Hyalinobatrachium carlesvilai, Castroviejo-Fisher, Santiago, Padial, José M., Chaparro, Juan C., Aguayo, Rodrigo & Riva, Ignacio De La, 2009

Castroviejo-Fisher, Santiago, Padial, José M., Chaparro, Juan C., Aguayo, Rodrigo & Riva, Ignacio De La, 2009, A new species of Hyalinobatrachium (Anura: Centrolenidae) from the Amazonian slopes of the central Andes, with comments on the diversity of the genus in the area, Zootaxa 2143, pp. 24-44 : 26-33

Hyalinobatrachium carlesvilai new species

( Figs. 1 A–C, 2A–C, 3A, 7A–B)

Holotype. MHNCP 5339 (IDL 4545), adult male from a point between Santa Rosa and San Juan del Oro (14º12'49.1" S, 69º08'09.5" W; 1135 m), Provincia Sandia, Departamento Puno, Peru, collected by I. De la Riva, S. Castroviejo-Fisher, J.C. Chaparro, J. Bosch and J. M. Padial on 12 February 2006.

Paratopotype. MNCN 43689 ( IDLR 4546), adult male, same data as holotype.

Paratypes. MNCN 43690–2 (IDLR 4557–9), adult males; MHNCP 5344 (IDLR 4560), adult female from a point three kilometers from the type locality (14º12'49.1" S, 69º08'09.5" W; 1135 m), Provincia Sandia, Departamento Puno, Peru, collected by I. De la Riva, S. Castroviejo-Fisher, J.C. Chaparro, J. Bosch and J.M. Padial on 12 February 2006; MNCN 44213 (IDLR 4759), adult male from a point placed fifteen kilometers from Quincemil towards Puerto Maldonado (13°12’03.6’’ S, 70°40’28.9’’ W; 572 m), Provincia Quispicanchis, Departamento Cusco, Peru, collected by I. De la Riva, S. Castroviejo-Fisher, J.C. Chaparro and J.M. Padial on 0 2 February 2007; MHNCP 6688, adult male from Cueva de los Guácharos (09°19'33.4'' S, 76°01'45.7'' W; 674m), Tingo Maria, Provincia Leoncio Prado, Departamento de Huánuco, Peru, collected by J.C. Chaparro, J.A. Ochoa and R. Gutiérrez on 0 2 November 2007; MHNCP 5434, adult male, first “ chacra ” (crop clearing) after the Puesto de Vigilancia 3 de Mayo (09º25'10.5" S, 75º58'15.0" W; 723 m), Parque Nacional Tingo María, Distrito Mariano Damazo Veraun, Provincia Leoncio Prado, Departamento Huánuco, Peru, collected by J.C. Chaparro on 15 September 2006; MNCN 42797 (JMP 920), adult male from Paractito-los Guácharos (17º03' S, 65º28' W; 500 m), Provincia Chapare, Departamento Cochabamba, Bolivia, collected by J.M. Padial on 0 1 March 2004; CBG 1139,1140, adult males from Río Leche (17º16' S, 64º45' W; 500 m), Provincia Carrasco, Departamento de Cochabamba, Bolivia, collected by R. Aguayo and R. Rivas on 24 October 2004; ZFMK 75238, adult male from 7 km on road south of Paractito (17°04' S, 65°29' W; 500 m), Provincia Chapare, Departamento Cochabamba, Bolivia, collected by J. Köhler on 0 3 February 1998.

Diagnosis. The new species is placed in the genus Hyalinobatrachium because of the following characters: (1) humeral spine absent (Savage 1967); (2) digestive tract and bulbous liver covered by white peritonea (Savage 1967); (3) completely transparent ventral parietal peritoneum (Savage 1967); (4) white bones in life (Savage 1967); (5) dorsal coloration in preservative white or cream (Savage 1967); (6) males lack conspicuous dorsal spinules during breeding season; (7) when present, nuptial pad small and restricted to the inner edge of Finger I in males (Type V of Cisneros-Heredia & McDiarmid 2007); (8) dentigerous process of the vomer and vomerine teeth absent ( Ruiz-Carranza & Lynch 1991; Savage 1967); (9) males usually vocalize from the underside of leaves, and females deposit one layer of eggs on the underside of leaves (Ruiz- Carranza & Lynch 1998).

The following combination of characters distinguishes Hyalinobatrachium carlesvilai sp. nov. from other species of the genus: (1) dentigerous process on vomer absent; (2) snout truncate in dorsal and lateral view; (3) tympanum absent, tympanic annulus indistinct; (4) dorsal skin finely shagreened in life and preservative; (5) ventral skin granular, cloacal ornamentation consisting of small enameled tubercles and folds, enlarged paired round tubercles below vent absent; (6) parietal peritoneum transparent, pericardial, hepatic and visceral peritonea white, all other peritonea transparent; (7) liver bulbous; (8) humeral spine in adult males absent; (9) finger webbing III 2 – – 1+ IV, absent between Fingers I and II and basal between Fingers II and III; (10) toe webbing I 1 – 2 – II 1 – 2 – III 1 – 1 1/2 IV 1 1/2 – 1 V; (11) fringe on postaxial edge of Finger IV present and enameled, metacarpal fold present and enameled, ulnar fold present and enameled; fringe on postaxial edge of Toe V present and enameled, metatarsal fold present and enameled, tarsal fold present and enameled; (12) nuptial excrescence formed by a group of glands on Finger I (Type V), extending to the lateral fringes and membranes of the other fingers; glands slightly visible in the webbing between toes IV and V; prepollex not enlarged; prepollical spine not projecting (spine not exposed); (13) when adpressed, Finger I longer than II; (14) diameter of eye about 2X width of disc on Finger III; (15) coloration in life: dorsal surface lime green with small yellow spots and minute melanophores, bones white; (16) coloration in preservative: dorsum cream with dark melanophores; (17) iris coloration in life: cream with black flecks and an incomplete pale yellow ring around pupil; (18) distribution of melanophores on digits constant, only present on the proximal edge of Fingers I and toes IV–V; in life, hands and feet light green, tips of fingers and toes orange; (19) males call from the under side of leaves; advertisement call consisting of a single note, the first third being pulsed and the second third tonal; call duration 0.102– 0.152 s, dominant frequency 4617.33–4915.36 Hz; (20) clutches deposited on vegetation overhanging streams on the under side of leaves, clutch size 27– 32 eggs (n = 2); clutches observed were guarded by males; (21) SVL in males 20.6–23.9 mm (n = 7); in females 23.2 mm (n = 1). Combat behavior and tadpoles are unknown for the new species.

Comparisons. All described species of Hyalinobatrachium known to occur in the Amazonian slopes of the Andes are endemic to this area. Because the new species described here inhabits this ecoregion, we constrict our comparisons to those Hyalinobatrachium species occurring in Amazonian slopes of the Andes. Hyalinobatrachium carlesvilai sp. nov. can be distinguished from H. bergeri (SE Peru and Bolivia), by (character of the former in parentheses) webbing formula on hand III 3 – 2 + IV (III 2 – – 1+ IV; Fig. 2 ), pericardium at least partially transparent (completely white), and presence of a dark grey ring around the pupil (absent; Fig. 3 ). Hyalinobatrachium esmeralda Ruiz-Carranza & Lynch (1998) , from the eastern Cordillera of Colombia, has a hand webbing formula III 2 + – 2+ IV (III 2 – – 1+ IV), transparent pericardium [but see Ruiz- Carranza & Lynch (1998; 4C)] (white pericardium), canthus rostralis not defined (defined), and round snout in dorsal and lateral view (truncate; Fig. 2 ). Hyalinobatrachium lemur ( Fig. 4 D), from Departamento San Martín in northern Peru (but see below), has a transparent pericardium (white), and has weak or absent humeral and tarsal folds (enameled ulnar and tarsal folds). Hyalinobatrachium munozorum ( Fig. 4 B), from the eastern Amazonian lowlands of Colombia, Ecuador and Peru (but see below for Peruvian localities), has a round snout in dorsal and lateral view (truncate), and lacks humeral and tarsal dermal folds (enameled ulnar and tarsal folds). Hyalinobatrachium pellucidum ( Figs. 4 A, C), from the Amazonian versant of the Andes in Ecuador (but see below for geographical extension), has a round snout in dorsal and lateral view (truncate), golden iris (creamy white; Fig. 3 ), webbing formula on hand III 2 + – 2 IV (III 2 – – 1+ IV), and a transparent pericardium (white). Hyalinobatrachium ruedai Ruiz-Carranza & Lynch (1998) , from the Amazonian slopes of the Andes in Colombia and Ecuador, has medium sized melanophores (absent), and an intense golden iris with a pupillary ring (cream and pupillar ring absent). We summarize in Table 1 characteristics that distinguish these species of Hyalinobatrachium .

Taxa SVL Snout Hand webbing Pericardium Iris

H. bergeri 20.3–22.4 Truncate III 3 – 2 + IV Pa r ti a l l y Dark grey ring around pupil

transparent

Description of the holotype. Adult male of small size, SVL 22.4 mm; head slightly wider than body, HW 36% of SVL; head slightly wider than long (HW/HL = 1.2); snout truncate in dorsal view and profile; ES/EL = 0.75 and ES/IOD = 1.0; loreal region slightly concave; nostrils slightly prominent, round; internarial region depressed; canthus rostralis defined; eyes small, directed antero-laterally; EL 47% of HL; EW/IOD = 0.8; tympanic annulus indistinct, tympanic membrane absent, supratympanic fold absent; dentigerous processes on vomers absent; choanae small, circular, widely 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; enameled ulnar fold remarkably evident; 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 – – 1+ IV; subarticular tubercles round and small; supernumerary tubercles slightly appreciable; palmar tubercle round and small, thenar tubercle small and elongated; nuptial excrescences Type V, glands present on the lateral fringes of fingers and the sides of membrane between fingers III and IV; hind limbs slender; SL 55% of SVL; enameled tarsal fold remarkably 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 – 1 1/2 IV 1 1/2 – 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.

Coloration in life. Dorsal surfaces light green dusted with minute black melanophores and with dull yellow spots. Enameled tarsal and dorsal folds. Cloacal ornamentation consisting of enameled warts and folds. Dorsal surface of tip of fingers orange. Dull creamy iris dotted with dark flecks. Parietal peritoneum transparent, pericardium, hepatic, and visceral peritonea white, peritonea covering the gall bladder and other internal organs (urinary bladder, gonads, kidneys) not mentioned before transparent.

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

Measurements. Holotype morphometrics are as follow: SVL = 22.4; HL = 6.8; HW = 8.1; IOD = 2.4; EL = 3.2; EW = 1.9; ES = 2.4; FIII = 1.3; TL = 12.3; SL = 12.2; FL = 10.8. Measurements of the complete paratype series but ZFMK 75238, which was not measured, are in Table 2 .

Variation. No significant variation was appreciated through the type series.

Distribution and ecology. Hyalinobatrachium carlesvilai sp. nov. is known from the Amazonian forest of the Andean slopes in Peru and Bolivia ( Fig. 5 ). In Peru it is known from Tingo María and the valleys of Marcapata and upper Tambopata, Departments of Huánuco, Cusco, and Puno respectively, and in Bolivia it has been found in the Provinces of Chapare and Carrasco (Departamento Cochabamba) and the Amboró National Park (Provincia Ichilo, Departamento Santa Cruz), covering an airline distance of approximately 1000 km ( Fig. 6 ). We have found specimens between 300 and 1200 m. We found specimens on leaves (1–3 m above water) along streams and rivers between dusk and midnight. All males collected were calling from the underside of leaves ( Fig. 7 A). The only female collected (paratype MHNCP 5344; Fig. 7 B) was on the same leaf as the paratype MNCN 43690, which was attending an egg clutch ( Fig. 7 C). The holotype MHNCP 5339 was found guarding a clutch of 27 eggs in advanced state of development ( Fig. 7 D), both the specimen and the clutch were in the same leaf. The egg clutch was collected and preserved in ethanol 70% (MNCN/DNA 8999). We did not visit egg clutches during the day. Hyalinobatrachium carlesvilai sp. nov. occurs in sympatry with H. bergeri , both occupy approximately the same altitudinal range and have the same reproductive strategy. Despite this apparent overlap in their niches, both species are abundant, suggesting little effect of potential competition. In neighboring areas occupied by H. carlesvilai sp. nov. in SE Peru, we collected other anurans, namely Atelopus sp., Teratohyla aff. amelie, Amereega simulans, Hypsiboas balzani, Hypsiboas boans, Pristimantis fenestratus, and P. cf. martiae.

Advertisement call. We recorded and analyzed a total of 23 notes from two males (MHNCP 5339 and MNCN 4557); air temperature was 21°C. The advertisement call consists of a single high-pitched note that was clearly audible at long distances, the first third being pulsed and the second third tonal ( Fig. 8 ). It lasts 0.102–0.152 seconds (‾ X = 0.134 ± 0.013) with a call repetition rate of 4.6 calls/minute. The dominant frequency is 4617.33–4915.36 Hz (‾ X = 4837.95 ± 85.79) and the call rises in frequency from 3606.40–4112.80 Hz (‾ X = 4019.6 ± 137.85) at the beginning of the call to 50693.30–5519.40 Hz (‾ X = 5225.33 ± 140.99) at the end. The first third of all recorded calls starts with a group of short pulses (2–4) that shows a fast rise in frequency. The following two thirds of the call consist of a tonal section at slightly increasing frequency. The maximum amplitude of the call is reached at the beginning of the tonal section.

We realized that the call here described is basically identical to that described by Márquez et al. (1996) and later published by De la Riva (2002) for Bolivian purported H. bergeri . Accordingly, we analyzed those recordings and compare them to our new recordings of H. bergeri . We summarize the results in Table 3 and Figure 9 . Our analysis shows that the call assigned to H. bergeri by Márquez et al. (1996) and De la Riva (2002) actually corresponds to the new species herein described. Both calls overlap in all the parameters studied and show the same structure. On the other hand, the calls emitted by H. bergeri (twenty eight calls corresponding to the vouchers MHNCP 5394 and MHNCP 5408, plus a non-collected specimen and recorded at 20–25°c) are remarkably different. The call of H. bergeri is completely tonal lacking the characteristic pulsed start of that of H. carlesvilai sp. nov. It is emitted at a constant frequency between 3775.20–4337.90 Hz (‾ X = 4084.68 ± 165.28) and 4788.10–5013.10 Hz (‾ X = 4876.36 ± 60.12) with a dominant frequency of 4489.60–4659.91 Hz (‾ X = 4599.08 ± 69.94).

Hyalinobatrachium carlesvilai * refers to the call previously assigned to H. bergeri by Márquez et al. (1996) and De la

Riva (2002) and that we identified as H. carlesvilai . Time is given seconds and frequency in Hertz. Phylogenetic relationships. Our genetic analyses ( Figs. 9–10 ) support the hypothesis of Hyalinobatrachium carlesvilae sp. nov. as an independently evolving lineage. Our results indicate that the sequences of the mtDNA gene analyzed are reciprocally monophyletic to all the other sequences analyzed, with little variation between and within populations (genetic distances 0–1 %) but clearly divergent from its sister clade H. fleischmanni + H. tatayoi (genetic distances 4.8–6.2 %). The topologies of the MP and NJ trees are congruent with our Bayesian tree ( Figs. 9–10 ). Additionally, Guayasamin et al. (2008a) provided genetic analyses of six loci that fully support recognition of H. carlesvilai sp. nov. (referred as Hyalinobatrachium aff. munozorum therein) as an independent evolving lineage that has been isolated from other recognized species of Hyalinobatrachium for a large time scale.

Etymology. The name is a patronym for Carles Vilà and is a noun in the genitive case. We take pleasure to dedicate this species to our dear friend in recognition of his scientific work on animal conservation, evolutionary biology, and domestication and for his continuous support on centrolenid research and companionship both in the field and the lab.

Figures

FIGURE 1. Hyalinobatrachium carlesvilai sp. nov. (A) paratype MNCN 44213 (photo JMP); (B) paratype MHNCP 5434 (photo JCC); (C) CET (adult male, specimen not yet catalogued, photo J. Ayarzagüena).

FIGURE 2. Details of the foot (A), hand (B), and head (C) of Hyalinobatrachium carlesvilai sp. nov. paratype MNCN 43691 (photos SCF). 1 = enameled tarsal fold; 2 = nuptial glands; 3 = enameled tarsal fold.

FIGURE 3. Irises of Hyalinobatrachium carlesvilai sp. nov. paratype MNCN 44213 (A) (photo JMP); H. pellucidum MHNCP 4880, adult male (B) (photo JMP); H. bergeri MHNCP 5713, gravid female (C) (photo JMP) and (D) (photo IDLR).

FIGURE 4. Hyalinobatrachium pellucidum, holotype (A) (photo Natural History Museum, University of Kansas); Hyalinobatrachium munozorum, holotype (B) (photo Natural History Museum, University of Kansas); Hyalinobatrachium pellucidum, adult male, MHNCP 4880 (C) (photo JMP); Hyalinobatrachium pellucidum, holotype of H. lemur (D) (photo Natural History Museum, University of Kansas).

FIGURE 5. Habitat of Hyalinobatrachium carlesvilai sp. nov. in Peru. Quincemil, Cusco (left and bottom right); between Santa Rosa and San Juan del Oro, Puno (top right).

FIGURE 6. Map showing the type localities of Hyalinobatrachium species from the eastern slopes of the Andes of Ecuador, Peru and Bolivia and the new locality of H. pellucidum in Peru. The star marks the localities of the holotype of H. carlesvilai sp. nov. 1 = Santa Cecilia; 2 = Río Azuela; 3 = Abra Tangarana; 4 = Cueva de los Guácharos; 5 = Parque Nacional Tingo María; 6 = Río Kimbiri; 7 = Quincemil; 8 = Santa Rosa and San Juan del Oro; 9 = Paractito-los Guácharos; 10 = 58.1 km SW Villa Tunari; 11 = Río Leche.

FIGURE 7. Calling male, paratype MNCN 43690 (A) and gravid female, paratype MHNCP 5344 (B) of Hyalinobatrachium carlesvilai sp. nov. Both were found in the same leaf together with an egg clutch not collected (C). Egg clutch, MNCN / ADN 8999, collected in the same leaf than the holotype (D).

FIGURE 8. Audiospectogram and oscillograms (top and down, respectively) of the advertisement calls of (A) Hyalinobatrachium bergeri, MHNCP 5394; (B) H. bergeri sensu Márquez et al. (1996) and De la Riva (2002), without voucher; and (C, D) H. carlesvilai sp. nov., holotype.

FIGURE 9. Bayesian majority rule consensus gene tree of 850 bp of the 16 S mtDNA gene. Numbers on branches indicate Bayesian posterior probabilities and bootstrap support of the Maximum Parsimony analysis respectively. Clades are labeled according to their general distribution (see main text for details). Type specimens of Hyalinobatrachium carlesvilai sp. nov. are in bold.

Tables

TABLE 1. Character states in species of the Hyalinobatrachium fleischmanni Group from the Amazonian lowlands and slopes of the Andes, plus the similar species H. fleischmanni and H. tatayoi. Males snout-vent length (SVL) in mm; snout shape in profile.

H. carlesvilai	20.6–23.6	Truncate	III 2– – 1+ IV	White	Creamy with dark flecks
H. esmeralda	21.2–22.4	Round	III 2+ – 2+ IV	White/Transparent	Golden with brownish flecks
H. fleischmanni H. munozorum	19.0–28.0 18.8–20.5	Subacuminated Round	III 2 1/ – 2 IV 2 III 2– – 1+ IV	White White/Transparent	Yellow with dark flecks Pale gold
H. pellucidum	20.4–21.4	Truncate	III 2 – 2 IV	Transparent	Yellow with dark flecks
H. ruedai H. tatayoi	20.2–22.6 21.5–22.4	Truncate Semiround	III 2 – 1 3/ IV 4 III 2– – 1 1/ IV 2	White White	Golden with dark flecks Yellow with dark flecks

TABLE 2. Measurements in mm of the type series (excluding paratype ZFMK 75238) of Hyalinobatrachium carlesvilai sp. nov.

SVL	MNCN 44213 23.9	MHNCP 5339 23.1	M N C N 43689 23.6	M N C N 43690 22.4	M N C N M N C N 43691 43692 22.7 20.6	M N C N 42797 23.5	MHNCP 5344 23.2	CBG 1139 21.92	CBG 1140 23.9
HL	6.5	7.1	6.9	6.8	7.2 6.2	6.7	6.6	6.9	7.2
HW IOD	8.5 2.3	8.2 2.3	8.3 2.7	8.1 2.4	8.4 8.1 2.6 2.5	8.7 2.7	8.5 2.7	8.4 2.3	8.7 2.5
EL	2.2	3.5	3.5	3.2	3.1 3.1	3.5	3.5	2.5	2.6
EW ES	2.1 2.4	2.3 2.3	2.2 2.2	1.9 2.4	1.9 1.7 2.4 1.7	2.0 2.8	1.8 2.2	2.0 1.7	2.1 1.8
FIII	2.4	1.1	1.4	1.3	1.2 1.2	1.1	1.4	1.1	1.4
TL SL	12.5 13.4	10.5 11.4	12.1 12.3	12.3 12.2	11.9 10.4 12.6 11.0	11.7 12.4	12.4 12.7	12.4 12.6	12.8 13.4
FL	10.6	10.0	10.8	10.8	11.0 9.8	10.5	10.3	9.9	10.6

TABLE 3. Characteristics of the advertisement call of Hyalinobatrachium bergeri and H. carlesvilai sp. nov.

	H. bergeri (n = 28)	H. carlesvilai * (n = 8)	H. carlesvilai (n = 23)
Structure	Completely tonal at constant frequency	First third pulsed and then tonal, increasing frequency	First third pulsed and then tonal, increasing frequency
M a x i m u m amplitude	Beginning of the call	Beginning of the tonal section	Beginning of the tonal section
Notes per call	1	1	1
Duration	0.129–0.198 (0.154 ± 0.019)	0.134–0.163 (0.146 ± 0.009)	0.102–0.152 (0.134 ± 0.013)
D o m i n a n t frequency	4489.6–4659.91 (4599.08 ± 69.94)	4489.60–4574.75 (4574.75 ± 45.52)	4617.33–4915.36 (4837.95 ± 85.79)
L o w e r frequency	3775.20–4337.90 (4084.68 ± 165.28)	3381.30–3775.20 (3648.60 ± 174.74)	3606.40–4112.80 (4019.06 ± 137.85)
U p p e r frequency	4788.00–5013.10 (4876.36 ± 60.12)	4788.00–4956.80 (4886.46 ± 58.23)	5069.30–5519.40 (5225.33 ± 140.99)
O th e r frequencies	9000; 13500; 18000	9000	–