3. Maximum likelihood phylogram (2lnL56722.3158) under a GTR+I+G model of evolution. Numbers above branches indicate posterior probabilities recovered from the Bayesian analysis (of the last 5000 trees). Phyllodytes spp. and Phytotriades auratus comb. nov. are labeled in bold type. Downloaded By: [University of Oxford] At: 16:10 24 February 2009 186 M. J. Jowers et al. important mating and reproductive differences from all other Phyllodytes species. Recently, a study on the concave-eared torrent frog (Amolops tormotus) has shown that this species’ advertisement call is emitted at an ultrasonic frequency, evolved to counteract the background noise of the torrents they inhabit (Feng et al. 2005). Although this aspect is beyond the scope of our study, montane bromeliad frogs might be subject to background noise during downpours and may use similar high-frequency communication. Striking differences in skin color (two iridescent yellow stripes), and restriction to only one species of cloud forest bromeliad (G. erectiflora) differentiates P. auratus from all other Phyllodytes. The genus Phyllodytes is classified mainly on the basis of odontoids and other weak morphological characters, with no clear characters unique to the genus. However, anuran fangs have evolved independently through parallelism and convergence, and large fangs are thought to have arisen as a consequence of sexual selection in several families (Fabrezi & Emerson 2003), suggesting that this character is not a reliable synapomorphy. The use of bromeliads for breeding and tadpole development has also evolved independently (e.g. Dendrobatidae and Hylidae) and is clearly not unique to the Hylinae. Thus, the monophyly of Phyllodytes is doubtful. Because the description of P. luteolus (Wied, 1824) predates that of P. auratus (Boulenger, 1917) and because no other name is available, we consider all species previously included in Phyllodytes, except the species originally described as Amphodus auratus, as Phyllodytes sensu stricto (with the following junior synonyms: Amphodus Peters, 1873 ‘‘1872’’; Lophyohyla Miranda-Ribeiro, 1923) and propose a new generic name for the species originally described as Amphodus auratus. Systematics Phytotriades gen. nov. Type species: Amphodus auratus Boulenger, 1917. Diagnosis The morphological description follows Boulenger (1917): head much depressed, a little broader than long; snout truncate, as long as the orbit, with distinct canthus and nearly vertical loreal region; nostril near the tip of the snout; interorbital space broader than the upper eyelid; tympanum hidden; a strong ridge above the temple. Fingers and toes moderately long, the tips dilated into well-developed disks. The subarticular tubercules very feeble; fingers free, first shorter than second; toes slightly webbed at the base. The tibio-tarsal articulation reaches the eye; tibia half the length of the head and body, longer than the foot. Skin smooth, coarsely granular on the belly and under the thighs. Brown above, with three golden-yellow longitudinal streaks on the back, the outer bifurcating on the head, the branches ending between and behind the upper eyelids; or head yellow, with brown spots and three brown streaks, the outer following the canthus rostralis and the supratemporal ridge. Large widely spaced vomerinelooking teeth in the lower jaw, decreasing in size from the symphysis, and small serrated teeth on the parasphenoid bone. These morphological characters distinguish Phytotriades gen. nov. to all other hylids except Phyllodytes (all species share the presence of vomerine teeth and different species share one or more of the morphological characters described). Phytotriades auratus gen. nov. is distinguished from all Phyllodytes species by the presence of two golden longitudinal stripes on its dorsum, lack of vocalization and molecular data (mitochondrial 12S and 16S rDNA partial sequences, Appendix 1). Distribution The new genus is known in habitats above 600 m on the summits of El Tucuche and Cerro del Aripo (Northern Range of Trinidad, Republic of Trinidad and Tobago). Species included Amphodus auratus Boulenger, 19175Phytotriades auratus (Boulenger, 1917) comb. nov. Remarks Phytotriades is strictly associated with the giant bromeliad Glomeropitcairnia erectiflora, two to five eggs are laid in the deposited rain water and hatched tadpoles develop by feeding on algae and infusora growing on the surface of the leaves until metamorphosis; males display territorial behavior and combat by biting with their ‘‘fangs’’. Intra-population differentiation Lack of genetic difference between the two P. auratus populations could indicate that El Tucuche and Cerro del Aripo were recently connected by cloud forest vegetation possibly during cooler Pleistocene conditions that would have favored the dispersal of G. erectiflora to colonize lower altitude localities. Thus, the G. erectiflora and P. auratus populations at Studies on Neotropical Fauna and Environment these two localities could be remnants of a once larger but now fragmented population. Etymology The new genus’ name is derived from ‘‘Phyto’’ (Greek meaning plant) and ‘‘triades’’ (Greek for trinity, making reference to the country to which the frog is endemic). Downloaded By: [University of Oxford] At: 16:10 24 February 2009 Conservation Our results confirm the continued presence of the Golden Tree Frog and the bromeliad Glomeropitcairnia erectiflora on El Tucuche and Cerro del Aripo, and their absence from Morne Bleu (Clarke et al. 1995). Although it was not an objective of our study to assess the Golden Tree Frog’s population size, our results from El Tucuche are similar to those of Clarke et al. (1995) (not significantly different: x251.96; P.0.05) but different from those at Cerro del Aripo. If our data are representative, they suggest a serious decline of this population. Factors underlying the possible decline at Cerro del Aripo are unknown. Although human disturbance could be a factor, this seems unlikely since access to Cerro del Aripo is much the more difficult of the two peaks. A possible explanation for P. auratus population decline may be linked to the chytrid fungus Batrachochytrium, known to be most infectious at altitudes above 1000 m (e.g. La Marca et al. 2005), where the high humidity, cloud cover, and diurnal and nocturnal temperatures are at the pathogen’s optimum growth range (Pounds et al. 2005; Puschendorf et al. 2006). Not more than 206 ha of montane rain forest remain around the summits of El Tucuche and Cerro del Aripo (Clarke et al. 1995) and preservation of these rain forest areas seems to be the only method for the frog’s conservation since its ecological specialization to one species of bromeliad makes a captive breeding program unlikely to succeed. Nevertheless, further study of the ecology and life history of both the Trinidad Golden Tree Frog and its host plant are desirable. Acknowledgements We wish to thank the Wildlife Section of the Trinidad Government for permission to carry out this work. This study was carried out with the help of several members of the University of Glasgow Trinidad Expedition 2003: in particular, Vicky Ogilvy, Celia Langhorne, Ellie Rotheray, Graham Stirling and 187 Rick Stiller. We thank Gabriel Egito for comments during the early stages. We are very grateful to Anne Zillikens for her valuable comments on the manuscript. M.J.J. was funded by a UK Natural Environmental Research Council postgraduate studentship. References Bokermann WCA. 1968. Notas sobre ‘‘Phyllodytes auratus’’ (Boulenger 1917) (Amphibia, Hylida). Rev Brasil Biol. 28:157–160. Boulenger GA. 1917. On a second species of the batrachian genus Amphodus. Ann Mag Nat Hist Series. 8(20):184–185. Caramaschi U, Peixoto OL. 2004. A new species of Phyllodytes (Anura: Hylidae) from the state of Sergipe, northeastern Brazil. Amph Rept. 25:1–7. Caramaschi U, Peixoto OL, Rodrigues MT. 2004. Revalidation and redescription of Phyllodytes wuchereri (Peters, 1873) (Amphibia, Anura, Hylidae). Arq Mus Nac Rio de Janeiro. 62:185–191. Clarke FM, Ward AI, Downie JR. 1995. Factors affecting the distribution and status of the golden tree frog, Phyllodytes auratus, in Trinidad. Br Herp Soc Bull. 54:3–9. Cruz CAG, Feio RN, Cardoso MCDS. 2006. Description of a new species of Phyllodytes Wagler, 1830 (Anura: Hylidae) from the Atlantic rain forest of the states of Minas Gerais and Bahia, Brazil. Arq Mus Nac Rio de Janeiro. 64:321–324. Fabrezi M, Emerson SB. 2003. Parallelism and convergence in anuran fangs. J Zool. 260:41–51. Faith DP, Cranston PS. 1991. Could a cladogram this short have arisen by chance alone? On permutation tests for cladistic structure. Cladistics. 7:1–28. Faivovich J, Haddad CFB, Garcia PCA, Frost DR, Campbell JA, Wheeler WC. 2005. Systematic review of the frog family Hylidae, with special reference to Hylinae: phylogenetic analysis and taxonomic revision. Bull Am Mus Nat Hist. 240:1–240. Farris JS, Källersjö M, Kluge AG, Bult C. 1995. Constructing a significance test for incongruence. Syst Biol. 44:570–572. Feller AE, Hedges SB. 1998. Molecular evidence for the early history of living amphibians. Mol Phylogenet Evol. 9:509–516. Feng AS, Narins PM, Xu C-H, Lin W-Y, Yu Z-L, Qiu Q, Xu Z-M, Shen J-X. 2005. Ultrasonic communication in frogs. Nature. 440:333–336. Gilbert D. 1993. Seqapp. Available by ftp from Bloomington: University of Indiana, Molecular Biology Software Archive (http://iubio.bio.indiana.edu). Hardy J. 2004. Phyllodytes auratus. In: IUCN 2006. 2006 IUCN red list of threatened species. Available from www.iucnredlist.org Hillis DM, Moritz C, Mable BK. 1996. Molecular systematics. Sunderland (MA): Sinauer Associates. 655 p. Huelsenbeck JP, Ronquist FR. 1999. MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics. 17:754–755. Kenny JS. 1969. The amphibian of Trinidad. Stud Fauna Curacao Carib Is. 29:1–78. Kocher TD, Thomas WK, Meyer A, Pääbo S, Villablanca F, Wilson AC. 1989. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc Natl Acad Sci USA. 86:6196–6200. La Marca E, Lips KR, Lötters S, Puschendorf R, Ibanez R, Rueda-Almonacid JV, Schulte R, Marty C, Castro F, Manzanilla-Puppo J, Garcia-Perez JE, Bolanos F, Chaves G, Pounds JA, Toral E, Young BE. 2005. Catastrophic population Downloaded By: [University of Oxford] At: 16:10 24 February 2009 188 M. J. Jowers et al. declines and extinctions in Neotropical harlequin frogs (Bufonidae: Atelopus). Biotropica. 37:190–201. Manzanilla J, Jowers MJ, La Marca E, Garcı́a Parı́s M. 2007. Taxonomic reassessment of Mannophryne trinitatis (Anura: Dendrobatidae) with a description of a new species from Venezuela. Herp J. 17:31–42. Murphy J. 1997. Reptiles and amphibians of Trinidad and Tobago. Malabar (FL): Krieger. 245 p. Nylander JAA. 2004. Mrmodeltest 2.0. program. Available from the author, Uppsala University, Evolutionary Biology Centre. Peixoto LO, Caramaschi U, Freire MX. 2003. Two new species of Phyllodytes (Anura: Hylidae) from the state of Alagoas, northeastern Brazil. Herpetologica. 59:235–246. Posada D, Crandall KA. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics. 14:817–818. Posada D, Buckley TR. 2004. Model selection and model averaging in phylogenetics: advantages of the aic and bayesian approaches over likelihood ratio tests. Syst Biol. 53:793–808. Pounds JA, Bustamante MR, Coloma LA, Consuegra JA, Fogden MPL, Foster PN, La Marca E, Masters KL, MerinoViteri A, Puschendorf R, Ron SR, Sánchez-Azofeifa GA, Still CJ, Young BE. 2005. Widespread amphibian extinctions from epidemic disease driven by global warming. Nature. 439: 161–167. Puschendorf R, Bolanos F, Chaves G. 2006. The amphibian chytrid fungus along an altitudinal transect before the first reported declines in Costa Rica. Biol Conserv. 132:136–142. Read VMJ. 1982. A new locality record for the bromeliad dwelling hylid Phyllodytes auratus (Boulenger) in Trinidad, the West Indies. Bull Chi Herp Soc. 18:12–29. Robinson M, Gouy M, Gautier C, Mouchiroud D. 1998. Sensitivity of the relative-rate test to taxonomic sampling. Mol Biol Evol. 15:1091–1098. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory. 999 p. Swofford DL. 2002. PAUP*. Phylogenetic analysis using parsimony (*and other methods). Sunderland (MA): Sinauer Associates. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The Clustal X windows interface: flexible strategies for multiple sequence alignment by quality analysis tools. Nucl Acid Res. 25:4876–4882. Appendix 1. Phytotriades auratus comb. nov. mitochondrial sequences Underlined bases were excluded from analysis. Numbering corresponds to Xenopus laevis.