A new species of bright-eyed treefrog (Mantellidae) from Madagascar, with comments on call evolution and patterns of syntopy in the Boophis ankaratra complex Author Hutter, Carl R. Author Lambert, Shea M. Author Cobb, Kerry A. Author Andriampenomanana, Zo Faniry Author Vences, Miguel text Zootaxa 2015 4034 3 531 555 journal article 10.11646/zootaxa.4034.3.6 6ebad0d3-65b4-4ace-9c18-2115b086ee6a 1175-5326 238090 7E826737-3324-4247-A483-E09CFDCEB9D9 Boophis boppa sp. nov. Suggested common English name. Boppa’s Bright-eyed treefrog Suggested common Malagasy name. Fity maso hazo Sahona ny Boppa Remarks. Previously referred to as Boophis sp. 1 ( aff. ankaratra ) ( Andreone et al. 2007 ), Boophis aff. ankaratra ‘Antoetra slow’ (Glaw & Vences 2007), and Boophis sp. 18 ( Vieites et al. 2009 ). Holotype ( Figs . 2–3A) . KU 336824, an adult male collected by Carl R. Hutter and Zo F. Andriampenomanana on 18 January 2014 , at Maharira within Ranomafana National Park ( 21°20'06.3"S , 47°24'28.31"E ; 1233 m , above sea level [a.s.l.]), Fianarantsoa province, Madagascar . Paratypes . Adult female KU 336829 and adult male KU 336826 collected on 21 January 2013 by Carl R. Hutter and Shea M. Lambert with same locality data as holotype . Adult males KU 336828, KU 336825, KU 336827, with same collection data as holotype . Referred specimens. UADBA-Uncatalogued ( CRH 080), UADBA-Uncatalogued ( CRH 168), UADBA- Uncatalogued ( CRH 178) with same collection data as holotype . Etymology . This species in named in honor of Nicholas Jay Pritzker, devoted grandfather, father and husband, brilliant businessman, philanthropist, amateur scientist, committed conservationist; and board member, supporter and long-time friend of Conservation International. The name ‘Boppa’ is an affectionate nickname used by his children and grandchildren. This dedication is courtesy of his youngest son Isaac, who has generously supported amphibian research in Madagascar , including this study and future taxonomic research as well. The species name is used as an invariable noun in apposition to the genus name. Diagnosis . Boophis boppa ( Figs. 2–4 ) is placed in the family Mantellidae , subfamily Boophinae , and genus Boophis , as diagnosed by Glaw & Vences (2006) . The new species shares the following combination of morphological traits with all other Boophis : presence of intercalary element between ultimate and penultimate phalanges of fingers and toes; presence of nuptial pads and absence of femoral glands in males; absence of gular glands in males; terminal discs of fingers and toes enlarged; lateral metatarsalia separated by webbing; and absence of outer metatarsal tubercle. Furthermore, phylogenetic analyses support the placement of the new species in the genus Boophis . Boophis boppa is placed in the Boophis albipunctatus group as supported by the phylogenetic analyses. Additionally, the following combination of characters provide additional evidence for the inferred phylogenetic relationships: small body size (male SVL < 27 mm ); tubercles or flaps on heel and elbow absent; webbing between fingers present; canthus rostralis indistinct; distinct white tarsal folds present; dorsal coloration translucent green in life and cream-colored in preservative; ventral skin partially transparent in life; red ventral coloration absent; single subgular vocal sac; and vomerine teeth present. FIGURE 2 . Dorsal view (right) and ventral view (left) of the Boophis boppa holotype (KU 336824) in preservative. Boophis boppa is a member of the monophyletic B. ankaratra complex and cannot easily be distinguished morphologically from other closely related species in the group, which includes B. ankaratra , B. haingana , B. miadana , and B. schuboeae ( Fig. 5 ). Species in this complex lack a dense and regular white-spotted pattern that is present in other members of the B. albipunctatus group; however, most individuals of the new species show some light yellow and white spotting. Boophis boppa differs from syntopic B. ankaratra by lacking a yellow parietal peritoneum, which was present in all live photographs and examined live individuals from two different populations for B. ankaratra (e.g. Fig. 3B and Fig. 5 A). Boophis boppa differs from allopatric B. miadana and B. haingana by having the smallest, non-overlapping body size among these three species (adult male SVL: B. boppa = 20.3–24.4 mm ; B. miadana = 25.5–26.8 mm ; B. haingana 24.8–29.0 mm). However, we could not discern any morphological differences between the new species and B. schuboeae , which occurs sympatrically at Ranomafana National Park, but has not been observed syntopic with B. boppa , instead occurring at lower elevations. While we find discernable morphological differences in life, our observations generally agree with previous studies suggesting that reliable morphological differentiation among specimens of these species is difficult (Glaw et al. 2010). Despite this difficulty, these species all can be easily diagnosed by advertisement call and/or molecular phylogenetics. Bioacoustically, Boophis boppa can easily be distinguished from all other species in this group by having the longest note duration ( Fig. 6 ) and longest intervals between notes and thus the slowest note repetition rate within calls ( Fig. 7 ; Table 2 ). The most similar species in call is B. miadana ( Fig. 6 B; Fig. 7 B), which has the second longest note duration and inter-note interval in the group, but does not overlap with the new species in these values. All other species in this complex have much shorter advertisement calls. Additionally, these differences cannot be attributed to temperature, which fall within 1ºC across the recordings of B. miadana and B. boppa . We did not correct the call of B. schuboeae for temperature (it was ~5°C warmer), as it has dramatically shorter note and internote interval durations (4-fold and 7-fold shorter, respectively) and a much faster note repetition rate than B. boppa , which are differences not likely due to this amount of temperature change. Furthermore, motivation of the calling male was unlikely a factor as the temporal differences remained consistent across hundreds of calls and a variety of motivational states and weather conditions. FIGU RE 3 . Ex-situ dorsal and ventral photographs of ( A ) Boophis boppa ( holotype , KU 336824) and ( B ) Boophis ankaratra (KU 336830) in life. FIGURE 4 . In-situ photograph of Boophis boppa . ( A ) in-situ, specimen not collected. ( B ) close-up view of the eyes from the same individual. Phylogenetically, Boophis boppa is reciprocally monophyletic to all other species in the complex with strong support based on DNA sequences of the mitochondrial marker 16S ( Fig. 8 ). The new species also shares no haplotypes of the nuclear exon DNAH-3 with other related species ( Fig. 9 ). Importantly, B. ankaratra is also monophyletic, which includes individuals from the southern parts of its range and a specimen from Ranomafana National Park that was syntopic with B. boppa . Although the sister group of B. boppa cannot be determined from the analyses undertaken here, close phylogenetic affinity is suggested with B. ankaratra , B. schuboeae , or B. miadana . Additionally, the low genetic distance among individuals within B. boppa and its monophyly is strong evidence that no genetic admixture is occurring with other species of the B. ankaratra complex. Notably, B. boppa and B. ankaratra occur syntopically and can commonly be found calling within a meter distance from each other, with consistent and strong call differences ( Figs. 6–7 ). Boophis boppa also occurs sympatrically with B. schuboeae (although calling males have thus far not been found together in syntopy) and the calls of these species are highly distinct ( Figs. 6–7 ). Finally, B. miadana is distributed allopatrically and has a genetic distance of 3.5–4.5% compared to B. boppa . Description of the holotype ( Fig. 2 ) . Adult male, SVL 23.4 mm . Body moderately slender; head wider than body, HW 36.3% of SVL; head slightly shorter than wide, HL 33.8% of SVL; snout of moderate length, ESD 15.8% of SVL; snout rounded in dorsal and lateral view; nostrils directed dorsolaterally, nearer to eye than to tip of snout; ED larger than END ; cranial crests absent; canthus rostralis indistinct, loreal region slightly concave; single subgular vocal sac; gular glands absent. Tympanic annulus distinct, round, TD 48.0% of ED; supratympanic fold barely distinct, tympanic membrane transparent. Vomerine odontophores distinct, well separated in two elongated patches, positioned posteromedial to choanae; choanae medium-sized, rounded. Tongue longer than wide; ovoid in shape, posteriorly bifid, free. Arms slender; subarticular tubercles single, round; metacarpal tubercles not recognizable. Nuptial pad on inner finger 1, barely distinguishable; not pigmented, slightly lighter than ground color; fingers moderately webbed and with lateral dermal fringes; webbing formula 1(1.5), 2i (1.5), 2e(1), 3i (2), 3e(1.5), 4(1); relative length of fingers 1<2<4<3, finger 2 distinctly shorter than finger 4, finger 1 slightly shorter than finger 2; finger discs enlarged. Hind limbs slender; femoral glands absent; white tarsal folds distinct; TIBL 52.6% of SVL; FOL 46.6% of SVL; lateral metatarsalia separated by webbing; inner metatarsal tubercle small, distinct, elongated; no outer metatarsal tubercle; toes broadly webbed; webbing formula 1(0.5), 2i (0.75), 2e(0.25), 3i (1), 3e(0.25), 4i (1.25), 4e(1.5), 5(0.5); relative length of toes 1<2<3<5<4; toe discs enlarged. Skin smooth on dorsal surfaces, finely granular on throat and chest, coarsely granular on belly and ventral surfaces of thighs. Enlarged tubercles absent in cloacal region. Muscle from right thigh and liver removed for tissue samples; abdominal cavity of right side opened. After one year in preservative ( Fig. 2 ), body coloration uniform cream-yellow. Dark pigments are present around nostrils. Dark pigmented spots are present on upper eyelids and along the dorsum. In life (Figs. 3A–4), dorsum lime green with numerous white-yellow flecks, dark brown spots present, skin translucent especially on limbs, groin turquoise; upper 3/4 visceral peritoneum white, lower peritoneum and flanks transparent; underside of limbs translucent green, joints turquoise. Pupil black; iris coloration reddish-brown around the pupil and outer edges lighter in color, non-uniform; iris outlined in black; iris periphery blue, posterior edges black; eye periphery black. Color sequence from pupil to eye periphery: black, reddish-brown, yellowish-brown, black, blue, and black. FIGURE 5. Comparative photographs of members of the Boophis ankaratra complex. ( A ) Boophis ankaratra ; ( B ) Boophis haingana ; ( C ) Boophis miadana ; and ( D ) Boophis schuboeae . FIGURE 6 . Comparative oscillograms (top) and corresponding spectrograms (bottom) of single notes of the advertisement calls on the same time scale in the Boophis ankaratra complex. Species shown are: ( A ) B. boppa (Maharira) ; ( B ) B. miadana (Andohahela) ; ( C ) B. ankaratra (Maharira) ; ( D ) B. schuboeae (Ambatolahy) ; and ( E ) B. haingana (Andohahela) . Spectrogram was produced in SEEWAVE using a Hanning window length of 512. Morphometry of type series . Measurements of the holotype and paratypes are shown in Table 1 . Variation ( Figs. 10–11 ) . In life, dorsal coloration is lime to a light-lime green, with a white venter that is transparent posteriorly and along the flanks. Some individuals of this species have a white spotting pattern, with some spots being light yellow upon closer examination. Given this, the morphological distinction between the Boophis ankaratra complex and other species in the B. albipunctatus group is weakened; however, these spots can be useful in distinguishing B. boppa when present. Brown dorsal spots are also variable, as some individuals may have dense, dark patches, several spots, or none at all ( Fig. 10 ). Additionally, the intensity of the iris coloration is variable ( Fig. 11 ). Bioacoustics ( Figs. 6 A–7A; Table 2 ) . We recorded and analyzed 171 notes from six individuals of Boophis boppa and 209 notes from two individuals B. ankaratra in syntopy with B. boppa . All individuals are adult males recorded at night with mist or light rain at a temperature of 16.9–18.2°C. The males were calling on the upper surfaces of leaves 2–4 m above the ground and were recorded 1 m or less from the male. The advertisement call of this species sounds like a long ‘groan’ to the human observer, emitted frequently in slow-paced succession. We define each groan as a note ( Fig. 6 A), which are arranged in series (each series we consider a call) of 5–16 notes separated by extended periods of silence. Each note in the call of Boophis boppa is strongly amplitude modulated, with the peak amplitude occurring in the last 50% of the note. The notes are strongly pulsed, with a rate of 135–263 (187 ± 31) pulses / s. The note duration is 379–526 (451.6 ± 42.4) ms with an inter-note interval of 989–1659 (1192 ± 167) ms. The dominant frequency measured at peak amplitude is 2929–3531 (3205 ± 139) Hz. The dominant frequency is within the fundamental frequency, which has a lower limit of 2500–2594 (2702 ± 101) Hz and an upper limit of 3001–3431 (3165 ± 91.8) Hz. The first harmonic is 5768–6384 (6073 ± 148) Hz. See Table 2 for a comparison among all identified species in the B. ankaratra complex. TABLE 2. Comparisons of advertisement calls recorded for the Boophis ankaratra complex. Calls used for comparison are from Glaw et al. (2010). Calls were recorded from males calling at night and were subsequently collected as vouchers. Note envelope is the ratio of the time of peak amplitude to note duration. RNP is an abbreviation for Ranomafana National Park. Data are the range and then the mean ± two standard deviations in parentheses, when appropriate. Species

Parameter	B. boppa	B.	B. ankaratra	B.	B. miadana	B.
ankaratra	haingana	schuboeae
Locality	Maharira, RNP	Maharira, RNP	Manjakatompo	Andohahela	Andohahela	Vohipara, RNP
Temp. (Cº)	16.9–18.2	17.9	18.0	17.6	17.6	23.0
n – individuals	6	2	1	2	2	2
n – calls (note series)	26	4	1	2	2	2
n – notes	171	209	66	150	144	108
Call duration (s)	3.9–22.12 (10.2 ± 3.9)	13.5–27.1 (19.7 ± 6.5)	25.8	16.4–28.4 (22.4 ± 8.5)	12.8–13.1 (13.0 ± 0.2)	13.9–14.8 (14.3 ± 0.6)
Note duration (ms)	379.0–526.0 (451.6 ± 42.4)	104.56– 130 (110.5 ± 8.076)	111.5–153.7 (134 ± 10.6)	46.6–85.8 (61.7 ± 12.7)	208.3–316.4 (249.7 ± 29.8)	73.17–169.3 (124.3 ± 20.9)
Inter-note	989–1659	204–341	222–318	232–352	591–910	114–248
interval (ms)	(1192 ± 167)	(269 ± 3)	(259 ± 37)	(238 ± 86)	(675 ± 102)	(162 ± 42)
Note
repetition	0.4–0.8	2.6–2.8	2.5	2.6–3.0	1.0–1.2	3.4
rate (/s)
Note envelope shape	0.582–0.954 (0.779 ± 0.087)	0.550– 0.881 (0.746 ± 0.072)	0.539–0.864 (0.72 ± 0.078)	0.173– 0.259 (0.216 ± 0.061)	0.417–0.807 (0.635 ± 0.094)	0.542–0.915 (0.752 ± 0.079)
Pulse rate	135-263	147-323	110-169 (143	155-173	76-203 (120	35-234 (119
(/s)	(187 ± 31)	(212 ± 29)	± 11)	(164 ± 13)	± 39)	± 46)
Dominant frequency (Hz)	2929–3531 (3205 ± 139)	2584–2756 (2671 ± 68)	2756–3015 (2905 ± 72)	2929	2756–3015 (2893 ± 71)	3359–3531 (3403 ± 45)
Lower
fundamental	2500-2972	2329-2525	2509-2746	2700-2733	2438-2563	3200-3281
frequency	(2702 ±	(2435 ±	(2659 ±	(2716 ±	(2506 ±	(3248 ±
(Hz)	101.046)	55.698)	48.378)	22.988)	29.911)	18.165)
Higher
fundamental	3001-3431	2636-2839	2802-3028	2936-2955	2818-2967	3289-3350
frequency	(3165 ±	(2757 ±	(2960 ±	(2946 ±	(2902 ±	(3318 ±
(Hz)	91.79)	48.063)	43.895)	12.989)	47.896)	12.698)
First	5768-6384	5930-7074	5843-6229	6172-6337	6128-6415	6054-6573
harmonic	(6073 ±	(6322 ±	(5996 ±	(6254 ±	(6268 ±	(6437 ±
(Hz)	148.285)	295.493)	83.465)	117.035)	78.665)	90.211)

FIGURE 7 . Advertisement call comparative oscillograms of five-second sections in the Boophis ankaratra complex. Species shown are: ( A ) B. boppa ; ( B ) B. miadana ; ( C ) B. ankaratra ; ( D ) B. schuboeae ; and ( E ) B. haingana . The localities are the same as Fig. 6. FIGURE 8 . Results of phylogenetic analysis of the concatenated 16S alignment for maximum likelihood (ML) and Bayesian (BA) analyses. Topology is a consensus tree from MrBayes. Significant support for ML and BA analyses are indicated by the black circles at the nodes (analyses were completely consistent). Note that Boophis boppa is strongly supported as reciprocally monophyletic with respect to all other species of the B. ankaratra complex. Phylogenetics ( Figs. 8–9 ). The phylogenetic results support the morphological diagnosis by placing Boophis boppa in the genus Boophis within the B. albipunctatus group. At the species level, B. boppa is monophyletic with strong support in all analyses (BS> 90%; PP> 0.95; Fig. 8 ). Uncorrected p-distances using the 16S fragment indicate that B. ankaratra has the lowest distance to the new species, at 1.9–3.7%. Additionally, we find that variation in genetic distance among B. boppa and B. ankaratra is not related to geographic proximity. The lowest distance of 1.9% (Imaitso Forest; FMGV 1697) does not correspond to proximate localities and the highest distance of 3.7% is from Ranomafana National Park (Ranomena; ZCMV 5989). This variability in distances is likely a result of varying amounts of sequence overlap being compared. Overall, these results provide strong evidence that the species is a separately evolving lineage and does not reproduce with other species in the complex. Distribution ( Fig . 1) . Boophis boppa is known from Ranomafana National Park (RNP), but has only been found at high elevation sites (~ 1046–1312 m , a.s.l.). In addition to the type locality of Maharira, DNA sequences also confirm that specimens FAZC 11454, 11462, and 11480 collected from Farihimazava forest near Antoetra ( 20°50'06"S , 47°19'57"E , 1380–1420 m , a.s.l.; ca. 55 km north west of the type locality; see Andreone et al. 2007 ) are conspecific. Additionally, tadpoles ZCMV 9739 collected at Imaloka (RNP: 21°14'32"S , 47°27'55"E , 1020 m , a.s.l.) and ZSM 1164/2007 from Sakaroa (RNP: 21°15'00.1"S , 47°24'53.6"E ) belong to Boophis boppa . The species has a known elevational distribution of ca. 1020–1400 m , a.s.l. FIGURE 9. Minimum-spanning haplotype network for the nuclear exon DNAH-3 in the B. albipunctatus group. Ticks on branches represent single nucleotide changes. Branch lengths are not meaningful. Natural history ( Fig. 12 ) . Boophis boppa was locally abundant but thus far only found in undisturbed, primary forests along fast moving streams and was occasionally found along slow flowing tributaries adjacent to fast streams. Males of the species typically were calling at night from the surfaces of vegetation less than three meters in height ( Fig. 12 A–B). Females of B. boppa were rarely encountered and were only observed while in amplexus ( Fig. 12 C). We also confirm that Boophis boppa is syntopic with Boophis ankaratra at Maharira and that the two species can be found calling less than a meter apart. This is also consistent with Andreone et al. (2007) , which found B. boppa (i.e. Boophis aff. ankaratra ) and B. ankaratra to occur sympatrically. Other syntopic species of Boophis at Maharira include: B. madagascariensis , B. majori , B. aff. marojezensis, B. aff. picturatus , B. popi , and B. reticulatus . Conservation status . The new species is known from Ranomafana National Park and its vicinity, extending into the Antoetra area. While it is known from a geographic area less than ~ 5,000 km 2, there are no known immediate threats, reductions in quality or extent of habitat, or observed declines as the species is well protected within Ranomafana National Park. Therefore, the new species meets only criterion (B1), and could become endangered in the future if the situation changes at Ranomafana, for instance through population declines from chytridiomycosis, which has been detected in the park ( Bletz et al. 2015 ). We recommend a conservation status of Near Threatened, following IUCN (2001) criteria.