Bioacoustics of Hyalinobatrachium carlesvilai

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.

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