Bioacoustics of Andinobates cassidyhornae

Andinobates cassidyhornae sp. nov. has an SVL of 19.03± 0.31 mm (mean±SD, N = 12 frogs), a bright red dorsum with the color extending onto the upper front and hind limbs; lower forearms and hind limbs are dark brown. Ventral coloration is black with bright red irregularly sized and spaced ovoid or ‘comma’ shaped blotches or spots. It can be externally distinguished from other species in the bombetes group by the distinctive color pattern: in A. cassidyhornae dorsum is bright red, and venter is black with well-defined bright red blotches or spots ( Figure 4 ) vs. (1) in A. opisthomelas dorsum is red often with a posterior suffusion to brown, and venter is black with numerous white spots or reticulation (white-venter form) or venter is chocolate brown sometimes with red suffusion from the flanks (brown-venter form); (2) in A. virolinensis venter is whitish or bluish with black reticulation; (3) in A. bombetes the anterior half of dorsum exhibits bright red, yellow or rarely orange longitudinal and broad dorsolateral stripes; (4) in A. tolimensis the head is yellow, fading to brown towards the dorsum; (5) in A. dorisswansonae the dorsum is black or brown with red blotches, and the venter entirely black or black with few white or yellowish blotches; and (6) in A. daleswansoni the head is entirely red, and the body dull gold or brown. Measurements of the holotype (mm). The holotype measurements correspond to an adult female with a SVL of 18.99 mm ; TL of 8.10; HaL of 4.58; HL of 4.85; HW of 6.29; GBW of 7.54; IOD of 2.31; HDT of 1.23; ED of 2.15; TSCN of 1.40; NED of 1.52; IND of 2.36; MTD of 0.75; W3FD of 0.78; W3F of 0.46; W3TD of 0.83; W3T of 0.47; W4TD of 0.87; and W4T of 0.49. The corresponding measurements of all specimens collected are shown in Table 2 . Measurements Andes- Andes- Andes- Andes- Andes- Andes- Andes- Median SD A1093 A1088 A1089 A1090 A1094 A1091 A1095 SVL 19.36 19.34 19.17 18.92 18.45 18.99 18.99 18.99 0.31 TL 8.59 8.52 8.23 8.51 8.50 8.66 8.10 8.51 0.20 HaL 4.59 4.67 4.67 4.60 5.14 4.91 4.58 4.67 0.21 HL 6.06 5.59 5.17 4.75 5.09 5.00 4.85 5.09 0.46 HW 5.38 5.99 5.94 5.75 6.20 5.90 6.29 5.94 0.30 GBW 8.50 8.85 8.13 7.11 7.56 7.20 7.54 7.56 0.66 IOD 2.38 2.30 2.12 1.85 2.26 2.14 2.31 2.26 0.18 HTD 1.41 1.17 0.98 0.96 1.05 0.97 1.23 1.05 0.17 ED 2.49 2.31 2.12 2.45 2.47 2.21 2.15 2.31 0.16 TSCN 1.78 1.34 1.26 1.22 1.54 1.35 1.40 1.35 0.19 NED 1.71 1.68 1.54 1.57 1.78 1.68 1.52 1.68 0.10 IND 2.47 2.33 2.32 2.42 2.84 2.56 2.36 2.42 0.18 MTD 0.84 0.72 0.67 0.60 0.89 0.91 0.75 0.75 0.12 W 3FD 0.94 0.99 0.85 0.95 1.12 1.10 0.78 0.95 0.12 W 3F 0.49 0.44 0.48 0.53 0.65 0.52 0.46 0.49 0.07 Description of the holotype : The head is slightly wider than longer, and is narrower than the body. Snout subovoid in dorsal view and truncated in lateral view. Canthus rostralis subovoid, loreal region flat and vertical. Nares situated much closer to the end of the snout than to the eyes, ovoid in shape and directed posterolaterally. Eyes large and prominent with a diameter of 11.3% of SVL. The pupil is rounded and horizontally elliptical. Tympana and tympanic rings are in the posterolateral regions, ovoid and wider dorsoventrally, measuring 57.2 % of the diameter of the eyes. Supratympanic fold absent. W4TD 0.98 0.83 0.64 0.71 1.01 0.93 0.87 0.87 0.14 W4T 0.55 0.52 0.43 0.41 0.60 0.56 0.49 0.52 0.07 W3TD 0.69 0.91 0.66 0.79 0.83 0.78 0.83 0.79 0.09 W3T 0.50 0.54 0.54 0.52 0.68 0.57 0.47 0.54 0.07 BM in grams 0.64 0.62 0.58 0.59 0.53 0.58 0.60 0.59 0.03 Rounded choanae, not visible in ventral view, as they are completely covered by the maxillary arch. Vomerine, maxillary and premaxillary teeth are absent. Tongue is elongated, almost two times longer than wide; the posterior margin of the tongue is not indented and its posterior third is not adhered to floor of mouth. Hand relatively large ( Figure 3 ), with a length equal to 24.1% of the snout-vent length. The relative length of the fingers in increasing order of size is: IV<II<I<III. The tip of the number IV digit reaches the middle of the second to last phalange of the number III digit and the distal extreme of the first digit reaches the base of the disc of the second digit. Finger discs moderately expanded on digits of the hand. Paired dorsal pads on dorsal surfaces of the discs are present. Outer metacarpal tubercle somewhat flat and rounded, inner metacarpal tubercles are elliptical and are located at the base of the first (I) fingers, basal subarticular tubercles are rounded and flat over fingers I and II. Two rounded and flattened subarticular tubercles appear on the number III and IV finger digits; the latter subarticular tubercle is not pronounced ( Figure 3 ). The relative length of the toes, in increasing size order is I<II<V<III<IV. The discs of toes are smaller than disks of fingers. Toes number II with the basal subarticular tubercle not protuberant. Toes III and V with two subarticular tubercles, and toes IV with three subarticular tubercles. Supernumerary plantar tubercles are absent. External metatarsal tubercle is smaller than the inner metatarsal tubercles ( Figure 3 ). Coloration of holotype in life: Iris very dark brown; almost indistinguishable from black pupil. Nares encircled by black; margins of upper and lower jaws are very dark brown; tympana are black. Dorsum primarily bright scarlet red, sharply defined along the margins with a few irregularly scattered black speckles and small irregular black markings; in other specimens, black marks may reflect attacks by predators and should therefore not be considered an element of the dorsal coloration. Flanks are red laterally and black ventrolaterally with no gradient merging of the two colors as is seen in A . opisthomelas . Venter ground color black with irregular sharply contrasting bright scarlet red irregularly shaped blotches or spots. Upper forelimbs bright scarlet red and tinged brown where the upper and lower colorations meet, with lower forearm dark brown below the elbow; wrists and hands brown with tips of toes and fingers beige. Thigh mostly red, irregularly speckled with brown; shanks mostly brown tinged with red irregularly ( Figure 2 ). Color in preservative (ethanol 70%): The dark brown and black colors turn dull black to dark olive in preservative. Discs and tubercules on hands and feet, pupil and cornea become grey or nearly white with time. The predominantly bright scarlet red dorsal coloration and ventral red blotches and spots turn metallic olive; pattern remains clearly distinguishable in preservative (ethanol 70%). Natural history: The natural history of this species is poorly known. We found individuals in areas covered with a thick layer of leaf litter and where abundant refuges were available. Males call regularly from the leaf litter or hidden amidst tree roots, throughout the day but prominently between 10h–14h and after periods of rain. Most calling males were observed accompanied by a female. We also encountered several courting pairs during a visit in March and April 2012 . Males carry 1–3 tadpoles ( Figure 5 ) on their dorsum. Males of other species of the bombetes group are known to release their tadpoles in bromeliad water tanks. Some tadpoles of the new species were found in water within the inflorescence husks of Wettinia palms. Whether this species displays biparental care is unknown. Molecular and phylogenetic analyses. The final alignment consisted of 1119 bp (700bp unambiguously aligned from the Cytb gene and 419bp from the 16S gene). The chosen partitioning strategy was as follows: 16S unpartitioned under the GTR+ Γ model, and Cytb partitioned by codon position with K80+I, HKY+I and GTR+ Γ for the first, second, and third positions, respectively. Both ML and Bayesian phylogenies placed A. cassidyhornae sp. nov. as an independent, well-supported clade, separate from other species, within the bombetes species group. The two sequenced populations, the white- and the brown-venter forms, of A. opisthomelas formed a monophyletic albeit poorly supported group ( Figure 6 ). The topology obtained is roughly consistent with the results of Brown & Twomey et al . (2011), but changes the relationships between A. virolinensis , A. tolimensis and A. bombetes , placing the first two as reciprocally monophyletic clades, and A. bombetes as the sister group of the two. However, the deeper phylogenetic relationships between the species in this group still require further study, since the obtained topology is highly polytomic and not well supported at this level. Within A. cassidyhornae sp. nov. , individuals from the Mesenia-Paramillo Natural Reserve, Antioquia, cluster together in a monophyletic group, whereas individuals from other localities do not show evidence of genetic structuring. The SH tests rejected, at a very high level of statistical significance, two out of the three null topologies (H0) that placed A. cassidyhornae sp. nov. within a clade with A. opisthomelas (P <0.00001), with A. virolinensis (P <0.00001), and with A. bombetes (P = 0.05134). Additionally, pairwise genetic distances (K2P) between A. cassidyhornae sp. nov. and other species within the bombetes species group ranged between 0.0320–0.0677, well within the range of interspecific distances ( Figure 7 ) observed for the group (0.0216–0.1053), and about tenfold the intraspecific distances observed (0–0.0091). Altogether, the reconstructed phylogenetic tree, the topology (SH) tests, and pairwise genetic distances offer strong support for A. cassidyhornae sp. nov. being a distinct species within the bombetes species group. Bioacoustic analyses. All species’ calls within the Andinobates bombetes species group consist of long and atonal series of pulses sounding like a ‘buzz’ or rattle, which is often longer than 1 sec. The advertisement call of A. cassidyhornae sp. nov. ( Figure 8 ) follows roughly the same pattern. Calls consist of 234.3±20.3 (mean±SD) pulses, last 1.94± 0.26 s , and are often uttered as series of calls separated by regular silent intervals of 10.1± 2.1 s . The rise time is 50.0±13.2 % of the call duration. The peak call frequency averages 4.32±0.14 kHz and the frequency bandwidth 0.81±0.40 kHz. Combining the calls of A. cassidyhornae sp. nov. , A. opisthomelas (two localities), A. bombetes , and A. virolinensis , we found that call duration (linear regression, R2=0.31, F=13.9, P=0.0008, N= 16 males ), inter-call interval (R2=0.17, F=6.5, P=0.0162), and the number of pulses per call (R2=0.12, F=4.3, P=0.0472) decreased at higher temperatures. After removing the temperature effect by calculating regression residuals, the calls of A. cassidyhornae sp. nov. were clearly separated from the calls of the other species in a two-dimensional discriminant space ( Figure 9 , above, discriminant analysis, Wilks’ Lambda approxim. F=13.9, P<0.0001). The first discriminant axis accounted for 87.7 % of variation and separated very well the calls of A. cassidyhornae sp. nov. from the call of any other species because the former were lower in peak frequency (F to enter=39.6, standardized discriminant coefficient=1.10, P<0.0001). The second discriminant axis accounted for 10.1% of variation and separated the calls of A. cassidyhornae sp. nov. from the call of A. bombetes because the former were longer in duration (F to enter=6.9, standardized discriminant coefficient=0.97, P=0.0013). Regarding pairwise species comparisons by univariate ( Figure 9 , below) tests, the advertisement call of A. cassidyhornae sp. nov. was lower in peak frequency than the call of A. opisthomelas (Tukey-Kramer HSD test, - 0.97 kHz, P<0.0001), A. virolinensis (- 0.71, P<0.0001) and A. bombetes (- 0.53, P<0.0001). It was also longer in duration (+ 0.55 s , P=0.0058) and consisted of more pulses (+ 48 pulses, P=0.0356) compared to A. bombetes . Finally, its frequency bandwidth was wider compared to A. virolinensis (+ 0.40 kHz, P=0.0220) and A. opisthomelas (+ 0.35 kHz, P=0.0246). The strong among-species variation in call peak frequency could be partly attributed to concomitant variation in body size. Larger frogs usually produce calls at lower frequency values (e.g. Erdtmann & Amézquita 2009 for dendrobatid frogs) due to allometric constraints in larynx size. Indeed, increasing peak frequency is roughly related to decreasing body size in our study species: A. cassidyhornae sp. nov. is the largest species (mean±SD, 18.73± 0.22 mm , N = 5 recorded males, this study), followed by A. bombetes (17.76±0.55, N = 28 males , Myers & Daly 1980), A. opisthomelas (16.80±1.24, N = 26 males , Silverstone 1975), and A. virolinensis (16.72±0.54, N = 127 males , Valderrama-Vernaza et al. 2009). We did not correct for body size effects on peak frequency, because we did not have all information on body size of recorded individuals. In any case, the difference in call frequency alone probably has important evolutionary implications. Across many frog species, ear sensitivity appears to match the peak frequency of the advertisement call (Capranica & Moffat 1983, see Amézquita et al. 2006, 2011 for examples on dendrobatid frogs). Thus, among-lineages differences in call frequency would imply a frequency mismatch between senders and receivers in the mate recognition signal, which could have promoted reproductive isolation between any pair of the Andinobates species we studied here.