Species diversity of Hyalinobatrachium glassfrogs

Zootaxa 3132: 1–55 (2011) www.mapress.com / zootaxa/ Copyright © 2011 · Magnolia Press

ISSN 1175-5326 (print edition)

Article

ZOOTAXA ISSN 1175-5334 (online edition)

Species diversity of Hyalinobatrachium glassfrogs (Amphibia: Centrolenidae) from the Guiana Shield, with the description of two new species SANTIAGO CASTROVIEJO-FISHER1,8, CARLES VILÀ2, JOSÉ AYARZAGÜENA3, 4, MICHEL BLANC5 & RAFFAEL ERNST6, 7 1

Department of Herpetology, American Museum of Natural History, Central Park West at 79th Street, 10024-5192 New York, U.S.A.. Estación Biológica de Doñana-CSIC, Avd. Américo Vespucio s/n, 41092 Seville, Spain 3 Asociación Amigos de Doñana, Panama 6, 41012 Seville, Spain 4 Museo de Historia Natural, Fundación La Salle de Ciencias Naturales, Av. Boyacá with Maripérez, 1930, Caracas, Venezuela 5 Pointe Maripa, RN2/PK35, Roura, French Guiana 6 Museum of Zoology, Senckenberg Natural History Collections Dresden A. B. Meyer Building, D-01109 Dresden, Germany 7 Department of Biodiversity Dynamics, TU Berlin, Rothenburgstr. 12, D-12165 Berlin, Germany 8 Corresponding author. E-mail: [email protected]. 2

Table of contents Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Bioacoustics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Species accounts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Hyalinobatrachium cappellei . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Hyalinobatrachium kawense sp. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Hyalinobatrachium mesai . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Hyalinobatrachium mondolfii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Hyalinobatrachium taylori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Hyalinobatrachium tricolor sp. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Appendix I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Appendix II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Abstract Basic information about the taxonomy, biology and distribution of Hyalinobatrachium glassfrogs of the Guiana Shield (GS) is scarce, ambiguous, and in many cases even contradictory. In this review we aim to clarify the current taxonomic status of this group by means of phenotypic (morphology, morphometrics and bioacoustics) and molecular (mitochondrial DNA sequences) comparisons. Eight species have previously been recognized for the GS: H. crurifasciatum, H. eccentricum, H. fleischmanni (initially described as Hylella cappellei in the GS), H. iaspidiense (with the putative synonym H. nouraguense), H. ignioculus, H. mesai, H. mondolfii, and H. taylori. Our data support the resurrection of H . cappellei from its synonymy with H. fleischmanni. Hyalinobatrachium crurifasciatum, H. eccentricum, and H. ignioculus are proposed as junior synonyms of H. cappellei. We show that none of the four paratypes of H. taylori belong to this species and we assign two to H. cappellei and two to H. mondolfii. Additional specimens previously identified as H. taylori are reassigned to H. cappellei, and hence H. taylori is redefined. Hyalinobatrachium nouraguense is confirmed as a junior syn-

Accepted by J. Padial: 21 Oct. 2011; published: 15 Dec. 2011

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onym of H. iaspidiense. We also describe two new species of Hyalinobatrachium from French Guiana: Hyalinobatrachium kawense sp. nov. and Hyalinobatrachium tricolor sp. nov. In addition, and in concordance with the new taxonomic rearrangements, we provide diagnostic characters for all species, known distributions and main sources of references for their biology. We also report new distribution records for H. iaspidiense and H. mondolfii, and describe the formerly unknown tadpole of the later. Consequently, we recognize seven species of Hyalinobatrachium for the Guiana Shield: H. cappellei, H. iaspidiense, H. kawense sp. nov., H. mesai, H. mondolfii, H. taylori, and H. tricolor sp. nov. We discuss the suitability of integrative taxonomy as an approach to identify taxonomic uncertainty and consider its significance for conservation purposes. We also address the implications of our results to understand phylogeographic patterns in this area. Key words: Brazil, French Guiana, Guyana, Hyalinobatrachium, Integrative Taxonomy, Mitochondrial DNA, Phylogeography, Synonym, Suriname, Venezuela

Introduction Glassfrogs (family Centrolenidae) have long attracted the attention of biologists working in the Neotropics because of their morphological and ecological characteristics, as well as their phylogenetic and biogeographic complexity (Señaris & Ayarzagüena 2005; Castroviejo-Fisher et al. 2007; Guayasamin et al. 2008a). At present, the roughly 150 recognized species of glassfrogs are divided among 12 monophyletic genera (Guayasamin et al. 2009). Their main habitats are rainforests of Central America, Chocó, northern Andes, Guiana Shield, Amazonia, and southeastern Brazil. The Guiana Shield (GS) or Guianan Region, sensu Gibbs and Barron (1993), with the biogeographic additions for amphibians of Señaris and MacCulloch (2005), is situated between the Amazon and Orinoco rivers (Fig. 1). It is an overlooked biodiversity hotspot and a priority area for conservation purposes (Rull 2005; Orme et al. 2005), encompassing the most intact (80–90%), least inhabited (0.6–0.8 people/km2) tropical rainforest region in the world (Huber & Foster 2003). Recent efforts aiming at the exploration of the amphibian biodiversity of the GS resulted in the description of various new species (e.g. MacCulloch & Lathrop 2002; Kok et al. 2006; Kok & Ernst 2007; Myers & Donnelly 2008), including members of the family Centrolenidae (e.g. Noonan & Bonett 2003; Barrio-Amorós & Brewer-Carías 2008). Eight species of Hyalinobatrachium are currently recognized for the GS: H. crurifasciatum Myers & Donnelly, H. eccentricum Myers & Donnelly, H. fleischmanni (Boettger, initially described as Hylella cappellei van Lidth de Jeude in Guyana), H. iaspidiense (Ayarzagüena, including the recently proposed synonym H. nouraguense Lescure & Marty by Yáñez-Muñoz et al. 2009), H. ignioculus Noonan & Bonett, H. mesai Barrio-Amorós & BrewerCarías, H. mondolfii Señaris & Ayarzagüena, and H. taylori (Goin). However, the taxonomic status of several of these species remains uncertain (Cisneros-Heredia & McDiarmid 2007; Kok & Castroviejo-Fisher 2008; BarrioAmorós & Castroviejo-Fisher 2008), creating a confusing situation in terms of actual species richness and distribution that could potentially lead to misguided conservation strategies or erroneous biogeographic interpretations. In the following paragraphs we introduce the main problems concerning the taxonomy of Hyalinobatrachium species from the GS. van Lidth de Jeude (1904) described Hylella cappellei from Suriname based on a single individual. However, the original description lacks essential details and is very rudimentary. Goin (1964) considered Hylella cappellei as a junior synonym of Centrolenella fleischmanni, now in the genus Hyalinobatrachium (Ruiz-Carranza & Lynch 1991). Several authors (Noonan & Harvey 2000; Cisneros-Heredia & McDiarmid 2007; Kok & Castroviejo-Fisher 2008) have questioned this taxonomic arrangement but no scientific publication has really addressed this taxonomic problem since Goin’s seminal work. To the best of our knowledge, the holotype of Hylella cappellei constitutes the only current vouchered specimen assigned to Hyalinobatrachium fleischmanni from east of the Andes and by implication from the Guiana Shield. The taxonomic history of Hyalinobatrachium taylori is complex and has created confusion among herpetologist working in the GS, to the point that at least two different species are referred to as H. taylori (Kok & Castroviejo-Fisher 2008). Hyalinobatrachium taylori was described by Goin (1968) from preserved material and on the basis of morphological characters. Despite including five specimens in the type series (four paratypes and the holotype), the author only provided a description of the holotype. Lescure (1975), Hoogmoed and Avila-Pires (1990), and Lescure and Marty (2000) assigned specimens from French Guiana and Suriname to H. taylori. Señaris

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and Ayarzagüena (2005) dispute this identification and argue that these specimens are most similar to H. crurifasciatum. On the other hand, Ayarzagüena (1992), Señaris and Ayarzagüena (2005), Kok and Castroviejo-Fisher (2008), and Kok and Kalamandeen (2008) identified specimens from Venezuela and Guyana that are morphologically different from H. crurifasciatum as H. taylori. We think that this contradictory situation could be rooted in the type series of H. taylori, which we suspected to include more than one species.

FIGURE 1. Limits of the Guiana Shield sensu Gibbs and Barron (1993). Numbered circles correspond to approximated localities of Hyalinobatrachium species for this area including the biogeographic additions for amphibians of Señaris and MacCulloch (2005). For clarity, in some cases a numbered circle could refer to several closely situated localities. For details on localities see Appendix II.

Hyalinobatrachium mesai and H. nouraguense have been suggested to be conspecific with H. iaspidiense (Ernst et al. 2005; Cisneros-Heredia & McDiarmid 2007; Kok & Castroviejo-Fisher 2008; Yáñez-Muñoz et al. 2009) because they are very similar in both morphology and mating calls (Lescure & Marty 2000; Señaris & Ayarzagüena 2005; Barrio-Amorós & Brewer-Carías 2008). Yáñez-Muñoz et al. (2009) consider H. nouraguense as a junior synonym of H. iaspidiense based on the low genetic divergence presented by Guayasamin et al. (2008a). However, Guayasamin et al. (2008a) only sequenced one specimen of each species. Within- and between-population variability was therefore not addressed. Also the putative specimen of H. nouraguense included in Guayasamin et al. (2008a) originated from a locality in Guyana that is actually closer to the type locality of H. iaspidiense than to that of H. nouraguense (French Guiana). For the reasons outlined above, we considered it necessary to revaluate the validity of H. iaspidiense, H. mesai, and H. nouraguense. The species Hyalinobatrachium crurifasciatum, H. eccentricum, and H. ignioculus are very similar in morphology, bioacoustics and genetics (Señaris & Ayarzagüena 2005; Cisneros-Heredia & McDiarmid 2007; Guayasamin et al. 2008a; Barrio-Amorós & Castroviejo-Fisher 2008; Kok & Castroviejo-Fisher 2008; Kok & Kalamandeen 2008) to the point that several authors have suggested that they might be synonyms (Cisneros-Heredia & McDiarmid 2007; Kok & Castroviejo-Fisher 2008; Barrio-Amorós & Castroviejo-Fisher 2008). Nonetheless, there has not been a detailed evaluation of their taxonomic status.

HYALINOBATRACHIUM SPECIES OF THE GUIANA SHIELD

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Finally, we address the status of two putative new species of Hyalinobatrachium for the GS reported as H. aff. iaspidiense and H. aff. mondolfii respectively (Guayasamin et al. 2008a). These candidate species were suggested on molecular data alone and have not yet been formally described or named. The overall goal of this work is to assess the taxonomic status of the species of Hyalinobatrachium from the GS with particular emphasis on the taxonomic problems outlined above. We used a cumulative integrative taxonomic approach (Padial et al. 2010) that considers multiple and independent criteria for detecting lineage divergence (Dayrat 2005; Will et al. 2005; Padial et al. 2009, 2010; Padial & De la Riva 2010) under the theoretical framework that species are segments of separately evolving metapopulation lineages (Simpson 1951, 1961; see also Wiley 1978, 1981; Frost & Kluge 1994; Mayden 1997, 2002; de Queiroz 1998, 2005a, 2005b, 2005c, 2007). We inferred lineage divergence using qualitative morphological traits, morphometrics, genetic distances, reciprocal monophyly of mtDNA sequences (phylogenetics), and bioacustic analyses of advertisement calls. Based on our results, we propose a new taxonomic classification, provide diagnostic features for all species, describe two new species, and compile the available data on the biology, larvae and distribution of the recognized species of Hyalinobatrachium for the GS. We briefly discuss the conservation and biogeographic implications of our results, and the advantages of using different criteria to solve taxonomic uncertainty and discover cryptic species.

Material and methods Nomenclature and conventions. This study is based on published information, analyses of museum specimens, and collection of life history and ecological data in the field. Specimens captured for this study were preserved in 70% ethanol and some of them fixed in formalin (4–10%). Specimens and data examined are listed in Appendix I. Institution abbreviations are as in Frost (2011) with the additions of Brice P. Noonan (BPN) field collection numbers. Unless otherwise noted, characters are reported for adults only. Ontogenetic status was determined by examination of the development of gonads and presence of secondary sexual characteristics (i.e. vocal slits, sacs and nuptial pads). Credits for photographs are as follows: Raffael Ernst (RE), Philippe Kok (PK), Michel Blanc (MB) and Santiago Castroviejo-Fisher (SCF). We followed Guayasamin et al. (2009) for the supraspecific classification. Morphology. For terminology and definitions of morphological and color characteristics, we followed Lynch and Duellman (1973), Señaris and Ayarzagüena (2005), Cisneros-Heredia and McDiarmid (2007), Kok and Castroviejo-Fisher (2008), Guayasamin et al. (2009), and citations therein. Terminology for webbing follows Savage and Heyer (1967), as modified by Guayasamin et al. (2006). Description of nuptial excrescences (= nuptial pads, glandular clumps or nuptial glands) and prepollical spines follows Flores (1985), with the additions of CisnerosHeredia and McDiarmid (2007) and Guayasamin et al. (2009). Color characteristics were noted from live individuals, color photographs and descriptions and photographs in the literature (van Lidth de Jeude 1904; Goin 1964, 1968; Lescure 1975; Ibáñez et al. 1999; Myers & Donnelly 1997, 2001; Lescure & Marty 2000; Savage 2002; Noonan & Bonett 2003; Señaris & Ayarzagüena 2005; Kubicki 2007; Barrio-Amorós & Brewer-Carías 2008; Barrio-Amorós & Castroviejo-Fisher 2008; Kok & Castroviejo-Fisher 2008; Guayasamin et al. 2009). All species studied in this work have been described and differentiated from each other on the basis of mainly qualitative morphological character comparisons. Thus, we study and compare the characters listed and described in detail by Cisneros-Heredia and McDiarmid (2007). Measurements were taken with a digital caliper to the nearest 0.1 mm. We used data from the literature for the holotypes of Hyalinobatrachium mesai and H. eccentricum (Barrio-Amorós & Brewer-Carías 2008; Myers & Donnelly 2001). Abbreviations for measurements are as follows: snout-vent length (distance from tip of snout to posterior margin of vent, SVL); head length (from occiput to tip of snout, HL); head width (largest distance, HW); interorbital distance (minimum distance between upper eyelids, IOD); eye length (horizontal diameter, EL); upper eyelid width (greatest transverse width, EW); eye to snout tip distance (from tip of snout to anterior margin of eye, ES); width of the terminal disk of the third finger (FIII); femur length (distance from the middle of the cloacal slit to the femur-tibia articulation, FEL); tibia length (from the femur-tibia articulation to the tibia-heel one, TL); foot length (distance from proximal margin of outer metatarsal tubercle to tip of Toe IV, FL). We consider a taxon to show divergence in morphology when exhibiting at least one fixed (qualitative) or nonoverlapping (quantitative) character or a unique combination separating it from each of the other taxa. The underly-



ing assumption is that fixed differences in morphology might be strong evidence of reduced or absent gene flow (Wiens & Servedio 2000); thus constituting evidence of independent lineages. Bioacoustics. Vocalizations were recorded in the field at air temperatures between 17.5 and 26.0°C (exact temperatures for each recording are specified in Results) using a Sony WM D6C tape recorder and a Sennheiser Me 80 directional microphone. Distance between microphone and calling individuals was kept at approximately one meter. Additionally, we analyzed the recordings of Hyalinobatrachium taylori (sensu Lescure 1975; Hoogmoed & Avila-Pires 1990; Lescure & Marty 2000) and Cochranella sp. (= H. nouraguense) from Marty and Gaucher (1999), H. sp. (= H. iaspidiense) from Cocroft et al. (2001), and a recorded call of H. mesai (kindly provided by C. L. Barrio-Amorós). Recordings were processed on an Apple Macintosh computer. The sounds were digitized and edited at a sampling frequency of 44.1 KHz and 16 bit resolution with a Delta 66 digitalizing board and Peak 3.2 (OSX) software. The software Raven 1.1 (Cornell University, Ithaca, New York) was used to obtain numerical information from audiospectograms and oscillograms. Frequency information was obtained through fast Fourier transformation (FFT; width, 1024 points). Audiospectograms and oscillograms shown in the figures were obtained with the software Praat 5.2.25 for MacOS X (Boersma & Weenick 2006). We measured the following quantitative call characteristics: number of notes per call, call duration, dominant frequency, and lower and upper frequency of the fundamental frequency. Additionally, we compared the following qualitative parameters: presence/absence of pulses (pulsed versus tonal calls), frequency modulation, and distribution of the maximum intensity. For detailed explanations of the studied parameters see Goicochea et al. (2010). Advertisement call differences are usually interpreted as evidence of lineage divergence that can be used to separate species (Bickford et al. 2007; Vences & Wake 2007; Padial et al. 2007, 2008a, 2008b). We considered that advertisement calls strongly indicate the existence of lineage divergence when they show structural differences (e. g. pulsed vs. non-pulsed) or do not overlap in quantitative parameters (note length, call length, number of notes per call, and dominant frequency). Genetic analyses. We used two criteria to delimit species boundaries using DNA data: reciprocal monophyly and genetic distances (reviewed in Vences & Wake 2007). The first criterion is based on the assumption that coalescent patterns in gene genealogies are related to historical processes that originate separate lineages (e.g. Avise 2000; Knowles & Carstens 2007). The second assumes that genetic divergence between populations within a species tends to be relatively small because of gene flow, whereas divergence between species increases with time; however, for the reasons exposed by Padial et al. (2009), we do not use thresholds to delimit species boundaries. A total of 46 sequences were included in the molecular analyses (Appendix I). We used Celsiella revocata (Rivero) and C. vozmedianoi (Ayarzagüena & Señaris) as outgroups following Guayasamin et al. (2009). Genomic DNA was isolated using a standard phenol-chloroform extraction protocol (Sambrook et al. 1989). A fragment of approximately 575 bp from the mitochondrial gene 16S was amplified using previously described primers (16Sar5’ and 16Sbr-3’) and PCR conditions (Hillis et al. 1996). This marker outperforms COI as a species identifier or DNA barcode for amphibians (Vences et al. 2005a, 2005b) and has been broadly used in amphibian phylogenies (e.g. Darst & Cannatella 2004; Frost et al. 2006). PCR products were purified and sequenced in a MegaBACE 1000TM (GR Health Care) instrument following manufacturer protocols. Each PCR product was sequenced twice using forward and reverse primers. Sequences from both strands were compared to generate a consensus sequence for each specimen using Sequencher 4.7 (Gene Codes Corp. 2006). Sequences were aligned in MAFFT (Katoh & Toh 2008) under the E-INS-i option. We performed maximum likelihood (ML) and Bayesian phylogenetic analysis (BA) using Garli 0.96 Beta (Zwickl 2006) and MrBayes 3.2.1 (Ronquist & Huelsenbeck 2003) respectively. We implemented the model of nucleotide substitution GTR+I+G selected by ModelTest version 3.7 (Posada & Crandall 1998) following the Akaike Information Criterion (AIC). For ML analysis we used default search parameters and performed a total of 100 runs to reduce the probability of inferring a suboptimal likelihood solution. Node support was assessed by 1000 bootstrap (BSS) replicates (Felsenstein 1985). For BA two independent analyses were run for 5 x 106 generations, sampling every 1000, under default options. Burn-in and convergence were evaluated by examination of the standard deviation of split frequencies and the log-likelihood, -lnL. Clades with Bayesian posterior probabilities (BPP) ≥ 0.95 are considered strongly supported, but it must be noted that relatively high posterior probabilities for short internodes may be over-estimates of confidence (Alfaro et al. 2003; Erixon et al. 2003). Bootstrap values ≥ 70% are considered to indicate strong support (Hillis & Bull 1993). Genetic distances between taxa were calculated using the “Maximum Likelihood” option



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in the software package PAUP* 4.0b10 (Swofford 1998) using the results obtained in the ML search with Garli 0.96 Beta.

Results Morphology Qualitative characters. Relevant results are summarized in Table 1. We found that the holotype of Hyalinobatrachium cappellei differs in the following characters from H. fleischmanni (characters of H. cappellei in parentheses): snout rounded in dorsal view (truncated), pericardium white (transparent), canthus rostralis not differentiated (distinct), hand webbing formula III 2–(11/2–1+) IV (III 2–2 IV). We did not find any qualitative morphological difference between and among the holotype of H. cappellei and specimens (including types) of H. crurifasciatum, H. eccentricum, H. ignioculus, H. taylori [sensu Lescure (1975), Hoogmoed & Avila-Pires (1990), and Lescure & Marty (2000)], and two paratypes of H. taylori (RMNH 11473–4). Specimens of Hyalinobatrachium iaspidiense and H. nouraguense (including type specimens) show no difference in the characters studied and H. mesai is only divergent in the color of bones in life [white (H. iaspidiense and H. nouraguense) versus green (H. mesai)]. Specimens of H. aff. iaspidiense are different from H. iaspidiense in dorsal skin coloration in life and from H. mesai in dorsal skin and bone coloration in life. Specimens of H. aff. mondolfii differ from all other specimens by a combination of characters (Table 1). The two other paratypes of H. taylori (BM 1939.1.1.64, RMNH 11472) show the same character states than specimens of H. mondolfii (including type material). However, we did not find divergent characters between specimens of H. mondolfii and two paratypes of H. taylori (BM 1939.1.1.64, RMNH 11472) and specimens of H. fleischmanni. Finally, character states of specimens of H. taylori [sensu Ayarzagüena (1992), Señaris & Ayarzagüena (2005), Kok & Castroviejo-Fisher (2008), and Kok & Kalamandeen (2008)] are in concordance with those of the holotype of H. taylori. We give detailed discussions of relevant characters used in the literature to differentiate among species in the Species account section. Quantitative characters. Measurements of the compared taxonomic units are summarized in Table 2. Ranges of all measured continuous characters broadly overlap. Therefore, we disregard our quantitative measurements as being potentially useful to discriminate among Hyalinobatrachium species of the GS in the context of this study.

Bioacoustics Results are summarized in Table 3 and Figure 2. The advertisement call of H. fleischmanni differs in structure from all other taxonomic units compared but H. aff. mondolfii. However, the dominant frequency and the range of frequencies exhibited by the basal frequency are lower in the calls of specimens of H. fleischmanni (see also descriptions of the call of H. fleischmanni by Greer & Wells 1980; Jacobson 1985; Ibañez 1999; Kubicki 2007). The calls of the different populations of H. crurifasciatum, H. eccentricum, H. ignioculus, and H. taylori [sensu Lescure (1975), Hoogmoed & Avila-Pires (1990), and Lescure & Marty (2000)] have the same structure and overlap in all variables measured. The calls of specimens of H. iaspidiense, H. mesai and H. nouraguense have the same structure and overlap in all the quantitative characters studied. The call of H. aff. iaspidiense is readily differentiable for all others by having four notes, each with a fast increase in energy at the beginning. The call of individuals of H. mondolfii is different in structure and quantitative characters from all the other calls analyzed. The calls of H. taylori [sensu Ayarzagüena (1992); Señaris & Ayarzagüena (2005); Kok & Castroviejo-Fisher (2008); and Kok & Kalamandeen (2008)] are readily differentiable from all others by having 5–9 notes per call (one or four in all other taxonomic units studied).





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FIGURES 2. Comparative oscillograms and spectograms of advertisement calls of Hyalinobatrachium species from the Guiana Shield and H. fleischmanni: (A) H. fleischmanni from Panama, audio from Ibañez et al. (1999); (B) H. kawense sp. nov. from French Guiana, MNCN 44825; (C) H. cappellei Venezuela, MHNLS 17328; (D) H. cappellei French Guiana, audio from Marty and Gaucher (1999); (E) H. iaspidiense from Venezuela, MHNLS 13495; (F) H. iaspidiense from French Guiana, audio from Marty and Gaucher (1999); (G) H. mesai, holotype; (H) H. tricolor sp. nov. from type locality in French Guiana, uncollected specimen; (I) H. mondolfii from Venezuela, MHNLS 17120; (J) H. taylori from Venezuela, MHNLS 13496.



FIGURE 3. Maximum likelihood phylogenetic tree of Hyalinobatrachium glassfrogs from the Guiana Shield (GS) and H. fleischmanni inferred from 575 bp of the 16S mtDNA. Bootstrap support values followed by Bayesian posterior probabilities are indicated over branches. Outgroups (Celsiella revocata and C. vozmedianoi) are not shown. Photos depict representative specimens of respective clades (not to scale). Hyalinobatrachium taylori: 1sensu Lescure (1975), Hoogmoed and Avila-Pires (1990), and Lescure and Marty (2000); 2sensu Ayarzagüena (1992), Señaris and Ayarzagüena (2005), Kok and CastroviejoFisher (2008), and Kok and Kalamandeen (2008).



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Genetics We identified seven non-nested main clades in our analyses (clades A–G of Fig. 3). Each of these clades is strongly supported and shows very limited internal genetic distances among its terminals (0–3 %) that do not overlap with among clades genetic distances (≥ 5 %) suggesting relatively long periods of genetic isolation and divergence. Clade A exclusively includes sequences of H. taylori [sensu Ayarzagüena (1992), Señaris & Ayarzagüena (2005), Kok & Castroviejo-Fisher (2008), Kok & Kalamandeen (2008)] and is strongly supported (BSS = 98, BPP = 100). The genetic distance between these sequences and all the others is 6–12%. The intraspecific genetic distances show differentiation between lowland and highland populations. The three Venezuelan highland sequences are identical despite being sampled in two localities separated by up to 100 Km, while the sequence from the lowlands of French Guiana shows a genetic distance of 3% to the highland ones. Clade B is well supported (BSS = 99, BPP = 100) and includes sequences of two topotypes of H. mondolfii plus one specimen from Guyana. The three sequences are identical and differ by 6–10% from all other sequences of Hyalinobatrachium included in the study. Clade C includes three sequences of H. aff. mondolfii and received maximum support in both analyses. The three sequences are identical and are divergent (genetic distance ≥ 7 %) from all other sequences of Hyalinobatrachium included in the study. Clade D is formed by the sequences of six specimens of Hyalinobatrachium fleischmanni from across its distribution (Ecuador, Honduras, and Mexico) and is well supported (BSS = 96, BPP = 100). Genetic distances are relatively low (0–1%) within clade terminals and substantially larger (≥ 6%) with respect to other sequences. Clade E includes all the sequences of specimens of H. crurifasciatum, H. eccentricum, H. ignioculus, and H. taylori [sensu Lescure (1975), Hoogmoed & Avila-Pires (1990), and Lescure & Marty (2000)] and is strongly supported clade (BSS = 99, BPP = 100). However, the genetic distances among these specimens are low (< 1 %) and their relationships are not resolved, resulting in a politomy. Clade F (BSS = 82, BPP = 100) groups sequences of specimens of H. iaspidiense and H. nouraguense from Brazil, Venezuela, Guyana and French Guiana. Although the Brazilian sequences and those from the Guiana Shield are reciprocally monophyletic, the genetic distances between them are minimal (< 1%) and could be the result of progressive isolation by distance. Clade G (BSS = 99, BPP = 100) contains the two sequences of H. aff. iaspidiense that show no genetic distance between them and are relatively divergent with regard to the other clades (genetic distances ≥ 5 %). Sequences from H. mesai were not available.

Species accounts Hyalinobatrachium cappellei (Fig. 4) Hylella cappellei van Lidth de Jeude, 1904: 94, bona species. Centrolenella cappellei Noble, 1926: 18. Centrolenella fleischmanni Goin, 1964: 1. Cetrolenella taylori Lescure, 1975: 390. Centrolenella taylori Hoogmoed and Avila-Pires, 1990. Hyalinobatrachium crurifasciatum Myers and Donnelly, 1997: 9, new synonym. Hyalinobatrachium taylori Lescure and Marty, 2000: 78. Hyalinobatrachium eccentricum Myers and Donnelly, 2001: 16, new synonym. Hyalinobatrachium ignioculus Noonan and Bonett, 2003: 92, new synonym. Hyalinobatrachium sp2 Ernst et al. 2005: 183. Hyalinobatrachium igniocolus Barrio-Amorós and Castroviejo-Fisher, 2008: 235, lapsus calami. Hyalinobatrachium aff. ignioculus Guayasamin et al. 2008a: 580.

Type locality. River Saramacca and neighboring areas, Suriname. Diagnosis. (1) Dentigerous processes on vomer and vomerine teeth absent; (2) snout truncate in dorsal and lateral view; (3) tympanum covered by skin, not visible through skin; (4) dorsal skin shagreened in life and preservative; (5) presence of small cloacal enameled warts; (6) parietal peritoneum transparent, pericardium from completely transparent to totally white with intermediate states, visceral and hepatic peritonea white, all other peritonea transparent; (7) liver bulbous; (8) humeral spine absent; (9) webbing formula of fingers III (2– – 2+) – (2 – 2+)



IV; (10) webbing formula of toes I (11/3 – 11/2) – (2+ – 11/3) II (1+ – 11/3) – (21/3 – 21/2) III (1+ – 11/2) – (21/2 – 23/4) IV (23/4 – 3–) – (1 – 1+) V; (11) low and weakly enameled ulnar and tarsal folds; (12) nuptial excrescences Type-V composed of a cluster of glands and situated in the medial, dorso-lateral internal side of Finger I, glands not present in other fingers, prepollex not evident from external view; (13) Finger I longer than Finger II; (14) eye diameter larger than width of disc on Finger III; (15) coloration in life: dorsum with yellow or pale green spots set in a light green reticulum dotted with dark small and/or minute melanophores, when the yellow spots on the hind limbs are sufficiently big they give a pattern of crossbars, bones white; (16) coloration in preservative: dorsum cream with purple melanophores, spots and crossbars on hind limbs may be lost; (17) iris golden with dark melanophores, with or without a complete or not complete ring (from brown to light red) around the pupil, pupillary ring could be partly or completely absent in some specimens; (18) minute melanophores not extending throughout fingers and toes except on the base of Finger IV and Toe V; in life, tip of fingers and toes white; (19) advertisement call composed by a single pulsed note lasting 0.13–0.29 s, dominant frequency of 3926.6–5081.8 Hz, males call from the underside of leaves; (20) fighting behavior unknown; (21) egg clutches deposited on the underside of leaves, males often on the same leaf than eggs; (22) tadpole with sinistral spiracle on the posterior third of the body, short vent tube; (23) medium size adult males, SVL = 18.6–24.9 (21.8 ± 1.4, N = 30) mm and one female 21.3 mm. Comparisons. The following unique combination of phenotypic characters differentiates Hyalinobatrachium cappellei from all other species in the genus: snout truncated in dorsal and lateral views, tympanic membrane and annulus not appreciable in life, pericardium from completely transparent to totally white with intermediate states, hand weebing formula III (2- – 2+) – (2 – 2+) IV, dorsal coloration in life yellow big spots on a light green reticulum dotted with small and/or minute melanophores, dorsal coloration in preservative pale cream dotted with small and/or minute melanophores surrounding big cream spots (could be lost in some specimens), iris coloration in life yellow with brown-red flecks and a red-brown ring (complete or not) encircling the pupil, light yellow pupillary ring from absent to complete, bones in life white, coloration of hands and feet in life white, and a single pulsed note advertisement call with frequency modulation (increasing and decreasing within each pulse giving the shape of a saw), lasting 0.13–0.29 s and with a dominant frequency of 3926.6–5081.8 Hz. Morphological, bioacoustic and genetic evidences allowing the differentiation between Hyalinobatrachium cappellei and all other species of Hyalinobatrachium from the GS are summarized in Figures 2, 3, 4 and Tables 1, 3. Remarks. This is the most common and ubiquitous species of Hyalinobatrachium in the GS. The historical circumstances of the holotype combined with the intraspecific variability of some characters have led to a confusing taxonomic situation. The species was described based on a single specimen from Suriname (van Lidth de Jeude 1904). The original description lacks essential details and is very rudimentary; furthermore, the holotype is not very well preserved. Before any other species of Hyalinobatrachium was described or cited for the GS, Goin (1964) considered H. cappellei as a junior synonym of H. fleischmanni. Unfortunately, this implied that till recently (Noonan & Harvey 2000; Cisneros-Heredia & McDiarmid 2007; Kok & Castroviejo-Fisher 2008), other authors have overlooked this species name. We have shown that H. cappellei is different from H. fleischmanni in various qualitative morphological traits and revalidate the name. Biogeographic patterns of centrolenid species also support the idea that they constitute different and independently evolving lineages. To the best of our knowledge, the holotype of H. cappellei was the last remaining vouchered specimen from a locality east of the Andes assigned to H. fleischmanni. Otherwise Hyalinobatrachium fleischmanni is restricted to Central America and the Pacific coast of Colombia and Ecuador and it is separated from the Guiana Shield by the Andes, which is an important barrier for glassfrogs (Guayasamin et al. 2008a). No other centrolenid species is known to occur on lowlands at both versants of the Andes. Our taxonomic results reconcile this biogeographic conundrum. In addition, we could not find evidences of lineage divergence among H. crurifasciatum, H. eccentricum, H. ignioculus, and H. cappellei. Thus we consider them to be the same biological entity. According to the principle of priority (ICZN 1999) we use the name Hyalinobatrachium cappellei. Below we provide a detailed discussion of why the characters used in the original descriptions to diagnose these species are not valid. When Myers and Donnelly (1997) described Hyalinobatrachium crurifasciatum, they emphasized the fact that the pericardium is partially transparent (Fig. 4I), the iris yellow (Fig. 4K) and the nuptial excrescence of Type-I (Flores 1985). In Señaris and Ayarzagüena (2005), the taxonomy of the species was evaluated. Following the original description and after the examination of type material and 35 new specimens from different localities, they concluded that the species is distributed basically throughout the entire Guiana Shield Highlands and some uplands of Venezuela, the pericardium was reported to be either totally transparent or completely white with intermediate



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states (Fig. 4H–I; see also Guayasamin et al. 2006: Fig. 14), SVL = 19.0–24.0 mm in males and 22.1–22.8 mm in females, and the nuptial excrescence Type-I is associated with glands (as described in Ayarzagüena 1992: Fig. 5B). However, Señaris and Ayarzagüena (2005) did not provide any comment on H. ignioculus, a similar species from a close locality in Guyana described two years before.

FIGURES 4. Adult males of Hyalinobatrachium cappellei, resurrected in this work from synonymy with H. fleischmanni. Note the variability in pupil and pericardium coloration. Specimens from French Guiana and Suriname were previously identified as H. taylori. Specimens from Guyana and Venezuela were previously identified as H. crurifasciatum, H. eccentricum, or H. ignioculus. In this work we consider these three species junior synonyms of H. cappellei (see Appendix I for previous identifications). (A) Holotype, SVL = 21.6 mm; (B) RMNH 37850, SVL = 23.6 mm; (C) MNHN 2011.0109, SVL = 25.0 mm; (D) MNHN 2011.0108, SVL = 25.0 mm; (E, H) MTD 48146, SVL = 24.0 mm; (F) MTD 48140, SVL = 24.0 mm; (G, J) MHNLS 17327, SVL = 21.0 mm; (I) MHNLS 17135, SVL = 20.3 mm; (K) MHNLS 17328, SVL = 21.7 mm; (L) MHNLS 17335, SVL = 22.3 mm. Photographs by SCF (A–B, G, I–J) and MB (C–F, H).

Noonan and Bonett (2003) described Hyalinobatrachium ignioculus from the uplands of Guyana. According to the original description of H. ignioculus, the species can be separated from H. crurifasciatum by (characters of the former in parentheses): green limb bands in life present (absent); absence of a reddish ring around the pupil (present); smooth skin on the dorsum in preserved specimens (pustulated); nuptial excrescences Type-I (Type-II); and



adult size in males SVL = 22.0–24.0 mm (20.8–23.0 mm). Although Noonan and Bonett (2003) stated that the pericardium of H. crurifasciatum is white, the original description presented it as partially transparent while white in H. ignioculus. Later, Cisneros-Heredia and McDiarmid (2007) and Kok and Castroviejo-Fisher (2008) suggested that both species might be conspecific. Barrio-Amorós and Castroviejo-Fisher (2008) reported Hyalinobatrachium ignioculus for Venezuela, described its advertisement call, and restricted the morphological differences with H. crurifasciatum to the presence of a reddish ring around the pupil and males not larger than 23.0 mm. When comparing the published data and the new data from this study, we observed that the five qualitative morphological characters (pupil coloration, pericardium, nuptial excrescences, green limb bands, dorsal skin texture, and differences in SVL) used by Noonan and Bonett (2003) to separate Hyalinobatrachium ignioculus from H. crurifasciatum represent either continuous variations with intermediate states or descriptive lapses. Moreover, different combinations of characters states were present in different individuals. Pupil coloration has been used as the key character to separate Hyalinobatrachium ignioculus from H. crurifasciatum. However, our field observations show that this character varies in color and intensity between individuals (Table 1 and Fig. 4D–G, 4J–L; see also Barrio-Amorós & Castroviejo-Fisher 2008: Fig. 4) and even the same specimen can change ring size and color intensity when exposed to light or if disturbed. Subsequently, the presence or absence of a red-brown ring around the iris cannot be used to separate these species. Although the pericardium of H. ignioculus was described as white, the authors pointed out that, in some specimens, small portions of the heart appear reddish after 18 months of preservation and visible through the ventral surface. The paratype of H. ignioculus studied here showed this pattern, which was also observed in type material of H. crurifasciatum. However, we are not aware of any similar preservation artifact in other specimens of H. crurifasciatum. The transparency of the pericardium has therefore to be considered a continuous trait for both taxa with intermediate states (Table 1, Fig. 4H–I) and cannot be used to distinguish between them. All the males examined had nuptial excrescences composed of a group of glands (more or less evident) as described in Ayarzagüena (1992: Fig. 5B), Myers and Donnelly (2001), and Type-V of Cisneros-Heredia and McDiarmid (2007). After careful examination of this character, we concluded that the nuptial excrescences Type-II described for H. ignioculus by Noonan and Bonett (2003), and Type-I for H. crurifasciatum according to Myers and Donnelly (1997) and Señaris and Ayarzagüena (2005) cannot be considered different character states, but just different terminology for the same character. This implies that the type of nuptial excrescences is an invalid character to separate H. ignioculus from H. crurifasciatum. However, the ability to observe nuptial excrescences varies dramatically with the preservation state and the sexual activity of the specimens, and in some cases these structures are not evident. Green limb bands are the product of the dorsal coloration pattern exhibited by H. crurifasciatum and H. ignioculus (yellow or pale green spots in life set in a light green reticulum dotted with dark small and/or minute melanophores) so that when the yellow spots on the hind limbs are sufficiently big they give a pattern of crossbars. Compare Fig. 4C–E (specimens without crossbars) with Fig. 4G (crossbars present). Furthermore, the presence or absence of these crossbars has been observed in both species (Barrio-Amorós & Castroviejo-Fisher 2008; this work); hence, this character is also of no utility to differentiate the species. Texture of dorsal skin varies in both species from slightly shagreened to shagreened (Barrio-Amorós & Castroviejo-Fisher 2008; this work). Moreover, this character is very sensitive to preservation conditions (Kok & Castroviejo-Fisher 2008) and in some species of glassfrogs has been shown to vary with reproductive condition (Harvey & Noonan 2005). The differences in SVL between H. ignioculus and H. crurifasciatum in the study of Noonan and Bonett (2003) seems to be an artifact due to i) the small sample size of H. crurifasciatum (N=3) in their study; ii) the paratype of H. crurifasciatum was the biggest individual found so far (SVL= 24.0 mm); and iii) only two populations were compared, so variability of the species across its distribution was not portrayed. When comparing the data compiled for this work, SVL completely overlapped for males of both species: H. ignioculus (N= 13), SVL= 18.6–23.1 mm; H. crurifasciatum (N= 6), SVL= 20.3–24.7 mm. Hyalinobatrachium eccentricum was described by Myers and Donnelly (2001) for Cerro Yutajé, Amazonas, Venezuela. It is very similar in morphology and vocalizations to H. crurifasciatum and H. ignioculus and is only differentiated by the presence of an “iris bicolored with a brown circumpupillary zone concealing a pupil (pupillary ring absent) and a golden yellow peripheral zone with grey flecks” and “nuptial excrescences not distinctively



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pigmented (not white), comprised of a gland of cells (barely discernible in the holotype) along edge of thenar tubercle on medial base of thumb and distally expanded as a larger and more evident patch of cells on dorsomedial surface of third joint” (Myers & Donnelly 2001: 16–17) and a “group of glands” in the area behind the tympanum which resemble the ones in the hands (Señaris & Ayarzagüena 2005). Careful examination revealed that none of these characters allow separating species. The nuptial excrescences correspond to Type-V (see above) and the “group of glands” in the area behind the tympanum appears in several individuals of H. crurifasciatum (a character that was overlooked, J. Celsa Señaris personal communication to SCF, and that we have observed in males of H. crurifasciatum and H. ignioculus). This type of glands seems to depend on the sexual status of the individual and its preservation. The “iris bicolored with a brown circumpupillary zone concealing a pupil (pupillary ring absent) and a golden yellow peripheral zone with grey flecks” represents a extreme character state in a continuum: from absence of a colored ring with presence of a pupillary ring to presence of a circumpupillary zone with absence of pupillary ring (see Table 1 and Fig. 4D–G, 4J–L). Moreover, two specimens, one from the summit of Auyan-tepui, Bolivar, Venezuela and another from French Guiana lowland rainforest (Fig. 4D, L; see also Barrio-Amorós & Castroviejo-Fisher 2008: Figs 1, 2 for other specimens coming from Venezuela) presented exactly the same kind of iris as described for H. eccentricum, even lacking the pupillary ring (a novel trait for frogs following Myers & Donnelly 2001), refuting the idea that this phenotype corresponds to an endemic from the northwestern tepuys. Señaris and Ayarzagüena (2005), after examining the holotype of H. eccentricum, concluded that it is exactly identical to other specimens of H. crurifasciatum except for having a dark brown iris. However, they assigned two individuals from Cerro Guanay (Bolívar, Venezuela), which present dark brown iris, to H. crurifasciatum, due to their doubts about the authenticity of H. eccentricum as a valid species. Our bioacoustics and genetic analyses are congruent with the morphological results presented here. Biology and tadpole. Data on the number of eggs per clutch is provided in Señaris and Ayarzagüena (2005). Position of calling males and amplectant pairs in Señaris and Ayarzagüena (2005), Myers and Donnelly (1997; 2001) and Noonan and Bonett (2003). Lescure and Marty (2001) and Ernst et al. (2005) report proximity of males to egg clutches. The authors have observed males resting nearby egg clutches during the day in Quebrada Jaspe, Bolivar, Venezuela. In general, males vocalize underneath leaves (but some individuals have been observed on the upper side of leaves at the beginning of calling activity), 1–5 m above the water, and very often close to one or more clutches of eggs (presumably belonging to the same male). Clutches from 12 to 26 eggs are always on the underside of leaves. Advertisement call has been described in Myers and Donnelly (1997; 2001), Lescure and Marty (2000), Señaris and Ayarzagüena (2005), Barrio-Amorós and Castroviejo-Fisher (2008), and this work (Table 3; Fig. 2C–D). The tadpole is described in Myers and Donnelly (1997), Noonan and Bonett (2003), and Señaris and Ayarzagüena (2005). Ecology and distribution. This species is known to occur in the Guiana Shield and the Amazon rainforest. It has a broad distribution, from the summits of several western tepuys (table mountains) to the Gran Sabana and lowland forests (50–2000 m). It has always been found in the vicinity of clear water and often fast flowing streams, perching on vegetation. Specimens have been recorded for Brazil (Rodrigues et al. 2010), French Guiana, Guyana, Suriname, and Venezuela. It is very likely to appear in the Brazilian Guiana Shield (see Myers & Donnelly, 2001: 23).

Hyalinobatrachium iaspidiense (Fig. 5) Centrolenella iaspidiensis Ayarzagüena, 1992: 23. Centrolene iaspidiensis Duellman, 1993: 50. Hyalinobatrachium iaspidiense Myers and Donnelly, 1997: 16. Hyalinobatrachium nouraguensis Lescure and Marty, 2000: 74, synonym. Hyalinobatrachium nouraguense Kok and Castroviejo-Fisher, 2008: 48.

Type locality. Quebrada de Jaspe, San Ignacio de Yuraní, (04°55’N, 61°05’W; 800–1000 m) Bolívar, Venezuela. Diagnosis. (1) Dentigerous processes on vomer and vomerine teeth absent; (2) snout truncate in dorsal and lateral view; (3) tympanum covered by skin, not visible through skin; (4) dorsal skin from smooth to shagreened in life and preservative, (5) presence of small cloacal enameled warts; (6) parietal peritoneum transparent, pericardium transparent, visceral and hepatic peritonea white, all other peritonea transparent; (7) liver bulbous; (8)



humeral spine absent; (9) webbing formula of fingers III (2– – 2) – (2– – 2+) IV; (10) webbing formula of toes I (1 – 1+) – (2+ – 21/3) II (1 – 11/3) – (2+ – 21/4) III (1 – 1+) – (21/2 – 23/4) IV (2+ – 21/4) – (1+ – 11/4) V; (11) enameled ulnar and tarsal folds; (12) nuptial pad Type-V composed by a group of packed glands and situated in the medial, dorso-lateral internal side of Finger I, glands not present in other fingers, prepollex not evident from external view; (13) Finger I longer than Finger II; (14) eye diameter larger than width of disc on Finger III; (15) coloration in life: dorsum light green with big irregular darker green patches, black dots, and minute melanophores, bones white; (16) coloration in preservative: cream with big irregular white patches and black dots; (17) iris yellow with dark grey flecks; (18) minute melanophores not extending throughout fingers and toes except base of Finger IV and Toe V; in life, tip of fingers and toes white; (19) advertisement call composed by a single pulsed note lasting 0.05–0.10 s, dominant frequency of 4220.5–5000.5 Hz, males call from the underside of leaves; (20) fighting behavior unknown; (21) egg clutches deposited on the underside of leaves, males often on the same leaf than eggs; (22) tadpole not described; (23) medium size adult males, SVL = 18.7–22.2 (20.3 ± 0.8, N = 25) mm and one female 22.0 mm. Comparisons. The following unique combination of phenotypic characters differentiates Hyalinobatrachium iaspidiense from all other species in the genus: snout truncated in dorsal and lateral views, tympanic membrane and annulus not appreciable in life, pericardium transparent, hand webbing formula III (2--2) – (2–-2+) IV, dorsal coloration in life light green with big irregular darker green patches and black dots, dorsal coloration in preservative cream with big irregular white patches and black dots, iris coloration in life yellowish, clearing out towards eyelids, with dark flecks, coloration of bones in life white, coloration of hands and feet in life white, and a pulsed single note advertisement call without frequency or amplitude modulation, lasting 0.05–0.10 s and with dominant frequency of 4220.5–5000.5 Hz. Morphological, bioacoustic and genetic evidences allowing the differentiation between Hyalinobatrachium iaspidiense and all other species of Hyalinobatrachium from the GS are summarized in Figures 2, 3, 5 and Tables 1, 3. Remarks. To address the question of whether or not there are morphological differences between Hyalinobatrachium iaspidiense and H. nouraguense, we compared the type material of both species plus additional specimens from Guyana (N = 8), French Guiana (N = 4), Suriname (N = 11), and Venezuela (N = 2). We could not find any difference in the studied characters. Furthermore, we found descriptive lapses in both Lescure and Marty (2000) and Cisneros-Heredia and McDiarmid (2007). These are not preservation artifacts as stated by YáñezMuñoz et al. (2009). Both works described the pericardium of H. nouraguense as white (versus transparent in H. iaspidiense); however, all the specimens examined (including the type series of H. nouraguense and other material from French Guiana) have a transparent pericardium (Fig. 5D). The morphological characters, therefore, do not allow us to separate two species. We also compared the morphology of those specimens with that of the holotype of Hyalinobatrachium mesai (the only known specimen for this species). The only consistent difference was that H. mesai has green bones in life and those of H. iaspidiense and H. nouraguense are white (see also the Remarks section for H. mesai). Our bioacoustic analyses show, in concordance with the morphological data, that there are no divergent features between the calls of H. iaspidiense, H. mesai and H. nouraguense. The description of the call of H. mesai by Barrio-Amorós and Brewer-Carías (2008) indicates that it is longer than that of H. iaspidiense and H. nouraguense. However, the audiospectogram pictured in their publication has too little resolution to be properly compared. Our own analysis indicates that there are no differences in the calls between H. iaspidiense and H. mesai. Cocroft et al. (2001) report an unidentified species of Hyalinobatrachium from the Amazonian foothills of the Peruvian Andes in Manu National Park. They provide a photograph and the advertisement call of a single male. The specimen fully corresponds to the description here provided for H. iaspidiense and the parameters and structure of its advertisement call falls within the variability of H. iaspidiense. Rivera and Knell (2006) report a photograph of a specimen from the Amazonian lowlands of Tapiche, Loreto, Peru, which shares all of its morphological characters with H. iaspidiense. Photographs of six specimens collected from Río Ituxi, Amazonas, Brazil fully agree with the morphological characteristics of H. iaspidiense (J.P. Caldwell, unpublished data). The sequences obtained from tissue samples of these Brazilian specimens included in our study further support their identification (Fig. 3) and corroborate the morphological analysis. Accordingly, we assign all these specimens to H. iaspidiense, extending its distribution from its most western record in Brazil and Peru.



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FIGURES 5. Adult males of Hyalinobatrachium iaspidiense. (A) RMNH 37389, SVL = 20.5 mm; (B) SMNS 12247, SVL = 20.4 mm; (C) MTD 48145, SVL = 22.0 mm; (D) MTD 48147, SVL = 21.0 mm. Photographs by SCF (A), RE (B), MB (C, D).

Biology and tadpole. Very little information is available. Position of calling males and advertisement call are described in Señaris and Ayarzagüena (2005) and Lescure and Marty (2000). Here we provide new information from the known locality in Guyana (Ernst et al. 2005, 2006). Calling males were always found on the underside of leaves, usually facing leaf axils. Up to three males were observed calling from the same tree usually during and after rain. Individuals of Hyalinobatrachium iaspidiense occupied lower forest strata (≈ 1.0–2.0 m) in vegetation overhanging fast flowing segments of the creek, as compared to the sympatric H. mondolfii, whose calling sites were located in higher strata (≈ 4.0–6.0 m). Calling males of H. iaspidiense were found near by (≤ 5 cm) clutches containing 22 (in all cases recorded, N = 4) relatively large white eggs. A clutch containing 22 semi-developed tadpoles (Gosner 1960; stages 21–22) attached to a leaf overhanging a medium sized black water creek was collected on March 15, 2004 (tadpoles deposited at SMNS under field number MABT0104, no collection number assigned), and transferred to a plastic aquarium. The leaf was fastened to the lid of the container, which was filled with 10 cm of creek water. Eleven tadpoles had dropped into the container the following day, nine additional tadpoles followed on March 17. It took an additional day before all remaining tadpoles had left the clutch. The tadpoles of H. iaspidi-



ense are typical exotroph, lotic, fossorial tadpoles (eco-morphological guild after Altig & Johnston 1989) with an elongate, vermiform habitus (Fig. 6B). Compared to the larvae of the sympatric H. mondolfii, tadpoles of H. iaspidiense do not exhibit a clearly ovoid body. Rather, the anterior half is markedly triangular in shape, narrowing towards the base of the tail. Tadpoles of H. iaspidiense are always much darker than those of H. mondolfii (Fig. 6). Ecology and distribution. Hyalinobatrachium iaspidiense inhabits the lowland and upland forests of eastern Guiana Shield (50–1000 m) and western Amazon. It has always been found associated with streams. It is known from Brazil (Cordeiro-Duarte et al. 2002; Yáñez-Muñoz et al. 2009; Avila-Pires et al. 2010; this work), Ecuador (Yáñez-Muñoz et al. 2009; Guayasamin & North 2009), French Guiana (Lescure & Marty 2000; this work), Guyana (Ernst et al. 2005), Peru (Yáñez-Muñoz et al. 2009; this work), Suriname (Kok & Castroviejo-Fisher 2008), Venezuela (Ayarzagüena 1992; Señaris & Ayarzagüena 2005; this work), and expected to occur in the Amazon areas between the Ecuadorian and Peruvian localities and the GS.

FIGURES 6. Tadpoles of (A) Hyalinobatrachium cappellei, (B) H. iaspidiense; (C, D) H. mondolfii. RE (A,B), MB (C,D).

Hyalinobatrachium kawense sp. nov. (Fig. 7) Hyalinobatrachium aff. mondolfii Guayasamin et al., 2008a: 580.

Holotype. MNCN 44825 (field code MB 241), adult male from Rivière de Kaw (04°36'33'' N, 52°03'25'' W; 1–10 m), French Guiana, collected by M. Blanc on February 06, 2005. Paratopotypes. MNCN 44825 (field code MB 252), MNHN 2011.0118 (field code MB 253), MNHN 2011.0119 (field code MB 254), adult males and MTD 48143 (field code MB 259), adult female collected by M. Blanc on April 15 (males) and 16 (female), 2005. Paratypes. MTD 48142 (field code MB 255), adult male from Canal de Kaw (04°30' N, 52°1' W; 1 m) collected by M. Blanc on April 17, 2005; MTD 48144 (field code MB 260), adult male from Crique Gabrielle (04°41' N, 52°18' W; 2–10 m), collected by M. Blanc on May 04, 2005. Diagnosis. (1) Dentigerous process of vomer and vomerine teeth absent; (2) snout truncate in dorsal and lateral view; (3) tympanum covered by skin (not visible through skin); (4) dorsal skin in life and preservative from smooth to slightly shagreened; (5) ventral skin granular, presence of small cloacal enameled warts; (6) parietal peritoneum transparent, pericardium, visceral and hepatic peritonea white, all other peritonea transparent; (7) liver bulbous; (8) humeral spine absent; (9) webbing formula of fingers III 2 – 11/2 IV; (10) webbing on feet I 1 – 2- II 1 – 2 III 2 – 2 IV 2 – 1 V; (11) low and weakly enameled ulnar and tarsal folds; (12) nuptial excrescences Type-V, comprised by a line of individual glands extended throughout the internal side of Finger I, glands not present in other fingers, prepollex not evident from external view; (13) Finger I longer than Finger II; (14) eye diameter larger than width of disc on Finger III; (15) color in life: dorsum with small yellow dots forming a light green reticulum dotted with dark small and minute melanophores, bones white; (16) color in preservative: dorsal surfaces cream with small and minute dark melanophores and white minuscule dots due to absence of pigments; (17) iris golden towards the pupil but with small white spots in external areas and black reticulation; (18) minute melanophores not extending



23

throughout fingers and toes except on the base of Finger IV and Toe V; in life, tip of fingers and toes white; (19) advertisement call composed by a single note, first half pulsed and second tonal with increasing frequency, lasting 0.08–0.09 s, dominant frequency of 5622.5–6065.9 Hz, males call from upper and underside of leaves; (20) fighting behavior unknown; (21) egg clutches deposited on underside of leaves, no parental care observed; (22) tadpole unknown; (23) adult size between 19.9–20.0 (N = 2) mm in males and 20.0 mm in one female. Comparisons. Following Guayasamin et al. (2009), we place the new species in the genus Hyalinobatrachium because of the following characters: humeral spine absent in adult males, digestive tract and bulbous liver covered by white peritonea, completely transparent ventral parietal peritoneum, white bones in life, cream dorsal coloration in preservative, males lack conspicuous dorsal spicules during breeding season, nuptial pad small and restricted to the inner edge of Finger I in males (Type V), dentigerous process of the vomer and vomerine teeth absent, males vocalize from the undersides of leaves, and females deposit one layer of eggs on the undersides of leaves. Hyalinobatrachium kawense sp. nov. most notably differs from all recognized species of Hyalinobatrachium in the following combination of morphological characters: snout truncate in dorsal and lateral views, bones white in life, dorsum with melanophores of two sizes (small and minute) in life and preservative, white pericardium, fingers and toes white, webbing formula of fingers III 2 – 11/2 IV, advertisement call composed by a single note, first half pulsed and second tonal with increasing frequency. Other relevant morphological, acoustic and genetic data are summarized in Tables 1, 3 and Figures 2, 3, 7. Additionally, Hyalinobatrachium kawense sp. nov. is similar in morphology and bioacoustics to H. carlesvilai, H. fleischmanni and H. tatayoi. However it can be differentiated from these three species due to the presence of white fingers and toes in H. kawense sp. nov. (yellow in H. carlesvilai, H. fleischmanni and H. tatayoi). Remarks. Our molecular data support Hyalinobatrachium kawense sp. nov. as a valid species because their sequences are reciprocally monophyletic with regard to all the other Hyalinobatrachium species analyzed (Fig. 3) and are relatively divergent (genetic distance ≥ 7 %). Guayasamin et al. (2008a) found strong support for H. kawense sp. nov. (referred therein as H. aff. mondolfii) to be a member of a clade also including H. carlesvilai (referred therein as H. cf. munozorum from Bolivia), H. fleischmanni, H. mondolfii, H. munozorum and H. tatayoi. Nonetheless, its exact phylogenetic position within this clade could not be inferred with strong support (Guayasamin et al. 2008a). Description of holotype. Adult male of small size, SVL 20.0 mm; head wider than body, HW 30 % of SVL; head wider than long (HW/HL = 1.3); snout truncate in dorsal view and profile; ES/EL = 0.9 and ES/IOD = 1.2; loreal region concave; nostrils prominent, round; internarial region depressed; canthus rostralis defined; eyes small, directed antero-laterally; EL 40 % of HL; tympanic annulus indistinct, tympanic membrane absent, supratympanic fold absent; dentigerous processes on vomers absent; choanae, circular, separated; tongue elongate, ovoid, not attached to mouth posteriorly for about one sixth of its length; vocal slits extending from the sides of the base of tongue to the level of the mandibular joints. Forearms slim; diameter of forearms about one and a half times the diameter of upper arms; low and enameled ulnar fold; humeral spine absent; relative length of fingers: II < I < IV < III; finger discs wide, truncated and larger than those of toes; FIII 40 % of EL; webbing absent between fingers I–II and basal between II–III, webbing formula on hand III 2 – 11/2 IV; subarticular tubercles round and small; supernumerary tubercles slightly appreciable; palmar tubercle round, thenar tubercle elongated; nuptial excrescences Type V, glands on the lateral fringes of fingers and the sides of membrane between fingers III and IV not observed; hind limbs slender; TL 50 % of SVL; low and enameled tarsal fold; discs of toes round, truncate in profile; inner metatarsal tubercle small and ovoid; outer metatarsal tubercle absent; supernumerary tubercles difficult to observe; webbing formula of feet I 1 – 2- II 1 – 2 III 2 – 2 IV 2 – 1 V. In preservative, dorsal skin scarcely covered with enameled granules, area around tympanum almost granular; skin on belly and thighs granular, other ventral surfaces smooth; cloacal opening directed posteriorly at upper level of thighs, concealed by a dermal fold and flanked by small and enameled irregular folds and warts. Color in life. Dorsal surfaces light green with dull yellow spots and dusted with melanophores of two sizes, minute and small. Low and enameled tarsal and dorsal folds. Cloacal ornamentation consisting of enameled warts and folds. Fingers and toes white. Iris golden with small white areas towards the eyelids and with greyish-brown dots spreading concentrically from pupil, where they appear at higher density. Parietal peritoneum transparent, pericardium, hepatic, and visceral peritonea white, peritoneum covering the gall bladder white, peritonea of all other internal organs not mentioned before transparent.



FIGURES 7. Adult males of Hyalinobatrachium kawense sp. nov. (A, B) holotype, MNCN 44825, SVL = 20.0 mm; (C) MNCN 44825, SVL = 19.9 mm; (D) MTD 48142; SVL = 19.0 mm; (E) MTD 48143, SVL = 20.0 mm; (F) egg clutch on the underside of a leaf.

Color in preservative. General appearance cream. Dorsal surfaces dotted by a coat of minute and small dark melanophores, which leave uncovered cream spots. Dorsum with a fine layer of iridiophores only appreciable under magnification. Low enameled tarsal, ulnar and cloacal folds. Iris white. Other surfaces cream. Peritonea as stated above.



25

Variation. Measurements and body proportions of the holotype and paratopotype MNCN 44826 are presented in Table 2. Transparent peritoneum covering the gall bladder in paratypes MNHN 2011.0118, MTD 48142 and MTD 48143 while white in the other specimens. Paratopotype MNHN 2011.0119 has a darker appearance in its dorsal coloration due to a higher concentration of melanophores. Advertisement call. A single high-pitched note that was clearly audible at long distances, the first half being pulsed and the second tonal (Fig. 2B). It lasts 0.08–0.09 seconds (0.09 ± 0.01). The dominant frequency is 5622.5– 6065.9 Hz (5825.5 ± 117.9) and the call rises in frequency from 4200.0–4992.0 Hz (4643.05 ± 214.1) at the beginning of the call to 6093.0–6482.0 Hz (6257.05 ± 118.2) at the end. The first half of all recorded calls starts with a group of short pulses (6–11) that shows a fast rise in frequency. The following half of the call consists of a tonal section at slightly increasing frequency. The maximum amplitude of the call is reached at the beginning of the tonal section. Etymology. The name refers to the type locality Rivière de Kaw, close to the Atlantic coast of French Guian. This is so far the only known locality where the species has been recorded. Biology and tadpole. Males generally call above leaves (occasionally on the underside) of moucou-moucous (Montrichardia sp.) 3–4 m above the water. One male was observed calling (recorded) from the upper side of a Pachira aquatica leaf. When a gravid female with white eggs approached the male, this attempted to amplect with the female without success. Males form groups of about 10 individuals dispersed approximately every 100 m along the Kaw River. Eggs are laid on the underside of leaves; a clutch containing 33 eggs was photographed (Fig. 7F). Ecology and distribution. This species has only been found in the flooded forest of Rivière de Kaw in French Guiana.

Hyalinobatrachium mesai Barrio-Amorós and Brewer-Carías, 2008: 18.

Type locality. Southern slope of Sarisariñama-tepui (04°25’ N, 64°7’ W; 420 m), Bolívar, Venezuela. Diagnosis. (1) Dentigerous processes on vomer and vomerine teeth absent; (2) snout truncate in dorsal and lateral view; (3) tympanum covered by skin, not visible through skin; (4) dorsal skin shagreened in life and preservative; (5) presence of small cloacal enameled warts; (6) parietal peritoneum transparent, pericardium transparent, visceral and hepatic peritonea white, all other peritonea presumably transparent (not dissected); (7) liver bulbous; (8) humeral spine absent; (9) webbing formula of fingers III 21/3 – 2+ IV; (10) webbing formula of toes I 1 – 22/3 II 1 – 21/2 III 1 – 2 IV 2 – 1 V; (11) low and enameled ulnar and tarsal folds; (12) nuptial pad inconspicuous, prepollex not evident from external view; (13) Finger I longer Finger II; (14) eye diameter larger than width of disc on Finger III; (15) coloration in life: dorsum light green with big irregular darker green patches, black dots, and minute melanophores, bones green; (16) coloration in preservative: cream with big irregular white patches and black dots; (17) iris white with black flecks; (18) minute melanophores not extending throughout fingers and toes except base of Finger IV and Toe V; in life, tip of fingers and toes unknown; (19) advertisement call composed by one to two notes, each lasting 0.075 s, dominant frequency of 4414.5 Hz, one male observed to call from the upper side of a leave; (20) fighting behavior unknown; (21) egg clutches unknown, parental care unknown; (22) tadpole unknown; (23) adult size 20.0 mm in one male, unknown in females. Comparisons. This species can only be differentiated from Hyalinobatrachium iaspidiense by the presence of white bones in the later (versus green in H. mesai). All other characters compared are identical (see below for more details). Remarks. This species is only known from the holotype, an adult male. Although the original description (Barrio-Amorós & Brewer-Carías, 2008) listed seven differences between Hyalinobatrachium iaspidiense and H. mesai the authors did not directly compare it with material of H. iaspidiense but rather follow descriptions in the literature. We have identified descriptive lapses in their report of character states of H. iaspidiense and their comparison with those of H. mesai (in parentheses character states used by the authors): both species show low and enameled ulnar and tarsal folds (H. iaspidiense has folds but not H. mesai); as in most species of glassfrogs H. iaspidiense has a thenar tubercle, although low and difficult to appreciate (thenar tubercle absent in H. iaspidiense but present in H. mesai); the ventral skin of H. iaspidiense is granular and not smooth, we did not observe differences



with H. iaspidiense when we examined the holotype of H. mesai (ventral skin smooth in H. iaspidiense but areolate in H. mesai); the canthus rostralis of both species is similar and we did not find differences that would suggest considering them different character states (well defined in H. iaspidiense not well defined in H. mesai); webbing formula of toes is basically identical (toes approximately two-thirds webbed in H. mesai versus three-fourths in H. mesai); we did not find differences in the shape of the choanae, which is approximately oval in both species (choanae trilobate in H. iaspidiense and oval in H. mesai). Furthermore, the re-analysis of the recorded call revealed that it is identical to that of H. iaspidienses (Fig. 2E–G). The only consisted divergent character is color of bones in life specimens, which is white in H. iaspidiense but green in H. mesai. Although there is no record in the literature of intraspecific polymorphism of bone colors in Hyalinobatrachium, the fact that H. mesai is just known from a single specimen for which there are no ventral photographs in live (color of bones is lost in preservative) does not allow us to evaluate the validity of this character. Unfortunately, we could not amplify DNA from a tissue sample of the specimen. Thus and till more data are available, we consider H. mesai as a valid species but its status is pending revaluation. Ecology and distribution. Only known from the type locality in the Venezuelan GS.

Hyalinobatrachium mondolfii (Fig. 8) Señaris and Ayarzagüena, 2001: 1084. Hyalinobatrachium sp. 1 Ernst et al.,2005: 183.

Type locality. First stream of Caño Acoima, tributary of Río Grande, slopes of Serranía de Imataca (08°22’N, 61°32’W; 15 m), Delta Amacuro, Venezuela. Diagnosis. (1) Dentigerous process of vomer and vomerine teeth absent; (2) snout round in dorsal and lateral view; (3) tympanum covered by skin (not visible through skin); (4) dorsal skin from smooth to shagreened in life and preservative; (5) ventral skin granular, presence of small cloacal enameled warts; (6) parietal peritoneum transparent, pericardium, visceral and hepatic peritonea white, all other peritonea transparent; (7) liver bulbous; (8) humeral spine absent; (9) webbing formula of fingers III 2 – (1+ –11/2 ) IV; (10) webbing on feet I (11/4 – 1) – (13/4 – 11/4) II (11/4 – 1) – (2 – 11/2) III (11/4 – 1) – (2 – 13/4) IV (2 – 11/2) – (11/4 – 1) V; (11) enameled ulnar and tarsal folds; (12) nuptial excrescences Type-V, formed by a line of individual glands extended throughout the internal side of Finger I, this glands are also present in the lateral fringes of all fingers except the internal one of Finger III, prepollex not evident from external view; (13) Finger I longer than Finger II; (14) eye diameter larger than width of disc on Finger III; (15) color in life: dorsum with small yellow dots forming a light green reticulum dotted with dark and minute melanophores that in some specimens are more concentrated in the upper eyelid and specially around and between the nostrils where they form a brownish irregular patch, bones white; (16) color in preservative: dorsal surfaces cream with very small purple melanophores and white minuscule dots due to concentration of iridiophores that may or may not be lost; (17) irisgolden with small white spots and greyish-brown dots; (18) minute melanophores not extending throughout fingers and toes except base of Finger IV and Toe V; in life, tip of fingers and toes yellow; (19) advertisement call composed by a single tonal note lasting 0.18–0.25 s, dominant frequency of 4989.6–5168.0. Hz, males mostly call from the underside of leaves; (20) fighting behavior unknown; (21) egg clutches deposited on the underside of leaves, males often on same leaf than eggs; (22) tadpole with sinistral spiracle, on longitudinal axis of the body and directed posteriorly; short and medial vent tube; (23) adult size between 19.9–22.8 (21.3 ± 0.8, N = 12) mm in males and 20.3–22.5 mm in two females. Comparisons. The following unique combination of phenotypic characters differentiates Hyalinobatrachium mondolfii from all other species in the genus (but see below for exceptions): snout rounded in dorsal and lateral views, tympanic membrane and annulus not appreciable in life, pericardium white, hand webbing formula III 2 – (1+-11/2) IV, dorsal coloration in life light green with small yellow dots and minute melanophores, dorsal coloration in preservative pale cream dotted with minute melanophores and cream dots (could be lost in some specimens), iris coloration in life yellowish reticulated by dark flecks, coloration of bones in life white, coloration of hands and feet in life yellow, and a tonal single advertisement call without frequency modulation, lasting 0.18–0.25 s and with dominant frequency of 4989.6–5168.0 Hz



27

FIGURES 8. Adult males of Hyalinobatrachium mondolfii. (A) Topotype, MHNLS 17122, SVL = 22.2 mm; (B) MNHN 2011.0126, SVL = 23.0 mm; (C) MTD 48148, SVL = 22.0 mm; (D, E) SMNS 12255, SVL = 20.7 mm; (F) egg clutch on the underside of a leaf. Photographs by SCF (A), MB (B–C, F) and RE (D–E).

Morphological, bioacoustic and genetic evidence allowing the differentiation between Hyalinobatrachium mondolfii and all other species of Hyalinobatrachium from the GS are summarized in Figs 2, 3, 8 and Tables 1, 3. The species H. munozorum (Lynch & Duellman) and H. ruedai Ruiz-Carranza & Lynch are, as far as we know,



morphologically undistinguishable among each other and with respect to H. mondolfii. Furthermore, their distributions are overlapping or adjacent with that of H. mondolfii. Castroviejo-Fisher et al. (2011) showed that H. munozorum and H. mondolfii might constitute independent evolving lineages on the basis of DNA sequences and, possibly, advertisement calls. Castroviejo-Fisher et al. (2011) also noted that their results should be interpreted with caution because sample size was particularly limited and the purported call of H. munozorum lacks a clear voucher specimen. Hyalinobatrachium ruedai is just known for the type series and there are no DNA sequences, tissue samples and/or bioacoustic information. With the data at hand we cannot resolve with enough confidence the taxonomic status of H. munozorum, H. mondolfii, and H. ruedai. Remarks. A group of specimens coming from Guyana (Ernst et al. 2005) shows morphological similarities with Hyalinobatrachium mondolfii from the type locality in Venezuela, but with variations in color (in life and preservative). The fragment of mtDNA studied shows that both phenotypes share the same haplotype and they cluster together forming a clade. Also, their advertisement calls show no differences. We consider both phenotypes to belong to the same species and consequently assign them to H. mondolfii. The new population differs in the following characters (in parentheses characters for this population): golden iris (iris golden with small white areas and with greyish-brown dots spreading concentrically from pupil, where they appear at higher density); without concentrations of melanophores in dorsal surfaces (melanophores more concentrated in the upper eyelid and specially around and between the nostrils where they form a brownish irregular patch). Señaris and Ayarzagüena (2005) described Hyalinobatrachium mondolfii as having granular dorsal skin and very granular skin around the tympanum. However, all specimens of H. mondolfii that we have studied (even those coming from the type locality of H. mondolfii) have slightly shagreened dorsal skin and granular skin in the tympanic area. This could indicate that skin texture varies between individuals or that this dorsal granular skin appears at particular ontogenetic/reproductive states, as shown for skin structures in some species of Cochranella (e.g. Harvey and Noonan 2005). Hence, dorsal granular skin does not appear to be a distinctive character to differentiate H. mondolfii from other species of Hyalinobatrachium from the GS as suggested by Señaris and Ayarzagüena (2005). Additional records of Hyalinobatrachium mondolfii in Guyana come from two localities in Iwokrama Forest: Turtle mountain (04° 43' 51.5" N, 58° 43' 27.2" W; MTD 47920, male) and the field Station (04° 40' 17.46" N, 58° 41' 06.56" W, MTD 47920, 48189–92, all males except MTD 48190, female). In January 2010 we found three specimens of Hyalinobatrachium mondolfii in Quebrada El Sufragio (04°0'11.42" S, 69°53'44.05" W; 116 m), Leticia, Amazonas, Colombia that represent the first record for the country. The three specimens were adult males that where calling (advertisement calls recorded) from the upper side of leaves. Although the data is not included in the analyses here presented, direct comparison with other specimens and calls of H. mondolfii allowed us to assign them to this species. Lynch (2005) also found this species in the surroundings of Leticia and reported it as Hyalinobatrachium sp. Specimens reported as Cochranella sp. for Para, Brazil (Avila-Pires et al. 2010) are here identified as members of Hyalinobatrachium mondolfii. This identification was based on photographs generously provided by M. S. Hoogmoed. Biology and tadpole. Señaris and Ayarzagüena (2005) provide information about the position of calling males, number of eggs per clutch and advertisement call in Venezuela. Description of the advertisement call from specimens from Guyana and Venezuela is provided in Table 3 and Fig. 2. Information from the new population from Guyana is as follows: males have frequently been observed next to their nesting sites. Clutches at varying stages of development (usually two) were found on the underside of leaves with a single calling male. Nest and associated calling sites were usually located at heights of 4–5 m in trees overhanging fast flowing segments of medium sized black water creeks, particularly Maiko Creek and its tributaries, Mabura Hill Forest Reserve (MHFR). There is no indication of males staying by the clutches during the day. In two cases calling sites were revisited during the day, however, only “abandoned” clutches without males were encountered. Whether or not males revisited these clutches in following nights was not confirmed. Up to five males were recorded calling from the same tree usually during and after rain. Clutches consist of transparent gelatinous circular masses containing eggs 17–24 (19.6 ± 2.7; N = 5). A calling male (SMNS 12256) recorded on April 26, 2003, at the MHFR, was observed in the proximity (≤ 5 cm) of two clutches of different developmental stages. One clutch contained 19 eggs at stages 18–19 (Gosner 1960), whereas the second clutch contained 17 undeveloped eggs. Egg and jelly color did not differ between these ontogenetic stages. The largest clutch recorded contained 24 eggs, at developmental stage 21–22 (Gosner 1960), and tadpoles were about to hatch. This clutch was located on the underside of a leaf in a small tree overhanging Maiko Creek and was guarded by a calling male (SMNS 12258). A clutch guarded by a calling male (SMNS 12257) was collected in March 2004 and



29

contained 19 eggs at stage 20–21. The clutch was transferred to a plastic terrarium and tadpoles were preserved in 70 % alcohol after hatching (no collection number assigned, SMNS 12257 also refers to the tadpole series). An amplectant pair (SMNS 12259–60) that was caught in May 2004 and subsequently transferred to a plastic aquarium, remained in amplexus for several hours before spawning. The clutch, containing 19 eggs, was attached to the wall of the container but was apparently not fertilized as eggs did not develop in consequent days and finally started molding. Tadpole description. All examined specimens (N = 18) belong to a single clutch (SMNS 12257); stages 25– 26 of Gosner (1960). Typical exotroph, lotic, fossorial tadpole (eco-morphological guild after Altig and Johnston, 1989). Elongate, vermiform habitus (Fig. 6). Body ovoid, longer than wide (length approximately twice the width; BL/BW = 2.0), depth less than width (depth approximately half the width; BH/BW = 0.6); snout rounded in dorsal and lateral view; eyes dorsal, near sagittal line; pupils visible in dorsal view; dorsal surface flattened, oral disc anteroventral; belly flat; nostrils not protuberant, spiracle sinistral, on longitudinal axis of body, hardly visible, directed posteriorly; spiracular tube short (approx. 1 mm) triangular; cloacal tube medial, short; caudal musculature robust; caudal fins about half the width of caudal musculature at widest point (ventrally and dorsally), narrower at origin of tail muscle, expanding distally. Tail tip slightly pointed. Measurements are presented in Table 4. TABLE 4. Morphological features (mean ± SD, range) of Hyalinobatrachium mondolfii tadpoles (N = 18, stages 25–26). TL= total length; BL = body length; TAL = tail length; BW = body width; BH = body height; ED = horizontal eye diameter; IOD = interorbital distance; ESD = eye snout distance; TMH = tail muscle height; TH = total tail height; TFH = dorsal tail fin height. Parameter

Average ± SD

Range

TL

14.9 ± 1.08

13.0–17.0

BL

4.7 ± 0.49

4.0–5.5

TAL

10.2 ± 0.69

9.0–11.5

TAL/TL

0.7 ± 0.02

0.66–0.71

BW

2.3 ± 0.14

2.1–2.5

BH

1.3 ± 0.14

1.1–1.5

ED

0.2 ± 0.02

0.2–0.3

IOD

0.6 ± 0.04

0.5–0.6

ESD

1.1 ± 0.09

1.0–1.2

TMH

1.0 ± 0.07

0.8–1.1

TH

1.8 ± 0.22

1.5–2.1

TFH

0.4 ± 0.10

0.3–0.6

FIGURE 9. Oral disc of the tadpole of Hyalinobatrachium mondolfii.



Oral disc large with well-developed anterior and posterior folds; posterior fold strongly fringed, anterior fold only distinctly fringed on lateral edges; both anterior and posterior jaw sheath wide and heavily serrated. Tooth rows are distinctly keratinized. Labial tooth row formula 2(2)/3. Labial papillae are large and irregularly fringed especially on posterior labium. Size reduced on anterior labium. Oral disc emarginated (Fig. 9). Tadpoles of Hyalinobatrachium mondolfii are easily distinguishable from tadpoles of the two sympatric centrolenid species (H. cappellei and H. iaspidiense) by their bright pink color in life (Fig. 6), probably due to high concentrations of haemoglobin visible through large blood sinuses, indication of an adaptation to fossorial lotic habits. Color in preservative yellowish-white. Dorsal surface of body covered with dark brown melanophores; entire length of dorsal and caudal fins clear, however, covered with irregular but distinct white-pigmented spots bordering tail musculature. Ventral skin transparent. Eyes appear as sickle-shaped black-pigmented ring; lens off-white. Ecology and distribution. The species inhabits lowland rain forest of the eastern Guiana Shield (15–200 m) and the western Amazon. It has exclusively been found in vegetation associated with streams. Here we showed that Hyalinobatrachium mondolfii has a wider distribution than originally thought, occurring in Bolivia (CastroviejoFisher et al. 2011), Brazil (this work), Colombia (this work), French Guiana (this work), Guyana, Suriname (this work) and Venezuela. Hyalinobatrachium mondolfii, like H. iaspidiense, has a broad distribution through the lowland Amazon rainforests.

Hyalinobatrachium taylori (Fig. 10) Centrolenella taylori Goin, 1968: 115. Hyalinobatrachium taylori Ruiz-Carranza and Lynch, 1991: 25.

Type locality. Along New River, 228.6 m, Guyana. Diagnosis. (1) Dentigerous processes of vomer and vomerine teeth absent; (2) snout round in dorsal view, sloping in lateral view; (3) tympanum small, appreciable in the lower portion; (4) dorsal skin from smooth to slightly shagreened in life and preservative; (5) ventral skin granular, presence of small cloacal enameled warts; (6) parietal peritoneum transparent, pericardium white with small transparent areas, visceral and hepatic peritonea white, all other peritonea transparent; (7) liver bulbous; (8) humeral spine absent; (9) webbing formula of fingers:III (11/2 – 2-) – (1+ – 11/2) IV; (10) webbing on foot: I (1 – 1+) – (2 – 2+) II (1 – 1+) – (2- – 2) III (1 – 1+) – (21/3 – 21/2) IV (2 – 21/4) – (1 – 11/3) V; (11) enameled ulnar and tarsal folds; (12) nuptial excrescences of Type-V composed of a cluster of glands and situated in the medial, dorsolateral internal side of Finger I and extended externally reaching the base of the disc, glands are also present in webs and fringes of fingers II, III and IV, prepollex not evident from external view; (13) Finger I longer than Finger II; (14) eye diameter larger than width of disc on Finger III; (15) coloration in life: dorsum dark green with pale green spots usually bearing a white fleck (iridiophores) in the center of each spot, bones green; (16) coloration in preservative: dorsum cream with very small purple melanophores making a “pale lavender coat” and white flecks (iridiophores); (17) iris grey with a black reticulate net; (18) minute melanophores not extending throughout fingers and toes, except base of Finger IV and complete Toes V and IV; in life, tip of fingers and toes orange; (19) advertisement call composed of 5–9 tonal notes lasting 0.652– 1.052 s, and a dominant frequency between 4010.00–4350.00 Hz, males call on the upper side of leaves; (20) fighting behavior unknown; (21) egg clutches deposited on the underside of leaves, parental care not observed in males; (22) tadpole with sinistral spiracle, on the last third of the body and directed posteriorly; very short and medial vent tube; (23) adult size between 18.1–19.4 (19.0 ± 0.5, N = 5) mm in males and 20.6 mm in one female.



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FIGURE 10. Adult males of Hyalinobatrachium taylori. (A) Holotype, BMNH 1939.1.1.65, SVL = 19.0 mm; (B) IRSNB 13987, SVL = 19.0 mm; (C, D) MNHN 2011.0112; SVL= 21.0 mm. Photographs by PK (A–B) and MB (C–D).

Comparisons. The following unique combination of phenotypic characters differentiates Hyalinobatrachium taylori from all other species in the genus: snout rounded and sloping in dorsal and lateral views respectivelly, tympanic membrane and annulus appreciable in life, pericardium white with transparent spots, hand webbing formula III (11/2-2-) – (1+-11/2) IV, dorsal coloration in life dark green with small white dots, dorsal coloration in preservative cream with minute purple melanophores making a “lavender coat” and cream dots, iris coloration in life grey with a black reticulated net, coloration of bones in life green, coloration of hands and feet in life orange, and a tonal 5–9 notes advertisement call without frequency modulation, lasting 0.65–1.05 s and with dominant frequency of 4010.0–4350.0 Hz. Morphological, bioacoustic and genetic evidences allowing the differentiation between Hyalinobatrachium taylori and all other species of Hyalinobatrachium from the GS are summarized in Figures 2, 3, 10 and Tables 1, 3. Remarks. The morphological study of the complete type series of H. taylori (N = 5) indicated the presence of three differentiated phenotypes. The holotype (Fig. 10A) is unique among the type specimens and is characterized by its slender appearance, snout round in dorsal view and sloping in profile, pericardium partially white (dissected), dorsal coloration pale lavender with small unpigmented areas, webbing formula between external Fingers III 2 – 1– IV, and presence of glands in fringes between fingers. This combination of characters is unique among all the known species of Hyalinobatrachium and is only found in those specimens identified by Ayarzagüena (1992), Señaris and Ayarzagüena (2005), Kok and Castroviejo-Fisher (2008), and Kok and Kalamandeen (2008) as H. taylori (Fig. 10B–D). Furthermore, we have identified three specimens from French Guiana (Fig. 10C–D, Appendix I) that show a phenotype concordant with that of H. taylori. All of these specimens for whom the character could be



observed, show green bones in life. The coloration patterns observed in life specimens from Guyana, French Guiana, and Venezuela clearly allows distinction from any other species observed in the area. It shows green bones in life, dark green dorsal surfaces with small clear green areas and white flecks due to concentration of iridiophores, partially transparent pericardium, and metallic lavender iris with dark brown reticulations and a few bronze punctuations (Fig. 10B–D). Also, males call exclusively from the upper side of leaves while in all other species but H. kawense sp. nov. males call predominantly from the underside of leaves. Two paratypes of Hyalinobatrachium taylori (RMNH 11473–4) show a more robust appearance, snout truncated in dorsal and lateral view, transparent pericardium (dissected in both), dorsal coloration cream, webbing formula between external Fingers III 2 – 2 IV, and absence of glands in fringes between fingers. This combination of characters corresponds to the one exhibited by the holotype of H. cappellei (see above and Fig. 4A); accordingly we assign RMNH 11473–4 to this species. Also, specimens from French Guiana (Fig. 4C–F, H) and Suriname, identified by Lescure (1975), Hoogmoed and Avila-Pires (1990), and Lescure and Marty (2000) as Hyalinobatrachium taylori, and those from Guyana referred to as H. sp2 by Ernst et al. (2005) and as H. aff. ignioculus by Guayasamin et al. (2008a) (see specimens under H. cappellei in the Appendix I) share with the holotype of H. cappellei all of the morphological characters studied. Finally, the two other paratypes (BM 1939.1.1.64, RMNH 11472) have a snout round dorsal and lateral view, white pericardium (dissected in both), dorsal coloration cream, and hand webbing formula III 2 – 1 IV. These specimens plus four from French Guiana and one from Suriname share all of the studied morphological characters with Hyalinobatrachium mondolfii (Fig. 8); thus we assigned them to this species. We have detected a genetic gap between the populations of Hyalinobatrachium taylori (sensu this work) of Gran Sabana in Venezuela and the ones from French Guiana (genetic distance = 3 %). If these two populations represent different species, most likely the ones from the lowlands (Guyana and French Guiana) would correspond to H. taylori while the ones from the uplands and highlands of Venezuela would need a name. However, our sample size is small (N = 4) and we miss sequences from intermediate localities (Guyana) so we cannot exclude that processes like isolation by distance could be causing the observed pattern. Hyalinobatrachium taylori is unique among species belonging to the same genus because has bones green in life, an iris grey with black reticulation, dorsum dark green, and males only call from the upper side of leaves. These characters are shared with species like Vitreorana antisthenesi, Teratohyla pulverata, and T. amelie and led Señaris and Ayarzagüena (2005) to place H. taylori outside the Hyalinobatrachium fleischmanni Group (Ruiz-Carranza & Lynch 1991, 1998). Guayasamin et al. (2008a, 2009) conducted phylogenetic analyses of six genetic markers and showed that H. taylori clusters within the diversity of Hyalinobatrachium species previously assigned to the fleischmanni Group. Biology and tadpole. Señaris and Ayarzagüena (2005) described the position of calling males, number of eggs per clutch, advertisement call, and tadpole. Field observations by SCF, MB, and P. Kok (Venezuela, French Guiana, and Guyana respectively) confirm these observations. Ecology and distribution. Normally uses the high strata of vegetation surrounding streams for reproduction (2–30 m over the water). Endemic to the Guiana Shield, it has a wide distribution from the top of western tepuys to the eastern GS (200–2000 m). It has been found in French Guiana, Guyana, and Venezuela. Most likely occurs in Suriname (see Noonan & Bonett 2003 for a discussion about the type locality) and adjacent Brazil.

Hyalinobatrachium tricolor sp. nov. (Fig. 11) Hyalinobatrachium nouraguense Lescure and Marty, 2000: 74. Hyalinobatrachium aff. iaspidiense Guayasamin et al., 2008a: 580.

Holotype. MNCN 44827 (field code MB 250), adult male from Crique Wapou (04°26' N, 52°9' W; 2 m), Kaw, French Guiana, collected by M. Blanc on April 11, 2005. Paratopotypes. MNHN 2011.0116 (field code MB 247), MTD 48141 (field code MB 249), adult males collected by M. Blanc on April 4 and 11, 2005, respectively. Paratype. MNCN 44828 (field code MB 326), adult male from Kaw Mountain (04°32' N, 52°13' W; 10 m), French Guiana, collected by M. Blanc on January 18, 2008. HYALINOBATRACHIUM SPECIES OF THE GUIANA SHIELD


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FIGURE 11. Hyalinobatrachium tricolor sp. nov. from Kaw, French Guiana. (A, B) holotype, MNCN 44827, SVL = 20.3 mm; (C) paratopotype MNHN 2011.0116, SVL = 21.0 mm; (D, E) paratype MNCN 44828, SVL = 20.7 mm; (F) egg clutch on the underside of a leafcollected with MNCN 44828. Photographs by SCF (A) and MB (B–F).

Diagnosis. (1) Dentigerous processes on vomer and vomerine teeth absent; (2) snout truncated in dorsal and lateral view; (3) tympanum covered by skin, not visible through skin; (4) dorsal skin shagreened in life and preservative, (5) presence of small cloacal enameled warts; (6) parietal peritoneum transparent, pericardium transparent, visceral and hepatic peritonea white, all other peritonea transparent; (7) liver bulbous; (8) humeral spine absent; (9) webbing formula of fingers III 2 – (2 – 2+) IV; (10) webbing formula of toes I 1 – 2 II 1 – 2 III 1 – 2 IV 2 – 1 V; (11) enameled ulnar and tarsal folds; (12) nuptial pad Type-V, composed by a group of packed glands and situated in the medial, dorso-lateral internal side of Finger I, glands not present in other fingers, prepollex not evident from external view; (13) Finger I longer than Finger II; (14) eye diameter larger than width of disc on Finger III; (15) coloration in life: dorsum light green with big irregular darker green patches, brown warts, and minute melanophores, bones white; (16) coloration in preservative: cream with big irregular white patches and black dots; (17) iris yellow



with dark grey flecks; (18) minute melanophores not extending throughout fingers and toes except base of Finger IV and Toe V; in life, tip of fingers and toes white; (19) advertisement call composed by four tonal notes but with a fast increase of energy at the beginning of each note lasting 0.19–0.20 s, dominant frequency of 4628.11–4903.07 Hz, males call from the underside of leaves; (20) fighting behavior unknown; (21) egg clutches deposited on the underside of leaves, number of eggs per clutch 22 (N = 2), no parental care observed; (22) tadpole unknown; (23) medium size adult males, SVL = 20.3–21.0 mm (N = 4), females unknown. Comparisons. Following Guayasamin et al. (2009), we placed the new species in the genus Hyalinobatrachium because of the following characters: humeral spine absent in adult males; digestive tract and bulbous liver covered by white peritonea; completely transparent ventral parietal peritoneum; white bones in life; dorsal coloration cream in preservative; males lack conspicuous dorsal spinules during breeding season; nuptial pad small and restricted to the inner edge of Finger I in males (Type V); dentigerous process of the vomer and vomerine teeth absent; males vocalize from the undersides of leaves, and females deposit one layer of eggs on the undersides of leaves. The following unique combination of phenotypic characters differentiates Hyalinobatrachium tricolor sp. nov. from all other species in the genus: bones white in life, coloration in life lime green patches over a light green dorsum and brown warts, and advertisement call composed by four tonal notes lasting 0.19–0.20 s and a dominant frequency of 4628.11–4903.07 Hz. Additional morphological, acoustic and genetic evidence supporting the status of this species could be found in Tables 1, 3, 5, and Figs 2, 3, 11. Nonetheless, in preservative is, to the best of our knowledge, indistinguishable from H. iaspidiense and H. mesai. TABLE 5. Measured parameters for each note of the call of Hyalinobatrachium tricolor sp. nov. from two uncollected specimens (number of calls = 16). Time is given in seconds and frequency in Hertz, range is followed by mean and standard deviation. First note

Note duration

0.015–0.025; 0.018 ± 0.003

Dominant frequency

4706.76–4859.01; 4761.327 ± 50.859

Lower frequency

3840–4232; 3981.125 ± 113.933

Upper frequency

5032–5290; 5116.875 ± 69.894

Time between notes Second note

0.018–0.03; 0.025 ± 0.003 Note duration Dominant frequency

4635.73–4935.13; 4790.738 ± 86.326

Lower frequency

3248.000–4075.000; 3813.688 ± 211.981

Upper frequency

5183.000–5635.000; 5372.000 ± 139.295

Time between notes Third note

0.049–0.550; 0.116 ± 0.170 Note duration

0.008–0.016; 0.011 ± 0.002

Dominant frequency

4672.220–4903.070; 4804.573 ± 75.012

Lower frequency

3150.000–4094.000; 3800.438 ± 265.126

Upper frequency

5290.000–5936.000; 5597.375 ± 175.162

Note duration

0.008–0.110; 0.028 ± 0.036

Dominant frequency

4732.140–4935.130; 4816.679 ± 69.292

Lower frequency

3023.000–4251.000; 3844.250 ± 316.712

Upper frequency

5290.000–5976.000; 5668.688 ± 185.800

Time between notes Fourth note

0.011–0.019; 0.015 ± 0.003

0.061–0.660; 0.105 ± 0.148

Remarks. Lescure and Marty (2000) cited H. iaspidiense (= nouraguense) for Kaw, although they did not refer to any museum material. We assume that those specimens belong to H. tricolor sp. nov. Description of holotype. Adult male of small size, SVL 20.3 mm; head wider than body, HW 41 % of SVL; head wider than long (HW/HL = 1.3); snout truncate in dorsal view and profile; ES/EL = 0.8 and ES/IOD = 1.2; loreal region concave; nostrils prominent, round; internarial region depressed; canthus rostralis defined; eyes small, directed antero-laterally; EL 50 % of HL; tympanic annulus indistinct, tympanic membrane absent, supratympanic fold absent; dentigerous processes on vomers absent; choanae circular, separated; tongue elongate,



35

ovoid, not attached to mouth posteriorly for about one sixth of its length; vocal slits extending from the sides of the base of tongue to the level of the mandibular joints. Forearms slim; diameter of forearms about one and a half times the diameter of upper arms; enameled ulnar fold evident; humeral spine absent; relative length of fingers: II < I < IV < III; finger discs wide, truncated and larger than those of toes; FIII 30 % of EL; webbing absent between Fingers I–II and basal between II–III, webbing formula on hand III 2– – 2+ IV; subarticular tubercles round; supernumerary tubercles slightly appreciable; palmar tubercle round and small, thenar tubercle small and elongated; nuptial excrescences Type V, glands on the lateral fringes of fingers not observed; hind limbs slender; TL 50 % of SVL; enameled tarsal fold evident; discs of toes round, truncate in profile; inner metatarsal tubercle small and ovoid; outer metatarsal tubercle absent; supernumerary tubercles slightly appreciable; webbing formula of feet I 1 – 2 II 1 – 2 III 1 – 2 IV 2 – 1– V. In preservative, dorsal skin scarcely covered with enameled granules, area around tympanum almost granular; skin on belly and thighs granular, other ventral surfaces smooth; cloacal opening directed posteriorly at upper level of thighs, concealed by a dermal fold and flanked by evident and enameled irregular folds and warts. Color in life. Dorsal surfaces light green with irregular lime green patches and dusted with minute black melanophores extending to the base of Finger IV and Toes IV and V. Presence of single and blotches of brown (coffee like color) warts over dorsal surfaces of body and posterior limbs. Tip of fingers and toes white to light green with traces of yellow. Enameled tarsal and dorsal folds extending to the tip of Finger IV and Toe I. Cloacal ornamentation consisting of enameled warts and folds. Iris yellowish, clearing out towards eyelids, and with dark flecks. Parietal peritoneum and pericardium transparent, pericardium, hepatic, and visceral peritonea white, peritonea covering all other internal organs not mentioned before transparent. Color in preservative. Background of dorsal surfaces cream and dotted by a coat of minute dark melanophores and black dots. Large and irregular white patches due to concentration iridiophores. Note that the brown warts of live specimens become black dots undistinguishable from those of Hyalinobatrachium iaspidiense and H. mesai. Enameled tarsal, ulnar and cloacal folds. Iris white. Peritonea as mentioned above. Variation. Measurements and body proportions of the holotype and paratype MNCN 44828 are presented in Table 2. The number and aggregation of brown warts varies between specimens from just two aggregated warts in MTD 48141 to 21 single warts in MNHN 2011.0116. Advertisement call. We recorded 16 calls of Hyalinobatrachium tricolor sp. nov. from its type locality from two not collected specimens. We assigned the recorded calls to the new species because they are identical to the ear to those emitted by specimens of the type series, which we observed calling. The call of Hyalinobatrachium tricolor sp. nov. is unique among all other species of the genus by having four notes per call (Fig. 2H, Tables 3, 5). All other described species of Hyalinobatrachium with known calls have a single note call except H. taylori (5–9 notes per call). Barrio-Amorós and Brewer-Carías (2008) mention that H. mesai had one and two notes calls; however, we think that this rather represent two calls within a short interval of time, which have been observed in topotypes of H. iaspidiense (J. Ayarzagüena, personal observation). Each note of the call of H. tricolor sp. nov. is tonal but with a high increase in frequency at the beginning from 3023.0–4251.0 Hz (3640.2 ± 555.5) to a maximum of 5183.0–5976.0 Hz (5551.7 ± 348.6). The call lasts for 0.19–0.20 s (0.20 ± 0.003) with a dominant frequency of 4628.1–4903.1 Hz (4777.3 ± 85.8). Values for the measured parameters for each note are presented in Table 5. Etymology. The specific epithet refers to its dorsal coloration, which is a combination of three colors: light green, dark green and brown in life, and cream, white and black in preservative. Biology and tadpole. Males call from the underside of leaves during night and close to egg clutches. Together with MNCN 44828 we collected two clutches of 22 eggs each. Ecology and distribution. Specimens were found in the Crique Wapou, Réserve Naturelle des Marais de Kaw-Roura, and in Kaw Mountain, French Guiana. These localities are at low elevation (2–10 m) and occupied by Amazon rainforest. Males were found on leaves on the vegetation over streams 0.5–1.5 m deep and at heights of 4– 5 m. This species is only known from these two localities in French Guiana and it is restricted to the Guiana Shield.



Discussion Hyalinobatrachium species richness in the Guiana Shield. Based on our assessment of previously available data, specimens and novel analyses of new material we now recognize seven species of Hyalinobatrachium in the GS: H. cappellei, H. iaspidiense, H. kawense, H. mesai, H. mondolfii, H. taylori, and H. tricolor. Our work provides detailed diagnoses of all these species, has contributed to resolve the long-standing taxonomic problems of H. cappellei and H. taylori, and revealed two new previously undescribed species (H. kawense and H. tricolor). Furthermore, we have summarized all information available to us including the distribution and biology of these species. We expect this information to be of particular interest for future taxonomic and biogeographic studies as well as conservation initiatives (e.g. IUCN http://www.iucnredlist.org/initiatives/amphibians). Nonetheless several taxonomic problems concerning these frogs are pending clarification. Hyalinobatrachium mesai is only known from one specimen and given the available information at the moment can only be differentiated from H. iaspidiense by one morphological character; hence, the status of H. mesai is unstable and pending re-evaluation based on additional data and therefore specimens. We also consider H. mondolfii as unstable with regard to H. munozorum and H. ruedai for the reassons outlined in the respective species account. Additional data and specimens are necessary to reach a more stable taxonomic conclusion on these species. Biogeography. The Guiana Shield is an important area of diversity and endemism within the Neotropics (Myers et al. 2000; Orme et al. 2005) and has been proposed as one of the major refugia (or a group of refugia) during the Quaternary for the Pan-Amazon region (Haffer 1969). Of the seven species of Hyalinobatrachium recognized in this study and known to occur in the GS only H. mesai, H. kawense, and H. tricolor have a restricted distribution. However, the current known distributions of these three species likely reflect sampling biases and/or taxonomic uncertainty rather than restricted distributions. The remaining four species show either a broad distribution throughout the GS (e.g. H. taylori) or throughout the GS and the lowland Amazon rainforests (H. cappellei, H. iaspidiense, H. mondolfii). Thus, both the GS and the Amazon exhibit a high overlap in species composition of Hyalinobatrachium and might not be considered two independent biogeographic areas for this frog genus. At a genetic level, the GS is a reservoir for genetic diversity as evidenced from phylogeographic studies of different taxa (e.g. Da Silva & Patton 1998; Ditchfield 2000; Nyári 2007; Patton et al. 2000). In spite of its important contribution to the Neotropical diversity, little is known about the patterns of genetic diversity within the GS and, to our knowledge, only five studies have focused on the phylogeography of animal species in the area (Cardoso & Montoya-Burgos 2009; Fouquet et al. 2007c; Noonan & Gaucher 2005, 2006; Steiner & Catzeflis 2004). Nonetheless, all these studies mainly or exclusively focused on the lowlands of parts of the GS located in French Guiana and Suriname. The only exception is the study of Steiner and Catzeflis (2004) that included a few samples from northern Guyana and Venezuela. However, it is important to include more complete samplings along the latitudinal gradient of the GS because there is also an elevation gradient increasing towards the north, where the Gran Sabana plateau and the vast majority of tepuys are located. Although our study was not designed to study phylogeographic patterns but to evaluate taxonomic hypotheses, some preliminary insights can be drawn from our data. For Hyalinobatrachium cappellei, H. iaspidiense, and H. taylori we have samples from widespread locations corresponding to different elevations. Figure 3 clearly indicates that there is a genetic gap between populations of different elevations in H. cappellei (1%) and H. taylori (3%), the two species with a broader altitudinal range (50–2000 m). On the other hand, H. iaspidiense, with an upper altitudinal limit at 900 m, is basically invariable for the studied marker from the Orinoco Delta to French Guiana (separated about 1000 Km), although specimens from Brazilian lowlands appear separated. Also H. mondolfii, a lowland species, shows identical sequences in Guyana and Venezuela. Therefore, a south to north altitudinal gradient seems to fit the diversity observed within H. cappellei and H. taylori, resulting in distinct lowland and highland haplotypes. Cryptic species of Dendropsophus minutus (Peters) and the species pair Vitreorana helenae (Ayarzagüena) and V. oyampiensis (Lescure) also seem to follow this pattern (Hawkins et al. 2007; Kok & Castroviejo-Fisher 2008). Integrative taxonomy: revealing cryptic species and taxonomic uncertainty. One of the main advantages of using an integrative taxonomy approach to evaluate species boundaries is the possibility to discover species that would be overlooked under single criteria approaches (see Padial & De la Riva 2009 for an example in another group of Neotropical anurans). The Guiana Shield seems to harbor several morphological cryptic species of anurans (Fouquet et al. 2007a, 2007b, 2007c; Hawkins et al. 2007) and we predict that herpetologists working in the area will benefit from applying such approaches. Our study illustrates this fact very well.



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Our study also reflects that glassfrog taxonomy includes a high number of synonyms. Since 2007, twelve synonyms and two resurrections have been recognized among glassfrogs (Cisneros-Heredia & McDiarmid 2007; Guayasamin et al. 2008b; Kok & Castroviejo-Fisher 2008; Castroviejo-Fisher et al. 2008; Castroviejo-Fisher et al. 2009a, 2009b; Yáñez-Muñoz et al. 2009; this work). The high level of taxonomic uncertainty among glassfrogs (at least 10% of the total number of species) unveiled during the last four years is probably not unique to this group and might reflect the situation in many other taxa. Its possible causes and solutions deserve further discussion. In many instances (≈ 21 %) new amphibian species are described based on only a few individuals originating from a single locality (Dubois 2004) and exacerbating the situation even more, type material of similar species is frequently not included in comparisons. This implies that there is a high risk of describing inter-population variation rather than different species. In addition, the majority of the taxonomic studies follow a single-criteria approach, mostly relying on external morphological characters alone or more recently exclusively using molecular evidence. In our study, the four proposed synonyms and the resurrected species correspond to species that were described: (1) without direct or even indirect (i.e. analyzing original descriptions) comparisons of type material of the most similar species; (2) from a reduced number of specimens (N ≤ 3) or from just one locality (H. ignioculus); and (3) using morphological characters alone as evidence to establish divergence. This pattern is mirrored to a great extend in all the other cases of synonymies and resurrections in glassfrogs. Of course, we can do little but lament on the limitations in sample size and number of localities studied because in many instances frogs occur at very low densities in remote areas of difficult access and show secretive behaviors (e.g. arboreal and fossorial species). Limited material is indeed also a restriction that we had to face in the present study. Here we describe two new species based on seven and four specimens respectively and each species collected at two localities. The two remaining causes of taxonomic uncertainty (comparisons to type material and a single line of evidence to determine divergence) are easier to overcome and if properly addressed would reduce the negative effects of small sample sizes. Visits to scientific collections and/or loan of type material might be in some cases a slow and expensive procedure but probably is a sine qua non condition for taxonomic revisions. Although some specific grants are available, the situation is particularly difficult for taxonomists from developing countries, where the largest diversity is located and where the research is most needed for the design of proper conservation and management strategies, limiting the access to scientific collections in wealthier countries where the vast majority of types are deposited. The use of different lines of evidence to determine species although not new, see the works of Myers and Daly (1976a,b, 1979, 1980) on dendrobatid frogs, is only slowly but progressively becoming more common (e.g. Alström et al. 2008; Groeneveld et al. 2009; Padial & De la Riva, 2009; Padial et al. 2009; Roy et al. 2009; Thum & Derry, 2008; Timm et al. 2008; Jansen et al. 2011). Such an approach has proved useful to solving complex taxonomic problems with a particular emphasis on new morphologically cryptic species (e.g. Bond & Stockman 2008; Cadena & Cuervo 2010; Depraz et al. 2009; Glaw et al. 2010; Goodman et al. 2009; Jordaens et al. 2009; Vieites et al. 2009). However, this study shows that detailed taxonomic revisions integrating independent lines of evidence could lead to a reduction of the number of recognized species, at least in some groups of anurans. Our study may imply two seemingly contradictive messages (1) species numbers have been overestimated at least in part due to the reasons exposed above and (2) species numbers have been underestimated due to cryptic diversity. In an extreme case, actual valid species numbers may not change because synonyms and new descriptions would be leveled out, leading to identical net-species numbers. Although species diversity might not change for a particular group or region, accurate species taxonomies are crucial to compile data on ecology and biogeography that can be assigned to the correct units of biodiversity. They are also crucial to answer more complex questions of changes in species-trait diversity patterns and they are a prerequisite for assessing the role of common evolutionary history versus more recent ecological processes in shaping biological communities (Ernst et al. in press). Sound and accurate taxonomies are therefore the backbone of many studies in such diverse fields as evolutionary biology, conservation and ecology.

Acknowledgements For help during fieldwork we thank: R. Antelo, D. González, J.M. Castroviejo, J. Ruíz, T–L-K Pen, T. Konrad, M. Hölting, M. Dewynter and J. P. Champenois. For assistance during the study of collections we thank: J.C. Señaris and F. Rojas (MHNLS), G. Köhler and J. Sunyer (SMF), B. Clarke (BMNH), A. Quintana, C. Barrio-Amorós (Fun-



dación Andígena), A. Schlüter (SMNS), A. Ohler (MNHNP), K. van Egmond, P. Arntzen, R. de Ruiter, M.E. Gasso and L.W. v.d. Hoek (RMNH), P. Kok (IRSNB), R. den Tex (Uppsala University), R. Márquez, I. De la Riva and J.M. Padial (MNCN), C.J. Franklin (UTA), M. Ayub Brasil and M. Caixeta M. Viana (CHUNB), J.P. Caldwell (OKMNH), D.L. Dittmann (LSUMZ). Material from Brazil was obtained thanks to loans of Universidade de Brasília, Instituto de Biologia, Departamento de Zoologia, Coleção Herpetológica da Universidade de Brasília (CHUNB) and from Louisiana State University Museum of Natural Science Collection of Genetic Resources. Material from Brazil was obtained with the support of National Science Foundation grants DEB-9200779 and DEB-9505518 to L.J. Vitt and J.P. Caldwell. Material from French Guiana was obtained thanks to all the crew of the Réserve Naturelle des Marais de Kaw-Roura, CBJ-Caïman and the Office Nationale des Forêts. This study is included in the “Contrato Marco de Acceso a Recursos Genéticos N° 0001, 11 Enero 2007” subscribed between Fundación La Salle de Ciencias Naturales and the Ministerio del Ambiente, Venezuela. Permission to conduct biodiversity research in Guyana was given by the Environmental Protection Agency Guyana. Working in MHFR was kindly permitted by R. Thomas, Guyana Forestry Commission. Iwokrama International Centre helped with transportation and various administrative services. This work was supported by grants from: Estación Biológica El Frío (SCF, CV and JA), Asociación Amigos de Doñana and its director Javier Castroviejo Bolíbar (SCF, CV and JA), Stiftelsen Sven och Lilly Lawskis (SCF), Helge Axelsson Johnsons Stiftelse Foundation (SCF), Lars Hiertas Minne Foundation (SCF), Sederholms för utrikes resor (SCF), the Royal Swedish Academy of Sciences (SCF), European Union Synthesis Projects NL-TAF-4090 and ES-TAF-2827 (SCF), the Swedish Research Council (CV and SCF), Fulbright/Ministry of Education post-doctoral research contract (SCF), Programa de Captación del Conocimiento para Andalucía (CV), a doctoral scholarship from the German Academic Exchange Service (DAAD) and a research grant DFG ER 589/2-1 from the German Research Foundation (RE).

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APPENDIX II. Known localities, ordered by country and state or region, of Hyalinobatrachium species in the Guiana Shield, following the classification presented in this study. For French Guiana we followed the division of geographical areas presented in Lescure & Marty (2000). Map number indicates the approximate position of the locality in the map of Figure 1. Asterisk (*) indicates type locality for a certain taxa. H. sp. corresponds to a specimen identified by Lescure (1975) as H. taylori. However, given that the author could not differentiate between H. cappellei, H. mondolfii and H. taylori and that we have not examined that specimen, its identification remains unsolved. Locality

Map Number

Taxa

Source

First stream of Caño Acoima, tributary of río Grande, slopes of Serranía de Imataca (08°22’ N, 61°32’ W; 15 m)

1

H. mondolfii*, H. iaspidiense

Señaris and Ayarzagüena (2001; 2005), this work

Caño Jotajama, tributary of Caño Cocuina (09°20’ N, 61°30’ W; 5 m)

2

H. mondolfii

Señaris and Ayarzagüena (2001; 2005)

3

H. mondolfii

Señaris and Ayarzagüena (2001; 2005)

Venezuela Delta Amacuro:

Monagas: Campamento MARNR, Guarapiche river. Bolivar: La Escalera, Kms 119, 122, 127 and 130 4 of the El Dorado–Santa Elena road (05°57’34’’ N, 61°23’30’’ W; 1050– 1360 m)

H. cappellei, H. taylori Cannatella and Lamar (1986), Ayarzagüena (1992), Duellman (1997), Gorzula and Señaris (1998), Señaris and Ayarzagüena (1993; 2005), this work

Gran Sabana (05°40’ N, 61°37’ W; 1250 5 m); 13 km S Las Claritas, on the road Las Claritas–Santa Elena de Uairén

H. cappellei

Ayarzagüena (1992), Gorzula and Señaris (1998), Señaris and Ayarzagüena (2005); Guayasamin et al. (2008a)

Quebrada de Jaspe, San Ignacio de Yuraní (04°55’ N, 61°05’ W; 800–1000 m)

6

H. cappellei, H. iaspidiense*, H. taylori

Ayarzagüena, (1992), Señaris and Ayarzagüena (2005), this work

Salto Karuay, Karuay river (05°41’27’’ N, 61°51’40’’, 900 m)

7

H. cappellei, H. taylori Señaris (1996), Ayarzagüena (2005), this work

8 Auyan-tepui: Central–West Sector of Auyan-tepui (05°56’ N, 62°33’ W; 1850 m); Campamento Guayaraca, ascent to Auyan-tepui from Uruyen (05°41’06’’ N, 62°31’32’’ W; 1005 m); Stream between Campamento Oso and ascent to the top of Auyan-tepui.

H. cappellei, H. taylori Señaris and Ayarzagüena (1994), Señaris and Ayarzagüena (2005), this work

Guaiquinima (05°47’ N, 63°48’ W; 1400 m)

H. cappellei

Ayarzagüena, (1992), Gorzula and Señaris (1998), Señaris and Ayarzagüena (2005)

Central Sector of Cerro Jaua (04°49’55’’ 10 N, 64°25’54’’ W; 1600 m)

H. cappellei

Señaris and Ayarzagüena (2005)

Southern slope of Sarisariñama–tepui (04º25’ N, 64º7’ W; 420 m)

11

H. mesai*

Barrio–Amorós and Brewer– Carías (2008)

Serranía of Guanay, upper Parguaza river (05°55’ N, 66°23’ W; 1650 m)

12

H. cappellei

Ayarzagüena (1992), Gorzula and Señaris (1998), Señaris and Ayarzagüena (2005)

North base of Pico Tamacuari, Sierra de 13 Tapirapecó (01°13’ N, 64°42’ W; 1160– 1200 m)

H. cappellei

Myers and Donnelly (1997), Señaris and Ayarzagüena (2005)

9

Amazonas:

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APPENDIX II. (continued) Locality

Map Number

Temiche, cerro Marahuaca

14

H. taylori

Cerro Duida, 1000 m.

15

H. taylori

Señaris and Ayarzagüena (2005)

H. cappellei

Myers and Donnelly (2001)

H. cappellei

Noonan and Bonett (2003)

18

H. cappellei, H. iaspidiense, H. mondolfii

Ernst et al. (2005), this study

19

H. taylori*2, H. mondolfii

Goin (1968)

Cerro Yutajé (05°46’ N, 66°08’ W; 1700 16 m)

Taxa

Source 1

Rivero (1961), Goin (1968), Señaris and Ayarzagüena (2005)

Guyana Cuyuni–Mazaruni: Peters Mountain, 3.6 km north of 17 Imbaimadai in the Pacaraima Mountains (05°44’ N, 60°18’ W; 600 m), Administrative Region 7. Upper Demerara–Berbice: Maiko creek (N 05° 09' 19.30", W 58° 41' 58.96"; 60 m), in the Mabura Hill Forest Reserve (MHFR) situated approximately 20 km south–east of the township Mabura Hill, Central Guyana (5°13’ N, 58°48’ W) East Berbice–Corentyne: Along New River, 750 m Potaro–Siparuni Kaieteur National Park (5°10’ N, 59°29’ 20 W; 500 m)

H. cappellei, H. taylori Kok and Castroviejo-Fisher (2008), Kok and Kalamandeen (2008)

Iwokrama Forest: Turtle mountain (04° 43' 51.5" N, 58° 43' 27.2" W) and the field Station (04° 40' 17.46" N, 58° 41' 06.56" W)

H. mondolfii

39

This work

Suriname Marowijne: Loëkreek, camp Hofwijks VII, 54 km. S 21 airstrip Oelemari

H. iaspidiense

Sipaliwini: Marowijne river

21

H. mondolfii

Goin (1968)

Marowijne river, Nassaugebergte.

22

H. cappellei

Goin (1968)

Langa Soela, Paleomeu river

23

H. cappellei

Goin (1968)

13km NW of Tafelberg airstrip

24

H. cappellei

Road to Amotopo, Kabalebo area, km 39: 1000 m in line NW direction; Kabalebo river, R bank, 10 km S of camp Keyzer; Vreedzaamkreek, 10 km N. of Lucieriver

25

H. cappellei, H. iaspidiense

Kok and Castroviejo-Fisher (2008), this work

Maratakka river

26

H. sp

Lescure (1975)

27

H. cappellei*2

van Lidth de Jude, (1904), Goin (1964)

Nickerie:

Saramacca: Saramacca river and its neighborhood

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APPENDIX II. (continued) Locality

Map Number

Taxa

Source

H. cappellei, H. iaspidiense, H. taylori

Hoogmoed and Avila-Pires (1990), Lescure and Marty (2000), this work

French Guiana Bassin de la Sinnamary: Camp de Saint Eugène, Courcibo river; 28 Mont Trinité; Aya / Trinité (4°37’ N, 53°25’ W; 150 m); Mont Courcibo; Petit Saut; Crique Grand Leblond (4°40'11'' N, 53°16'99'' W; 50 m) Bassin de la Comté:

29

Crique Trésor (04°35' N, 52°18' W; 20 m)

29

Lescure and Marty (2000) H. cappellei

This work

H. cappellei, H. kawense*, H. tricolor*

Lescure and Marty (2000), this work

Montagne et Marais de Kaw: Crique Patawa (4°30'20'' N, 52°5'30'' W; 30 105m); Rivière de Kaw (04°36'33'' W; 52°03'25'' N; 1–10 m); Canal de Kaw (04°30' N, 52°1' W; 1 m); Crique Wapou (04°24' N, 52° 9’ W; 2 m); Kaw Mountain (04° 32’ N, 52° 13’ W; 10 m) Bassin de l’Approuague: Petit montagne Tortue près de Régina

31

H. cappellei

Lescure and Marty (2000)

Saut Arataye (environs du camp de base), Réserve des Nouragues

32

H. cappellei, H. iaspidiense


Bassin de l’Oyapock:

33

H. cappellei


Montagne St Marcel

34

H. cappellei

Lescure (1975), Lescure and Marty (2000), this work

35

H. cappellei

This work

H. kawense, H. mondolfii

This work

H. iaspidiense

Cordeiro–Duarte et al. (2002)

H. iaspidiense, H. mondolfii

Avila–Pires et al. (2010)

Others: Montagne des Singes (5°05 N, 52°38' W, 30 m)

Crique Gabrielle (04°41' N, 52°18' W; 2 36 m) Brazil Amazonas: Presidente Figueiredo (02°09’3’’ S, 60°02’06.2’’ W; 120 m)

37

Para: Óbidos: ESEC Grão–Pará Centre (0° 37’ 38 49.01” N, 55° 43’ 42.60” W; 350 m) 1 2

Most likely identification according to Señaris and Ayarzagüena (2005) Position in our Fig. 1 according to Lescure (1975: Fig. 1)



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