Cruziohyla (Anura: Phyllomedusidae), with Description of a New Species

Zootaxa 4450 (4): 401–426 ISSN 1175-5326 (print edition) http://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2018 Magnolia Press ISSN 1175-5334 (online edition) https://doi.org/10.11646/zootaxa.4450.4.1 http://zoobank.org/urn:lsid:zoobank.org:pub:54B89172-7983-40EB-89E9-6964A4D4D5AC

Review of the genus Cruziohyla (Anura: Phyllomedusidae), with description of a new species

ANDREW R. GRAY The Manchester Museum, The University of Manchester, England. E-mail: [email protected]

Abstract

The presented work summarises new and existing phenotypic and phylogenetic information for the genus Cruziohyla. Data based on morphology and skin peptide profiling supports the identification of a separate new species. Specimens of Cruziohyla calcarifer (Boulenger, 1902) occurring in Ecuador, Colombia, two localities in Panama, and one in the south east Atlantic lowlands of Costa Rica, distinctly differ from those occurring along the Atlantic versant of Central America from Panama northwards through Costa Rica, Nicaragua, to Honduras. A new species—Cruziohyla sylviae sp. n.—(the type locality: Alto Colorado in Costa Rica)—is diagnosed and described using an integrated approach from morphological and molecular data. Phylogenetic analysis of DNA sequences of the 16S rRNA gene confirms the new species having equal minimum 6.2% genetic divergence from both true C. calcarifer and Cruziohyla craspedopus.

Key words: Amphibia, Variation, Taxonomy, Cruziohyla, northern South America, Central America, Middle America, Cruziohyla calcarifer, Cruziohyla craspedopus, Cruziohyla sylviae sp. nov., new species

Resumen

Éste trabajo reúne la información fenotípica y filogenética, tanto nueva como conocida, del género Cruziohyla. Datos de la morfología y el perfil de péptidos de la piel apoyan la identificación de una nueva especie: los especímenes de Cruzi ohyla calcarifer (Boulenger, 1902) encontrados en Ecuador, Colombia, dos localidades en Panamá y una en las tierras bajas del Atlántico sudeste de Costa, difieren claramente de los encontrados a lo largo de la vertiente atlántica de América Central, desde Panamá hasta Honduras, incluyendo Costa Rica y Nicaragua. Una nueva especie—Cruziohyla sylviae sp. n.—(localidad tipo: Alto Colorado, Costa Rica)—es diagnosticada y descrita utilizando una aproximación integral de da tos morfológicos y moleculares. El análisis filogenético de las secuencias de ADN del gen 16S rRNA confirman que la nueva especie tiene al menos 6.2% de divergencia genética tanto de C. calcarifer como de Cruziohyla craspedopus.

Palabras clave: Anfibios, variación, taxonomía, Cruziohyla, norte de América del Sur, América Central, América Medio, Cruziohyla calcarifer, Cruziohyla craspedopus, Cruziohyla sylviae sp. nov., nueva especie

Introduction

The taxonomy of frogs in the family Phyllomedusidae Günther, 1858 has received a great deal of scientific attention, with a variety of approaches being utilised in investigating this family’s complex systematics (Funkhouser, 1957; Savage & Heyer, 1967; Duellman, 1968; Duellman, 1974; Maxson, 1976; Cannatella, 1980; Cannatella, 1982; Cruz, 1990; Faivovich et al., 2010; Luna et al., 2012; Ron et al., 2013, Baêta et al., 2016; Duellman et al., 2016). The highly commendable level of morphological, ecological, and behavioural work carried out with this group of frogs continues to provide a valuable framework for contextualising more recent phylogenetic studies. Details noted in some of the earliest work with these species still have clear inference in supporting our developing understanding of their phylogeny, complimented by the use of new scientific

Accepted by V. Orrico: 8 Jun. 2018; published: 26 Jul. 2018 401 techniques. The importance of phylogenetic work in clarifying amphibian systematics is clearly crucial. It is well recognised that an integrative approach that includes the continued gathering of information on the phenotypic variations found within each genus is important in helping us achieve a fully-informed understanding (Dayrat, 2005; Faivovich et al., 2010). The genus Cruziohyla Faivovich et al., 2005 currently consists of two species (Faivovich et al., 2005, 2010; Duellman et al., 2016): Cruziohyla calcarifer (Boulenger, 1902) commonly known as the Splendid Tree Frog, and Cruziohyla craspedopus (Funkhouser, 1957) commonly referred to as the Fringed Tree Frog in reference to the extensive skin flanges it possesses on its limbs. Cruziohyla calcarifer is reported as occurring in the lowlands of Ecuador, Colombia, Panama, Costa Rica, Nicaragua, and Honduras (Frost, 2018). Cruziohyla craspedopus occurs in the tropical lowlands of South America, in Colombia, Ecuador, Peru and Brazil (Hoogmoed & Cadle, 1991; Meneghelli et al., 2011; Nunez et al., 2015; Frost, 2018). Both species are characterised by having some degree of black barring on the flanks and legs, pronounced calcars, and a bicoloured iris with a silvery-gray centre and yellow-orange coloured surround (Duellman, 1970; Hoogmoed & Cadle, 1991). Cruziohyla calcarifer was first described as Agalychnis calcarifer (Boulenger, 1902), and later transferred from the genus Agalychnis Cope, 1864 to Phyllomedusa, Wagler, 1830 by Lutz (1950). Cruziohyla craspedopus was described in 1957 and subsequently designated Phyllomedusa craspedopus (Funkouser, 1957). Both species were attributed to the genus Agalychnis (Duellman, 1968) prior to being placed in the genus Cruziohyla by Faivovich et al. (2005). Since 2005 several phylogenetic studies have corroborated the establishment of Cruziohyla (Moen & Wiens, 2008; Gomez-Mestre et al., 2008; Faivovich et al., 2010; Duellman et al., 2016) and have highlighted the close relationship between the genus Cruziohyla and Phrynomedusa Miranda-Ribeiro, 1923. Both genera are identified as being the most basal of Phyllomedusidae (Baêta et al, 2016) and it is proposed that the origin of todays Central American Phyllomedusids derived directly from ancestral Cruziohyla stock from the western Amazon Basin (Duellman et al, 2016). Cruziohyla craspedopus being paired with C. calcarifer as a sister species in the genus Cruziohyla has followed assumptions based on their similar morphology and reproductive strategy (Duellman, 1970; Hoogmoed & Cadle, 1991), although the relationship between these species has never been confirmed from a genetic perspective. Results from the limited phylogenetic work comparing specimens of C. calcarifer have indicated significant intraspecific difference (Crawford et al., 2010) and that the taxonomy of populations currently under the name C. calcarifer may be in need of addressing (Favovich et al., 2010). Cruziohyla calcarifer was first described by George E. Boulenger in 1902, from a single specimen collected by William F. H. Rosenberg, a British Naturalist, near the Rio Durango, Northwest Ecuador [BM.1902.5.27.26]. In 1913, a second C. calcarifer specimen was collected by Dr H. G. F. Spurrell in Condoto, the Choco, Colombia [BM 1913.11.12.126]. Both specimens are held in the British Museum of Natural History in London. In 1927, Emmett R. Dunn, an American herpetologist, collected a specimen in Panama [MCZ A15610] returning with it the USA. Over the following four decades, only a handful of specimens were collected, including three from the Serranía del Darién, Panama, in 1963, by William E. Duellman and Charles W. Myers. These were deposited at Kansas University, and specimen KU77415 (Fig. 11) used to first define C. calcarifer from a chromosomal perspective (Duellman & Cole, 1965). By 1970 the number of C. calcarifer specimens in museums totaled only nine (Duellman, 1970) and until as recently as the 1990s there were fewer than twenty museum specimens worldwide (Caldwell, 1994). In more recent years a greater number of Cruziohyla specimens have been recorded, not least through commendable efforts at the Costa Rican Amphibian Research Centre and Centro Jambatu in Ecuador to support the species’ conservation. Further specimens collected have confirmed occurrences in northwestern Costa Rica and that the distributional range extends northwards to Nicaragua and Honduras (Caldwell, 1994; Köhler, 2001; McCranie & Wilson, 2002; Köhler et al., 2004; McCranie, 2015). Currently, the number of museum specimens listed as C. calcarifer worldwide totals at least 160 specimens. As past sample sizes have been very limited, few opportunities for direct comparative studies between the more geographically distant populations have been afforded. This work aims to provide a taxonomic review the genus Cruziohyla, evaluating variation between specimens of C. calcarifer from localities throughout the species’ known range in order to clarify their taxonomic status. A second aim is to describe the resulting new species from Panama, Costa Rica, Nicaragua, and Honduras.

Examinations of wild specimens from specific geographical populations, and museum specimens (live and preserved) from confirmed localities, took place periodically between 1996 and 2018. An integrated approach, combining morphological and molecular characters, was used to perform the diagnosis of each Cruziohyla species. The reference list to species provided is restricted to only works or publications containing evidence, illustrations, or photographs that allow clear identification of Cruziohyla species. Referred specimens relate to those the author has been able to re-examine (see Appendix II). DNA sampling. Buccal swabs were taken from three live captive C. calcarifer specimens at Manchester Museum originating from Esmeraldas, Ecuador, and one C. craspedopus individual originating from near Nasuta, Loreto, Peru. Tissue samples were excised from four preserved Cruziohyla specimens in the collection of Manchester Museum, originating from Alto Colorado, near Guayacan, Costa Rica. Two preserved specimens from Comadre, Costa Rica, were also kindly provided by Dr Karl-Heinz Jungfer. In addition, two specimens [GQ366229; GQ366230] referenced in Faivovich et al. (2010) one originating from Comadre, Costa Rica, and one from Carretera Lita, San-Lorenzo, Ecuador, were resampled and sequenced for 16S. In phylogenetic analysis these sequences were aligned against additional sequences from Genbank (see Appendix III). Amplification and Sequencing of 16S. Genomic DNA was extracted from ethanol-preserved tissues and buccal swabs using the QIAamp DNA mini-kit (Qiagen, Valencia, CA, USA). The ~560 base-pair fragment of the ribosomal 16S gene was amplified and sequenced using the primers 16sAR-L and 16sBR-H described by Palumbi et al (1996) as implemented by Crawford et al (2010). Amplification was carried out in a 25-µL reaction using Bioline MyFi™ DNA polymerase (Bioline, London, UK), following the protocol outlined in Meyer (2003) with an annealing temperature of 58-60ºC. PCR amplification products were purified using EXO/FastAP (ThermoFisher Scientific, Waltham, MA, USA) and were sequenced at the GATC Biotech facility in Cologne, Germany (GATC Biotech AG). All samples were sequenced in both directions to eliminate the possibility of error and chromatograms were screened for quality using Chromas (Technelysium). Phylogenetic analysis. Screened gene sequences for all samples were aligned with sequences of the 16S region from GenBank using MUSCLE (Edgar, 2004). Sequences from Genbank incorporated in the analysis included those C. calcarifer specimens collected in 2003 from Omar Torrijos, Panama [KRL 0800; KRL 1218] and one specimen collected from the same locality in 2004 [KRL 0781] included by Crawford et al (2010). In addition, the two sequences [GQ366229; GQ366230] referenced in Faivovich et al. (2010) were also included. Litoria caerulea [AMNH A-168409] and Phrynomeduas marginata [GQ366234] were included as distant and closely related outgroups respectively (see Appendix III for all accession numbers). Analysis was conducted on a total of 17 sequences with 577 positions included in the final dataset. All evolutionary analyses were conducted using MEGA7 (Kumar et al., 2016). Initial trees were obtained using the Neighbour-Join and BioNJ algorithms applied to a matrix of pairwise distances, estimated using the Maximum Composite Likelihood (MCL) approach and selecting the topology with highest log likelihood. Evolutionary rate differences among sites were modeled using a discrete Gamma distribution (5 categories (+G, parameter = 0.3375)). The model test feature of MEGA7 (Kumar et al., 2016) was used to identify the GTR+G model as the best-fit model of molecular evolution. Evolutionary history was inferred using the Maximum Likelihood method based on the General Time Reversible model (Nei and Kumar, 2000). The initial tree was constructed through the application of Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree with the highest log likelihood was compared with the Bootstrap consensus tree inferred from 1000 replicates. There were a total of 577 positions in the final dataset. Peptide barcoding. It has previously been demonstrated that the peptides secreted by frogs are species specific and that mass spectrometry derived fingerprints of the peptides can be used to distinguish between closely related species (Goraya et al., 2000; Zheng et al., 2004). In this study, mass spectrometry was carried out at the Bio-MS Facility at the University of Manchester. Secreted peptides of live specimens held at Manchester Museum were collected, non-invasively, using distilled water. The specimens consisted of three C. calcarifer originating from Alto Tambo Ecuador [MM1019–1021], three specimens from Guaycan, Costa Rica [MM1022–1024], and three C. craspedopus from Loreto, Peru [MM1014–1016]. A volume of 100µl of each secreted peptide sample was acidified to 0.1% formic acid, isolated on 10mg of POROS R2 beads (ThermoFisher Scientific), washed with

REVIEW OF THE GENUS CRUZIOHYLA Zootaxa 4450 (4) © 2018 Magnolia Press · 403 500µl of 0.1% formic acid in water and eluted in 100µl of 50% acetonitrile in water. The samples were dried completely in a vacuum centrifuge and then reconstituted in 2µl of matrix (1mg/ml of α-Cyano-4-hydroxycinnamic acid (Sigma) in a 50/50 mixture of acetonitrile and ethanol). An aliquot of 1µl of each sample was spotted on to a steel target plate and analysed on an Ultraflex II MALDI-TOF system (Bruker). For fingerprinting purposes the resultant peak intensities were binned into 1 Da units and only signals that were present in all three examples of each species and had a relative intensity of 10% of the maximum intensity in each spectra were used for comparison. Call recording and analysis. An Olympus LS11 digital recorder using a built-in microphone and Marantz 74 PMD-201 cassette recorder with Sony ECM-F8 microphone were used for call recording. A total of twenty-eight advertisement calls were analysed. Cruziohyla specimens from near Guayacán, Costa Rica, C. calcarifer from Esmeraldas, Ecuador, and C. craspedopus recorded at Estación Científica Yasuní, Provincia Orellana, Ecuador, were assessed and the original field recording of C. calcarifer [AMNH reel 226] by Dr C. W. Myers in Changuinola, Panama, on April 21st, 1980, was kindly made available by the American Museum of Natural History. Air temperatures at the time of the new recordings were 22.4–25°C, humidity levels ranged from 85–87%. Only frog calls matching the criteria of general advertisement calls (Littlejohn, 1977; Wells, 1977) were used. The recording was resampled using Raven 2.0.0. software at a sampling rate of 44.1 kHz and a frequency resolution of 10.8 Hz. Morphometrics. Measurements were made in accordance to the methodology described by Duellman (1970). These were taken using either dial callipers accurate to the nearest 0.1 mm or digital callipers accurate to the nearest 0.01mm then rounded to the nearest 0.1mm. A Zeiss Stemi 305 microscope was used to facilitate detailed close-up examination of key characters. Where measurements of the tympanum relative to the eye are given (TY/ E), the tympanum and eye were always measured along their greatest horizontal diameter. Colour and pattern descriptions are of specimens in life, unless otherwise noted. Colour references are obtained directly from live specimens, colour photographs taken in the field, or field notes taken during daytime in recognition that all the species concerned are capable of considerable metachrosis. Other images included a number of colour photographs taken, in life, of key museum specimens included in this study prior to their preservation: e.g. colour photos of C. calcarifer from Changuinola, Panama, courtesy of Dr C. W. Myers, and from Laguna, Panama, courtesy of Professor W. E. Duellman. Colour quantification follows Köhler (2012): where possible colours are also stated numerically (e.g. Leaf Green - GK122), utilising colour coding system developed for herpetological use by Köhler (2012). Skin pattern definitions follow Savage (2002): specifying markings as either: (a) blotches; (b) spots; (c) speckles. Webbing formulae follow Savage and Heyer (1967, 1997) as modified by Myers and Duellman (1982). Morphological terminology follows Duellman (1970), and taxonomy is based on Amphibian Species of the World (Frost, 2018). I also examined the holotype of C. calcarifer (BM.1902.5.27.26 [BM.1947.2.24.22]) at the British Museum of Natural History, where the type and paratype for C. sylviae are now deposited. Abbreviations of institutions and collections. The following acronyms are used in the text for collections providing access to live or preserved specimens and associated data used in this study: American Museum of Natural History, USA (AMNH); Academy of Natural Sciences Philadelphia, USA (ANSP); British Museum of Natural History, London, UK (BMNH); Costa Rican Amphibian Research Centre, Costa Rica (CRARC); Banco de Vida/Centro Jambatu, Ecuador (CJ); Manchester Museum, The University of Manchester, UK (MM); Florida Museum of Natural History, USA (FM); Universidad Nacional de Colombia, Colombia (ICM); Karl-Heinz Jungfer field series, Germany (KHJ); Field series of K. R. Lips, USA (KRL); University of Costa Rica, Costa Rica (UCR); Museum of Comparative Zoology, University of Harvard, USA (MCZ); Museo de Vertebrados de la Universidad de Panamá, Panamá (MVUP); Smithsonian Institution, National Museum of Natural History, USA (NMNH); Pontificia Universidad Católica del Ecuador, Ecuador (PUCE); Senkenberg Research Institute, Germany (SMF); Stanford University, USA (SU); University of Michigan Museum of Zoology, USA (UMMZ); University of Southern California, USA (USC); United States National Museum, USA (USNM).

Results

Diagnosis of Cruziohyla species.

Agalychnis calcarifer—Dunn (1931): [MCZ 15610] (misidentified). Phyllomedusa calcarifer—Lutz (1950): Transferred from Agalychnis. Phyllomedusa calcarifer—Funkhouser (1957): in part [MCZ 15610] (misidentified). Phyllomedusa calcarifer—Duellman and Cole (1965): [KU77415] (misidentified). Agalychnis calcarifer—Duellman (1968): Transferred from Phyllomedusa. Agalychnis calcarifer—Duellman (1970): (misidentified). Agalychnis calcarifer—Hoogmoed & Cadle (1991): in part [JEC 9414] (misidentified). Agalychnis calcarifer—Caldwell (1994): (misidentified). Agalychnis calcarifer— Savage (2002): (misidentified). Agalychnis calcarifer—Kubicki (2004): (misidentified). Cruziohyla calcarifer— Faivorvich et al (2005): Transferred from Agalychnis. Cruziohyla calcarifer—Sunyer et al (2009): (misidentified). Cruziohyla calcarifer— Faivorvich et al (2010): in part [GQ366229;GQ366230] (misidentified). Cruziohyla calcarifer—Crawford et al (2010): in part [KRL 0781] (misidentified).

Holotype. BMNH 2018.5509. Adult female (88.2 mm) from Alto Colorado: Guayacán: District of Siquirres: Limón Province: Costa Rica, 700 m a.s.l. (N 10 ̊ 02' 8'' W 83 ̊ 31' 17''), collected on July 15th, 1998, by Andrew Gray and Miguel Solano. Paratype. BMNH 2018.5510. Adult male (67.0 mm) collected with the holotype from the type locality: Alto Colorado: Guayacán: District of Siquirres: Limón Province: Costa Rica., 700 m a.s.l. (N 10 ̊ 02' 8'' W 83 ̊ 31' 17''). Diagnosis. The new species Cruziohyla sylviae sp. n is diagnosed on the basis of the results of phylogenetic analysis (Fig. 2) and can be distinguished by a combination of the following characters: (1) it is a moderate to large sized species, SVL in males 44.3 mm to 67.0 mm and 73.5 mm to 88.2 mm in females; (2) the absence of dark brown patches on each side and below the vent; (3) large distinct tympanum, diameter size more than 70% that of the eye; (4) fingers and toes extensively webbed: modal webbing formula (fingers): I 2—2 II 1+—2 III 11/2—11/4 IV; modal webbing formula (toes): I 1—11/2 II 1—2 III 1—2 IV 2—1+ V; (5) sloping snout in profile; (6) in life, the presence of small irregular-shaped pale green lichenose speckles to whole of dorsal surface area; (7) bold black barring extending to lateral surfaces of the flanks, often interconnecting; (8) triangular dermal flap on heel, faces inward when limb closes; (9) lack of calcar terminating on heel projecting outward. Description (based on the holotype). An adult female of 88.2 mm SVL. Body is elongated, robust for genus. Head is wider than long, broader than the body. Eyes large, pupil vertically elliptical. Palpebral membrane is transparent to partially-transparent, not reticulated. Tympanum is large, distinct, more than two-thirds diameter of the eye. Faint supratympanic fold present. Snout sloping in profile, nostrils having minimal protuberance, distanced slightly nearer to the upper lip than to the eye, distanced from each other by an interval about the same distance as from the upper lip. Canthus rostralis not distinct. Loreal region slightly concave. Vomerine teeth in two short oblique series located between the choanae. Tongue oval-shaped, longer than wide, more than half the width of the mouth, deeply notched anteriorly. Dorsal surface smooth, ventral surface slightly granular; skin on the thighs and surrounding vent coarsely granular. Upper arm slender. Forearm robust, having distinct dermal fold or ridge (3.7mm), almost ovoid in shape, extending along the anterior edge of the forearm, tapering from widest point of forearm to meet disk of the fourth finger. Fingers and toes possess large rounded disks (to 9.3mm). Subarticular tubercles on fingers and toes prominent and round. Fingers and toes approximately ¾ webbed, webbing extending to the base of the disc on the fourth finger and penultimate phalange on the fourth toe: modal webbing formula (fingers): I 2—2 II 1+—2 III 11/2—11/4 IV; modal webbing formula (toes): I 1—11/2 II 1—2 III 1—2 IV 2—1+ V. A distinct dermal ridge extends along posterior ventrolateral edge of tarsus, paralleling a less distinct inner tarsal fold extending along medial edge of tarsus. Triangular dermal flap present on heel, which when hindlimb is withdrawn faces inward toward the vent. In life, dorsal surface colouration combines two shades of green, small pale lichenose speckling present throughout. Concealed surfaces of flanks, thighs, hands and feet, orange-yellow. Extensive bold black bars to flanks, black barring to the thigh upper surfaces and anterior surface of tibia. Ventral surfaces pale orange-yellow. Eye contains two separate and contrasting colours: having a silvery-grey centre with a yellow periphery. Lower eyelid opaquely mottled, consistent with skin pattern of dorsal surface. In preservative, dorsal

REVIEW OF THE GENUS CRUZIOHYLA Zootaxa 4450 (4) © 2018 Magnolia Press · 405 ground colour dark greenish-grey; lichenose markings pale, as much emphasised as in life; orange colour in life changes to cream colour.

FIGURE 1. Members of the genus Cruziohyla: (a) C. sylviae sp. n.; (b) C. calcarifer (Boulenger, 1902); (c) C. craspedopus (Funkhouser, 1957).

Measurements of holotype (in mm). SVL = 88.2; Head length = 25.6; Head width = 27.7; Eye diameter (horizontal) = 8.4; Tympanum diameter = 7.4 (TY/E = 88%); Tibia length = 51.4; Foot length = 31.3. Variation. Measurements of the paratype (mm): SVL = 67.0; Head length = 20.1; Head width = 21.8; Eye diameter (horizontal) = 7.9; Tympanum diameter = 6.3 (TY/E = 80%); Tibia length = 33.3; Foot length = 25.7. Etymology. The specific name sylviae is a patronym given in adoration of my first grandchild, Sylvia Beatrice Gray. Sylvia originates from the Latin word for forest Silva meaning ‘Spirit of the Forest’. Distribution. Occurs at low-mid elevation localities (below 750m) along the Cordillera Central of Central America, primarily along the Atlantic versant, from the Darien Province in southern Panama through Costa Rica and Nicaragua to southern Honduras (Fig. 10). See discussion. Phylogenetic analysis. Analysis of the DNA sequences shows that C. sylviae sp. n. is highly divergent from both other members of the genus Cruziohyla. The tree selected, based on highest log likelihood (-1622.41), is shown (Fig. 2). The percentage of trees in which the associated taxa clustered together (based on 1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths drawn according to number of substitutions per site. The interspecies differences in genetic distances ranged from 6.2% to 7.5% between the three species (Fig. 3). As comparison, these genetic distances are similar to those reported by Rivera-Correa et al. (2013) between Agalychnis species (5.69-13.0%).

FIGURE 3. Uncorrected genetic distances among Cruziohyla species at the 16S gene.

Uncorrected intraspecies genetic distances at the 16S gene were greatest among C. calcarifer individuals, and ranged from 0–2.5% (Fig. 3). The high level of divergence corresponds to the large geographic range of this species, extending from northwestern Ecuador to southeastern Costa Rica. All sequences resulting from this study have been deposited in GenBank: 16s DNA sequences for C. sylviae sp. n. [MH377068—377071]; C. calcarifer [MH377063—377067]; C. craspedopus [MH377072].

Cruziohyla calcarifer (Boulenger, 1902) (Figs. 1b, 5, 6b, 7b, 9b, 11b). Splendid Tree Frog / Rana de arból Esplendida

Agalychnis calcarifer—Boulenger (1902).

REVIEW OF THE GENUS CRUZIOHYLA Zootaxa 4450 (4) © 2018 Magnolia Press · 407 Phyllomedusa calcarifer—Lutz (1950): Transferred from Agalychnis. Phyllomedusa calcarifer—Funkhouser (1957): in part [BM.1902.5.27.26]. Agalychnis calcarifer—Duellman (1968): Transferred from Phyllomedusa. Agalychnis calcarifer—Cochran and Goin (1970). Agalychnis calcarifer—Duellman and Myers (1982). Cruziohyla calcarifer— Faivorvich et al (2005): Transferred from Agalychnis. Cruziohyla calcarifer—Frost et al (2006). Cruziohyla calcarifer—Faivovich et al (2010): in part [GQ366229, GQ366230]. Cruziohyla calcarifer—Crawford et al (2010): in part [KRL 0800].

Holotype: BM.1902.5.27.26 [BM.1942.2.24.22]. Female from Rio Durango, Northwestern Ecuador. Diagnosis. Cruziohyla calcarifer is a moderate to large species of the genus Cruziohyla, attaining a maximum SVL in males of 56.4 mm to 80.5 mm, and 57 mm to 88.8 mm in females, distinguished by a combination of the following characters: (1) prominent dark brown ventral thigh markings present either side of the vent in both sexes; (2) small tympanum, diameter 40–60% that of the eye; (3) fingers and toes moderately webbed, approximately two-thirds: modal webbing formula (fingers): I 2—2- II 1+—2 III 11/2—2- IV; modal webbing formula (toes): I 1— 1+ II 1—2 III 1+—3 IV 3—2- V; (4) in life, upper surfaces uniform green, often having small white or pale blue spots; (5) in life, yellow-coloured spot on the posterior rim of the eye when open; (6) short black bars to lateral surfaces of the flanks; (7) colour of undersides, hands, feet, concealed surfaces of thighs and flanks, orange; (8) short sloping snout; (9) calcar on heel, often terminating in two dermal processes with one projecting inward and one projecting outward. Variation. Variation is noted in the extent of the outward facing calcar found on the heel: in all specimens from Costa Rica and Panama this feature is large, extensive, and marked with dark pigmentation; in some specimens from Colombia and Ecuador the outward-facing calcar on the heel is highly reduced and more rounded. Distribution: Highly restricted populations occurring in northwest Ecuador, western Colombia, Panama, southeastern Costa Rica (Fig. 10). See discussion.

FIGURE 4. (a) Peptide fingerprint of Cruziohyla (b) Significant individual and shared peptides.

Cruziohyla craspedopus (Funkhouser, 1957) (Figs. 1c, 6c, 7c, 9c). Fringed Tree Frog / Rana Arbol Franja

Phyllomedusa craspedopus—Funhouser (1957). Agalychnis craspedopus—Duellman (1968): Transferred from Phyllomedusa. Cruziohyla calcarifer—Faivorvich et al (2005): Transferred from Agalychnis.

408 · Zootaxa 4450 (4) © 2018 Magnolia Press GRAY Holotype. SU 10310. Adult female from Chicherota, Rio Bobonaza, Napo-Pastaza Province, eastern Ecuador. Diagnosis. Cruziohyla craspedopus is a moderate-sized species of the genus Cruziohyla, SVL in males 53 mm to 66 mm and 68.3 mm to 75.5 mm in females, distinguished by a combination of the following characters: (1) snout is sharply truncate in profile; (2) lacking dark ventral thigh markings either side of the vent; (3) modal webbing formula (fingers): I 2-—2- II 2-—2 III 2-—2- IV; modal webbing formula (toes): I 1—1 II 1—2 III 1—3 IV 3—1 V; (4) in life, dark green background colour to very smooth dorsal surface, overlaid with a network of large irregular pale blue-grey blotches often with a thin cobweb-like pattern; (5) head is slightly wider than long; (6) narrow black vertical bands extend on lateral surfaces of the flanks to margin of underside; (7) in life, undersides and concealed surfaces of the flanks and legs are yellow; (8) sharply truncate snout; (9) presence of extensive dermal appendages forming irregular spurs along the posterior margin of the tarsus; (10) dermal fold to fifth toe and on forearm; (11) presence of a dermal fold around margin of lower jaw. Distribution: East of the Andes in Colombia, Ecuador, Peru and Brazil. Comparison of C. sylviae sp. n. with the congeners. The morphological features of C. sylviae sp. n., have been previously covered by Duellman (1970), and Savage (2002) under the name C. calcarifer. However, Cruziohyla sylviae sp. n. can be distinguished from both C. calcarifer and C. craspedopus by the following: Peptide fingerprinting. In examining the signals it can be seen that the majority of these appear unique to each species, with the greatest commonality seen between C. sylviae sp. n. and C. calcarifer (Fig. 4a). Although the number of replicates included are not sufficient to determine a confident diagnostic pattern, the differences do display a clear compositional difference between C. sylviae sp. n. and C. calcarifer (Fig. 4a). Ventral markings. Cruziohyla sylviae sp. n. can be distinguished from C. calcarifer in lacking dark pigmented markings found either side of the vent (Fig 5b). This feature is a key character of C. calcarifer and is not present in any other species of Cruziohyla. All specimens of C. calcarifer carry the feature to some degree, with it extending to the ventral parts of the thighs in larger specimens: A juvenile [KHJ884] from Esmeraldas, Ecuador, at 25 mm SVL, has small dark markings either side of the vent, whilst in those collected from Bocas del Toro, Panama, by Myers and Duellman (1982), 71.5–87.0 mm SVL, the markings extend over a wide surface area of the thigh region (Appendix I). All eleven specimens of C. calcarifer collected and detailed by Myers and Duellman (1982) [AMNH A107238–107248] carry the characteristic dark ventral markings to a high degree. The feature is also detailed in the field notes of Dr Myers at the time of specimen collection (April 21st, 1980: AMNH unpublished). The largest female specimen [AMNH A107238] has the most extensive thigh markings seen, thus it can be confirmed that this character is as strongly marked in females as in males. The dark markings, noted by Cochran and Goin (1970) in reference to the second C. calcarifer specimen detailed by Boulenger (1913) [BM:1913.11.12.126] from Colombia, fade little with age in preservative and are clearly seen as distinct patches either side of the vent on the holotype, and on the specimen from Colombia where they extend to the thighs (Appendix I). The exclusive presence of this feature in association with key characters seen in all genetically defined C. calcarifer specimens confirms it as a strong and reliable taxonomic character for that species. Therefore, the evidence of differences in ventral markings clearly distinguish C. sylviae sp. n from C. calcarifer. Close up examination with a Zeiss microscope at x 40 magnification clearly highlights gland ducts throughout the darkly pigmented ventral granulations (Fig. 5c). However, further work is required to establish the function of this unusual feature. Head morphology and tympanum width to the diameter of eye (TY/E). C. sylviae has a sloping snout when viewed in lateral profile compared to a sharply truncated snout possessed by C. craspedopus (Funkhouser, 1957), and in dorsal profile the snout of C. sylviae sp. n. is more rounded compared with that of C. calcarifer, which is broadly triangular and has an almost vertical loreal region (Cochran & Goin, 1970). Cruziohyla sylviae sp n. has a noticeably large tympanum, ranging between 70–100% the diameter of the eye compared with a tympanum size of 40–60% the diameter of the eye in C. calcarifer: the first C. sylviae sp. n. specimen collected, from Barro Colorado Island, Panama, has a tympanum diameter approximately the same size as the eye [ZA (MCZ) 15610]; one specimen from La Mosquitia region [SMF 86287], and two other specimens from Gracias a Dios, Honduras [USNM 559748;USNM 563933] also have tympani approximately the size of the eye (Moen, 2012). The TY/E of C. sylviae sp. n. specimens from the Guayacán, Limon Province, and Puerto Viejo de Sarapiqui, Heredia Province, Costa Rica are comparable: male TY/E 0.728–0.833 (mean 0.784; n = 17); female TY/E from 0.878–0.974 (mean 0.933; n = 10). These sizes are also comparable with the measurements described

REVIEW OF THE GENUS CRUZIOHYLA Zootaxa 4450 (4) © 2018 Magnolia Press · 409 by others for C. sylviae sp. n. from Costa Rica, having TY/E of 0.750–0.965 (Savage, 2002; Duellman, 1970), the latter being a female of 78.5 mm [USC 496] having a tympanum diameter approximately the same diameter as the eye (Duellman, 1970). The holotype of C. calcarifer (BM.1902.5.27.26), a small female (57mm) from Esmeraldas, Ecuador, has a tympanum half the diameter of the eye (Boulenger, 1902), as does a large female (87 mm) from Changuinola, Panama [AMNH 107238]. A wild male C. calcarifer specimen from Alto Tambo, Esmeraldas Province, Ecuador, has TY/E 0.540, and male specimens from Esmeraldas province in captivity at Manchester Museum [MM 1019– 1021] have TY/E 0.533–0.548 (mean 0.541, n = 3). Specimens from Colombia have TY/E to 60% (0.600): two live specimens, from near San Cipriano and Buenaventura, Valle de Cauca, Colombia, have TY/E of 0.417 and 0.567 respectively, the latter being comparable to the 60% ratio given by Cochran and Goin (1970) for the first specimen collected from Colombia [BM 1913.11.12.126]. Allowing for any sexual dimorphism within the overall TY/E ranges identified (C. sylviae sp. n: 70–100% TY/E; C. calcarifer: 40–60% TY/E), the differing TY/E support the identification of C. sylviae sp. n. Hands and feet. Cruziohyla sylviae sp. n. and C. calcarifer can both be distinguished from C. craspedopus in lacking the broad, well-developed, fringes found along the posterior margin of the distal part of the foot and fifth toe in that species (Funkhouser, 1957; Duellman, 1970; Hoogmoed & Cadle, 1991; Meneghelli et al., 2011). Distinctions between C. sylviae sp. n. and C. calcarifer include C. sylviae sp. n. having webbing that extends to the penultimate phalanx on the 4th toe (Duellman, 1970) whereas C. calcarifer has more moderate webbing that extends to the antepenultimate phalanx on the 4th toe (Cochran & Goin, 1970) (Fig. 6). Colouration comparison. All Cruziohyla species are capable of considerable metachrosis, however overall dorsal colouration and patterning is fully consistent when compared intraspecifically and distinctly different when compared interspecifically: C. sylviae sp. n. has a dorsal surface colouration consisting of two shades of green, with an areolated texture, giving a very finely granulated appearance, and also having small pale green lichenose speckles throughout; C. craspedopus has a uniform dark green dorsal surface background colouration with irregular-shaped large pale blue-grey lichenose blotches throughout; C. calcarifer has a consistent uniform green background colouration, often with a fine scattering of white or pale-blue spots (Fig. 7), and possesses a small yellow coloured spot directly posterior to the eye on the very rim. This marking, which is concealed when the frog is at rest, is lacking in both C. craspedopus and C. sylviae sp. n. Flank barring. All members of the genus Cruziohyla have skin colourations that include a degree of black barring to the orange-yellow flanks, also having similar colourations on the thighs, foot, forearm and outer finger (Funkhouser, 1957, Duellman, 1970; Savage 2002; Kubicki, 2004). Cruziohyla. craspedopus has narrow black lines on the flanks (Funkhouser, 1957), C. sylviae sp. n. has barring that form black vertical bands (Kubicki, 2004), and C. calcarifer has black barring that is short and limited (Boulenger, 1902). All the examined specimens of C. calcarifer from northwestern Ecuador to southeastern Costa Rica have black barring down the side lateral surface, forming short vertical finger-like bars extending approximately halfway down the flanks. By comparison, specimens of C. sylviae sp. n. and C. craspedopus have black vertical band patterning on their flanks extending further than halfway down the lateral surface, often extensive and interconnecting (Fig. 11). Dorsal colouration. Cruziohyla sylviae sp. n. is considered a dark green frog (Savage, 2002). Small green (GK113-114) lichenose speckles are present throughout the whole of the dorsal surface, extending to the forearm, shanks, tarsi, fourth fingers, and fifth toes (Duellman, 1970) (Fig. 7a). The pale green speckling on the dorsum is similar, but smaller, to the larger pale blue-grey lichenose blotches found on C. craspedopus (Hoogmoed & Cadle, 1991). Hoogmoed and Cadle (1991) make specific reference to the dorsal coloration of a C. sylviae sp. n. specimen from near Bribri, Limón Province, Costa Rica [ANSP 32501], separately to that of specimens of C. calcarifer (e.g. AMNH 16248) collected in Changuinola, Panama, by Myers and Duellman (1982). In the description of the C. calcarifer specimen collected by Dr Spurrell in Condoto, Chocó, Colombia, Boulenger (1913) used the collector’s field notes to highlight the dorsal colour of the specimen concerned (BM.1902.5.27.26 [BM. 1947.2.24.22]) as it was found in life: that the skin above is smooth and sage-green (Boulenger, 1913, pg.1023). There are no discernible differences between that specimen and the type, collected from the Rio Durango, Esmeraldas Province, northwest Ecuador (Cochran & Goin, 1970). All the specimens of C. calcarifer assessed from Ecuador, Colombia, and western Panama have a uniform green (GK106–107) dorsal skin surface which lacks the lichenose markings found on the other members of the genus (Fig. 7b). Specifically, the smooth green dorsal surface of C. calcarifer is most similar to that observed in members of the genus

410 · Zootaxa 4450 (4) © 2018 Magnolia Press GRAY Phynomedusa, the closest known relative of Cruziohyla (Duellman et al, 2016). Cruziohyla calcarifer does however have a scattering of very fine white or pale blue spots (GK191) to the dorsal surfaces, previously recognised by Myers and Duellman (1982) and reported on by Ibáñez et al. (1999). The white or pale-blue spots found on C. calcarifer contrast significantly when the consistent green dorsal surfaces darken through metachrosis (Fig. 11).

FIGURE 5. Dark pigmented ventral markings of C. calcarifer (a) Juvenile originating Esmeraldas, Ecuador (b) Ventral marking detail (c) Ventral marking x 40 magnification.

Cruziohyla craspedopus has very smooth uniform dark green background coloration to the dorsal surfaces together with a network of large, highly-contrasting, pale greyish-blue coloured lichenose markings. These vary from thin cobweb-like patterns to large irregular blotches (Funkhouser, 1957; Hoogmoed & Cadle, 1991; Nunez et al., 2015). The dorsal markings of specimens from Yasuní, Ecuador, and specimens from near Nasuta, in Loreto, Peru [MM1014–1018] are similarly marked, with large pale grey-blue lichenose blotches (GK287) contrasted against a dark green (GK132) dorsal background (Fig. 7c). Pattern development. The development of the skin colourations from juvenile to adult in all species of Cruziohyla varies considerably: Juvenile C. craspedopus show typical lichose markings on their dorsal surface before metamorphosis, and their juvenile colouration is similar to that of the adults; juveniles of C. calcarifer show bright yellow markings above the mouth and on the lower eyelid, which changes to green as adult; Juvenile C. sylviae sp. n. show no yellow markings above the mouth or on the lower eyelid, being brown initially and eventually changing green with age (Kubicki, 2004: sub C. calcarifer). The extensive barring to the flanks seen in C. sylviae sp. n. and C. craspedopus develops as vertical bands on the sides of both species amid the yellow-orange

412 · Zootaxa 4450 (4) © 2018 Magnolia Press GRAY flank background colour. By comparison, the reduced barring seen in C. calcarifer develops gradually with age with each finger-like bar extending slowly down the flanks from the very margin of where the green dorsal surface meets the orange-yellow flank. These differences in pattern development allow for the clear identification of juvenile C. sylviae sp. n (Fig. 5a).

FIGURE 7. Selected morphological characters of Cruziohyla: (a) C. sylviae sp. n. (MM1029); (b) C. calcarifer (MM1020); (c) C. craspedopus (MM1016).

Call comparison. Advertisement calls of Cruziohyla consist of single low-pitched notes repeated at regular intervals. C. sylviae sp. n. has a call (note) duration of 170–215 m/secs (x̄ = 190 ± 3.7 m/secs, n = 14) (Fig. 8) compared with the call duration of 105–120 m/secs (x̄ = 112.5 ± 2.1 m/secs, n = 6) for C. craspedopus, and 135– 212 m/secs (x̄ = 171.1 ± 10.1 m/secs, n = 7) for C. calcarifer. Variances in different recording methods and qualities make overall comparisons of frequency band ranges limited. However, the highest intensity peak (maximum energy output) within the dominant frequency band range for C. sylviae sp. n. is located around 750Hz, compared with C. craspedopus at around 700 Hz and C. calcarifer at around 860 Hz. C. calcarifer also has a similarly intense output at around 950 Hz (Fig. 9a–c).

FIGURE 9. Audiospectragrams of the advertisement call of members of the genus Cruziohyla: (a) C. sylviae sp. n.; (b) C. calcarifer (courtesy AMNH); (c) C. craspedopus (courtesy Morley Read).

Distribution. Cruziohyla craspedopus occurs east of the Andes and is found in Colombia, Ecuador, Peru and Brazil (Funkhouser, 1957; Hoogmoed & Cadle, 1991; Ruiz-Carranza et al., 1996; Meneghelli et al., 2011; Venâncio et al., 2014; Moraes et al., 2017). The species is primarily associated with Amazonia and has a distributional range that does not overlap with either C. calcarifer or C. sylviae sp. n. C. calcarifer occurs from Esmeraldas Province in northwest Ecuador (Boulenger, 1902; Cisneros-Heredia, 2005; Coloma et al., 2008), through western Colombia at several reported localities (Boulenger, 1913; Cochran & Goin, 1970; Ruiz-Carranza et al., 1996). In Colombia the species has been recorded in Municipal Mecana in the Chocó and Valle de Cauca (Vargas & Bolaños, 1999; Lynch & Suárez, 2004; Castro-Herrera & Vargas-Salinas, 2008) and Panama (Myers & Duellman, 1982; Crawford et al., 2010), to the most southerly part of Costa Rica, where it is found in the Fila Carbon area of Comadre, Limón Province (Faivovich et al., 2010). Thus, there is considerable overlap in the distribution of these two species (Fig. 10).

All specimens of C. calcarifer collected and reported on by Myers and Duellman (1982) from Changuinola Bocas del Toro, northwestern Panama, have been examined as part of this study and are confirmed as C. calcarifer [AMNH A107238–107248] (Fig. 11). Two specimens from the same locality [KHJ150;KHJ151], one specimen from “Omar Torrios”, El Cope, Panama [USNM 572743 (KRL 800)], and two specimens from Comadre, Costa Rica [KHJ30;KHJ31], are also in agreement with all the features of C. calcarifer specimens from all localities highlighted for the species (Fig. 10). These align with specimens of C. calcarifer from Esmeraldas, Ecuador, e.g. KHJ831, a specimen previously sampled by Faivovich et al. (2010), which may be considered genetically representative of the name bearing population (Faivovich et al., 2010). Cruziohyla sylviae sp. n. has a distribution range extending from the Gracias a Dios Province of southeast Honduras, southwards through Nicaragua and Costa Rica to Laguna in the Darien Province of southern Panama (Fig 11). Alto Colorado, near Guayacán, Costa Rica, is the type locality for C. sylviae sp. n., and live specimens observed at the nearby CRARC possess all the characteristics of the species, as do specimens from Alajuela and near Bribri, Costa Rica [UCR15601/ANSP 32501]. The collection of the latter C. sylviae sp. n. specimen, approximately 10 km south from that of C. calcarifer specimens collected at Comadre, Fila Carbon, Costa Rica [KHJ150/KHJ151], suggests that in Costa Rica both species may occur sympatrically (Fig 10). In Panama, both species do occur sympatrically: a specimen of C. sylviae sp. n. is observed (iNaturalist, 2018) 3.5 km from the collection site of C. calcarifer [USNM 572743 (KRL 0800)], at “Omar Torrios”, Panama, where specimens of both species were collected from the very same locality and included in the work of Crawford et al. (2010) and this work (C. calcarifer: USNM 572743 [KRL 0800] and C. sylviae sp. n. (USNM 572744 [KRL 0781]).

Discussion

This work has presented the significant and consistent differences between the new species and both other

REVIEW OF THE GENUS CRUZIOHYLA Zootaxa 4450 (4) © 2018 Magnolia Press · 415 members of the genus Cruziohyla. The molecular analyses verify early observations made by Crawford et al (2010) that C. calcarifer and C. sylviae sp. n show molecular divergence beyond commonly accepted amphibian barcode thresholds. For their specimens from Omar Torrios, Panama, Crawford et al (2010) detected 12.5% divergence at mitochondrial gene CO1, having an arbitrary threshold value of 10%, which lends support to the result of a minimum genetic divergence of 6.2% at the 16S gene, having a usual threshold of 3–5% (Vences, 2005 & Fouquet et al, 2007). Despite morphological similarities, phylogenetic results place C. craspedopus as the sister species of C. sylviae sp. n., rather than C. calcarifer as presumed. The genus Cruziohyla is confirmed as monophyletic with the present composition. The taxonomic history of the new species explains how it remained hidden in plain sight for so many years. The first Cruziohyla specimen to be collected in Panama (ZA[MCZ]15610) was found by Emmett Reid Dunn on Barro Colorado Island on December 18th, 1927 (Dunn, 1931). Unfamiliar with the species, and others in the genus occurring in the area at that time, Dunn concluded that the specimen was C. calcarifer based on it being ‘Green with concealed surfaces orange with black bars’ (Dunn, 1931: p.420). The specimen [MCZ A15610] was deposited in the Museum of Comparative Zoology at Harvard University, USA. Here it was accessioned as C. calcarifer, potentially without the curator making his own direct comparison with the holotype after being told by Dunn that it was the third C. calcarifer specimen to be collected (Curatorial note in the amphibian catalog of Harvard University Museum (1928, pg. 65). The third specimen of C. calcarifer collected was actually the one found by Mr R. Johnson at the Rio Atrato in Colombia, on the 31st October 1920 [AMNH A13604]. The male specimen Dunn had collected in Panama possesses all the features of C. sylviae sp. n.. George Lee then collected the second Cruziohyla specimen found in Panama, on 6th May, 1951, [FMNH 67446] from the same locality. This specimen also agrees with all the diagnostic features of C. sylviae sp. n., the white venter of this specimen also being noted by Funkhouser (1957). Seemingly, Emmett Dunn and George Lee unknowingly collected the very first specimens of the new species, and further specimens collected in Central America based on their identification may have inadvertently prevented later distinctions between the two species being made. The confusion made is understandable, as both species share many striking characters of the genus and to the untrained eye may look similar. The first specimens of C. calcarifer found in Costa Rica were almost certainly discovered by Miguel Solano in the Fila Carbon area in the mid 1980s (Karl-Heinz Jungfer, pers. comm.), “Comadre” being the general locality name used as reference for specimens collected in the area at that time (Miguel Solano, pers. comm.). The specimen [KHJ 831] from Comadre, Costa Rica, which provided genetic material for this study and also that of Faivovich et al. (2010), relates to specimens from that early collection. Reports of two forms of C. calcarifer occurring in Costa Rica have been based on Solano’s experience of discovering a population that differed from those normally encountered, where ‘males were the size of normal females’ (Miguel Solano, pers. comm.) This undoubtedly refers to Solano’s early experience with the true C. calcarifer compared with C. sylviae sp. n., the type specimen of the latter being collected as a result of his assistance in 1998. The ventral markings described for C. calcarifer in this work are a highly unusual feature to be found on any hylid frog. The presence and distinctiveness of the ventral markings suggest a special function for this feature. Appearing to be bilaterally symmetrical, and developing into distinctive triangular patterning on the thighs, this prominent characteristic develops soon after metamorphosis and is distinct before juveniles attain adult colouration (Fig. 5a). Therefore, it would be interesting to study the ontogeny of this feature in order to try and establish the function it serves in the life history of C. calcarifer. The feature may be glandular in nature, with external pores leading to specific exocrine glands. It is as prominent in females as in males, so a possible role in anti-predator defense may not be ruled out. The dark ventral markings seen in C. calcarifer are not found on any other Cruziohyla or Central American Phyllomedusid. However, at least one species belonging to the closest genus Phrynomedusa, P. dryade (Baêta et al, 2016) from southeastern Brazil, has very small black markings either side of the cloacal region. These markings, described as ‘black and white marbling’ (Baêta et al, 2016), may upon further investigation be comparable. Both genera possess distinct triangular calcars on the tarsus, and an eye that contains a bicoloured iris and which lacks a reticulated palpebral membrane (Baêta et al, 2016). All members of the genus Phrynomedusa also have a relatively small tympanum to eye diameter size ratios (50% or less). It is interesting to note that although female C. calcarifer and C. sylviae sp. n. grow to a similar approximate size, with male C. sylviae sp. n. attaining a considerably smaller size than that of their male C. calcarifer

416 · Zootaxa 4450 (4) © 2018 Magnolia Press GRAY counterparts, C. sylviae sp. n. retain proportionally larger tympani. Caprinica (1977) suggest any such changes to the amphibian ear require a very gradual evolutionary process. It is considered that tympanum size in anurans notably influences their auditory sensitivity (Loftus-Hills, 1973), with those having larger tympani exhibiting greater sensitivity (Fox, 1995). That the notably soft calls of both species are quite similar suggests that possibly a variation in sexual selection may be involved in constraining tympanum size. Limited reports suggest that their reproduction may differ, with C. calcarifer potentially using open terrestrial pools to breed, where males call together are more easily heard, compared with C. sylviae sp. n, where males will call singularly within the forest and pairs use water holes in trees to reproduce (Kubicki, 2004; Karl-Heinz Jungfer, pers. comm).

FIGURE 11. Size comparison of two adult female Cruziohyla specimens from Panama: (a). C. sylviae sp. n. [KU 77415] from Laguna, Darien (Courtesy William E. Duellman) (b). C. calcarifer [AMNH A107238] from Chanquinola, Bocas del Toro (Courtesy Charles W. Myers). * Note darkening of skin through metacrosis.

The reproduction of C. sylviae sp. n. and C. craspedopus are well described (Hoogmoed & Cadle, 1991; Gray, 2001; Savage, 2002; Kubicki, 2004 (sub: C. calcarifer). However, a re-examination of the tadpole described for C.

REVIEW OF THE GENUS CRUZIOHYLA Zootaxa 4450 (4) © 2018 Magnolia Press · 417 calcarifer by Donnelly et al (1987) is required as only adult specimens of C. sylviae sp. n. are confirmed from the collection locality, La Selva Biological Research Station, Puerto Viejo de Sarapiqui, Costa Rica. The collection site is approximately 80 km from the type locality of C. sylviae sp. n., and more than 2000 km from the type locality of C. calcarifer. The conservation status of C. calcarifer is currently Least Concern (Coloma et al., 2008). With a reduction in distributional range associated with the new species, a conservation status review for members of the genus may now be appropriate. Cruziohyla calcarifer is a particularly rare frog and populations are highly restricted in all countries in which it is known to occur: In Ecuador the species is recorded very infrequently and where it occurs is under considerable human pressure (Coloma et al., 2008); In Colombia, the species has been recorded only at very few localities, mainly in the Chocó and Valle de Cauca (Castro-Herrera & Vargas-Salinas, 2008) and information on the level of potential threats faced by the species there is limited; in Panama, only thirteen specimens are known (Duellman & Myers, 1982; Crawford et al., 2010), the last two being found over 15 years ago; in Costa Rica only two specimens are currently known [KHJ30/KHJ31], found over 30 years ago. The identification of the new species, incorporates both ‘Integrative’ and ‘Forensic’ taxonomy (Dayrat, 2005; Mendleson, 2011), combining a number of disciplines in the gathering of evidence to piece together a taxonomic discovery that has eluded herpetologists for over 90 years. This highlights the value of historical museum specimens and the descriptions produced by pioneering herpetologists of the past in supporting the present recognition of species, which may be in real need of conservation in the future.

Acknowledgements

I am indebted to Franziska Elsner-Gearing for her help in genetic work, Karl-Heinz Jungfer for providing samples and related information, and David Knight, Georgina Drury, and Emma-Jayne Keevil for support with peptide profiling. I am grateful to Miguel Solano for his assistance in the collection of specimens. Access to key specimens and related material was kindly provided by D. Dickey, D. Kizirian, C. Raxworthy (AMNH) G. Köhler (SMF), F. Bolaños and G. Chaves (UCR), J. Streicher (BMNH), A. Resetar (FMNH), J. Rosado and J. Martinez (MCZ), N. Gilmore (ANSP), N. Camacho (LACM), S. Gotte, and J. Poindexter (USNM), A. Sosa, N. Geisler, L. Guarderas (CJ). Images for incorporation were kindly provided by David Dickey (AMNH) on behalf of Charles Myers, Lauren Vonnahme (AMNH), Matthew Wilson, Matthew O’Donnell (MM), Pete Oxford, Addison Wynn (USNM). I thank Robert Jehle, Dmitri Logunov, and Amanda Bamford, as well as Victor Orrico, and two anonymous reviewers, for providing valuable comments on previous drafts of the manuscript. Alex Villegas and Morley Read supported call comparisons. I am grateful to William Duellman for providing his personal material and encouragement. I thank Brian and Aura Kubicki for their hospitality and allowing me to observe specimens on their property. Thanks to Dan Wand and Patrick Reynolds for providing graphics support and Francisco Herrerias-Azcue for translating the abstract. I would also like to thank Javier Guevara and the Costa Rican Ministry of Energy, the Environment, and Telecommunications (MINAET) for providing scientific collecting and export permits (109-98- OFAU; DGVS-166-98).

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