Corresponding author: Gonçalo M. Rosa (
Academic editor: Johannes Penner
A new species of treefrog of the
Penny SG, Andreone F, Crottini A, Holderied MW, Rakotozafy LS, Schwitzer C, Rosa GM (2014) A new species of the
The genus
The Sahamalaza Peninsula in north-western Madagascar has undergone only two previous amphibian surveys (
The Sahamalaza Peninsula is in the province of Mahajanga, northwest Madagascar (
The Sahamalaza Peninsula in northwest Madagascar, indicating the study sites of (
The climate is sub-humid and has two distinct seasons: a cooler, drier season from May to November; and a hotter, wetter season from December to April. Monthly mean maximum temperature ranges from 28.5 ± 3.61 °C in July to 39.1 ± 2.11 °C in February; and monthly mean minimum temperature ranges from 13.2 ± 0.81 °C in October to 21.8 ± 0.81 °C in January (
Sahamalaza supports a unique type of transitional forest that harbours plant species from both the wetter Sambirano domain and drier western domain (
Fieldwork took place over two separate periods: a three month survey between October 2011 and January 2012, and an additional survey between January and February 2013. This ensured the coverage of most of the wet season when rain is more abundant and the individuals are expected to be more active. Surveys were conducted in Ankarafa Forest, Anabohazo Forest and around the villages of Antafiabe and Betsimpoaka.
Surveys were conducted by opportunistic searching and directed towards vocalising males using headlamps and torches. Transects were made across a range of habitats and degradation levels, covering the four previously mentioned areas of the peninsula. Searches were repeated during the day and night to account for any diel differences in activity, taking place in the morning and evening. Searching took place approximately two metres either side of the transect and up to two metres in height. Most searches in Ankarafa were repeated at least once both in the dry and wet season (during the 2011–2012 period) following the same routes where possible. The sites in Anabohazo Forest, Antafiabe and Betsimpoaka village were sampled only once. Sites were sampled in a randomised order and all searches were conducted by the same two individuals to avoid systematic observer bias. The frogs’ frequency of occurrence was estimated by dividing the total number of frogs encountered during each transect search by the respective length.
The vouchers were photographed to document life colouration and calls were recorded whenever possible. Acoustic description follows that outlined in the acoustic analysis methods below. The tissue samples (fourth digit of the left toe removed with scissors) were stored in 70% ethanol or 96% ethanol for genetic analysis. Location was logged using a handheld GPS receiver (Garmin eTrex Vista HCx; Garmin International Inc., Olathe, United States). Microhabitat was noted and vertical position from the ground measured using a tape measure.
Specimens were collected both day and night, euthanized in a chlorobutanol solution, fixed in 90% ethanol or 5% formalin, and preserved in 70% ethanol. Specimens are deposited in the collections of Museo Regionale di Scienze Naturali, Torino, Italy (MRSN; (snout-vent length) (greatest head width) (head length) (horizontal eye diameter) (eye-nostril distance) (nostril-snout tip distance) (nostril-nostril distance) (horizontal tympanum diameter) (tibia length) (hand length) (foot length) (foot length including tarsus) (forelimb length) (hindlimb length) (reaching of tibiotarsal articulation when hindlimb is adpressed along body)
Morphometric measurements (in mm) of preserved specimens of
MRSN | A6973 | A6974 | A6975 | A6976 |
STATUS | HOLOTYPE | PARATYPE | PARATYPE | PARATYPE |
SEX | male | female | male | male |
LIFE STAGE | adult | adult | adult | adult |
SVL | 24.0 | 28.5 | 23.7 | 22.9 |
HW | 9.1 | 12.0 | 8.4 | 8.5 |
HL | 7.8 | 11.5 | 8.2 | 8.1 |
ED | 4.1 | 4.7 | 3.6 | 3.6 |
END | 2.1 | 3.3 | 2.6 | 2.7 |
NSD | 2.6 | 2.8 | 2.2 | 2.0 |
NND | 3.0 | 3.8 | 2.7 | 2.3 |
TD | 1.5 | 1.6 | 1.7 | 1.2 |
TL | 12.7 | 17.2 | 12.0 | 10.8 |
HAL | 8.1 | 9.2 | 7.5 | 6.1 |
FOL | 11.2 | 14.1 | 10.2 | 9.6 |
FOTL | 17.2 | 22.8 | 16.4 | 15.6 |
FORL | 15.1 | 21.3 | 14.1 | 11.5 |
HIL | 41.1 | 53.7 | 37.9 | 37.5 |
Tissue samples were available for four individuals. Total genomic DNA was extracted from the tissue samples using proteinase K digestion (10 mg/ml concentration) followed by a standard salt-extraction protocol (
Sequences were checked by eye, edited and aligned using the BioEdit sequence alignment editor (version 7.0.5.3;
To assess genetic distinctness of the new species from all other Malagasy frogs and ascertain its belonging to the
Most amphibian calls are species-specific and it is usually possible to identify syntopic calls to the species level. Sound recordings were taken to obtain detailed information on the acoustic parameters of the species and investigate any intraspecific variability and overnight temporal patterns in activity. Acoustic recordings were made continuously from dusk until dawn on sixty nights between October 2011 and January 2012. Data were collected from 37 different locations; the majority of these locations were within Ankarafa Forest (29), followed by Anabohazo Forest (7) and a single location on the Vavan’aneno River near the village of Antafiabe. Nineteen locations had recordings made on two or more nights, separated between 9 and 79 days.
Calls were recorded in the field using a Song Meter SM2 digital recorder (Wildlife Acoustics Inc, Concord, United States) at a 16-bit resolution and 16 kHz sampling rate using two side-mounted SMX-II microphones. The digital recorder was placed one to two metres above the ground or water by securing it to deadwood or a protruding branch using bungee cords. Continuous recordings split into sections of 120 minutes each were saved in the standard uncompressed .WAV format. Preceding analysis, these were split using a custom-written MATLAB (The Mathworks, Natick, USA, v7.14.0.739) script into minute long segments to allow for more efficient analysis. Spectrograms were viewed individually as a dual channel output using Avisoft SASlab Pro (Berlin, Germany, v5.2.06); frequency resolution of 512 FFT, a 100% frame rate, Hamming window and an intensity threshold of 50%.
From each night-long recording where
From each of these notes spectral and temporal characteristics were measured, and the minimum, maximum and average values (with SD) calculated (Avisoft SASlab Pro; Berlin, Germany; v5.2.06). To remove interspecific and abiotic noise outside of the
Minute by minute changes in activity were recorded for all detected species from dusk (sunset) until dawn (sunrise) for 60 nights. The activity index per minute was 1 for one caller and 2 for more than one. The time period between dusk and dawn was split into percentiles for each night; this controlled for the slight variations in night length experienced across the sampling period, enabling the summation of data across multiple nights for each analogous percentile. The number of minute periods containing calling activity within each percentile was calculated and the values for each analogous percentile across all nights summed. The totals for each percentile were then divided by the total activity to determine the proportional change in activity.
The term
MRSN A6973, adult male (
Life colouration of
Breeding activity of
MRSN A6974 adult female (
A treefrog assigned to the genus
MRSN A6973, adult male in a good state of preservation. SVL 24.0 mm (see
The paratypes (MRSN A6974-6976) closely match the holotype but with slightly different patterning of pigment patches. Finer regular black spots were observed on the dorsum and limbs (possibly single melanophores); a feature similarly observed in the related
The molecular data confirms the attribution of
Genetic divergence in the analysed 16S rRNA mitochondrial gene fragment of the
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4.9% |
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9.0% | 8.0% |
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11.4% | 11.6% | 11.9% |
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9.9% | 9.6% | 9.7% | 9.1% |
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11.1% | 10.2% | 12.0% | 13.0% | 12.1% |
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The genetic distance between
Vocalisations of
Detection of
Nocturnal variation in calling activity of
The acoustic repertoire consists of two note types: a multi-pulsed trill (type 1;
Representative call of
Acoustic measurements of two note types of
Note | Parameter | Section | Mean±SD | Range |
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Entire Note (s) | 4.29 ± 1.29 | 1.61–8.09 |
Broadband Pulse (ms) | 3.47 ± 0.485 | 2.50–5.06 | ||
Narrowband Pulse (ms) | 17.9 ± 2.56 | 11.2–25.1 | ||
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20–30 Broadband Pulses (pulses/s) | 15.0 ± 1.19 | 11.7–17.3 | |
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40–60 Pulses (kHz) | 4.14 ± 0.131 | 3.69–4.48 | |
Broadband Pulse (kHz) | 4.19 ± 0.122 | 3.91–4.34 | ||
Narrowband Pulse (kHz) | 4.15 ± 0.108 | 4.00–4.31 | ||
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Entire Note (ms) | 50.9 ± 5.20 | 40.0–68.7 |
Pulse one (ms) | 6.37 ± 3.04 | 2.18–15.7 | ||
Pulse two (ms) | 6.61 ± 2.99 | 1.75–15.9 | ||
Interpulse Interval (ms) | 37.9 ± 6.28 | 20.0–50.7 | ||
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Entire Note (ms) | 3.95 ± 0.162 | 3.57–4.30 |
The trill note measured 4.29 ± 1.29 s (1.614–8.091 s, n = 55) and is composed of alternating broadband and narrowband pulses. These notes had a broadband pulse rate of 15.0 ± 1.19 pulses/s (11.7–17.3 pulses/s, n = 25) and peak frequency of 4.14 ± 0.131 kHz (3.69–4.48 kHz, n = 55). Broadband pulses measured 3.47 ± 0.485 ms (2.50–5.06 ms, n = 50) whereas narrowband pulses were slightly longer at 17.9 ± 2.56 ms (11.2–25.1 m; n = 50). The broadband pulse had a peak frequency of 4.19 ± 0.122 kHz (3.91–4.34 kHz; n = 50), whereas the narrowband pulse measured 4.15 ± 0.108 kHz (4.00–4.31 kHz, n = 50). Bandwidths were 0.288 ± 0.169 kHz (0.120–0.780 kHz, n = 50) for the broadband pulse compared to just 0.155 ± 0.036 kHz (0.129–0.280 kHz, n = 50) for the narrowband pulse when measured at a -10 dB threshold. The majority of click notes consisted of two pulses, although singular and triple pulses were also observed. Click notes had a total duration of 52.9 ± 5.20 ms (40.0–68.7 ms; n = 55) and peak frequency of 3.95 ± 0.162 kHz (3.57–4.30 kHz; n = 50). Bandwidth of the click note measured 0.538 ± 0.234 kHz (0.120–0.143 kHz) at a -10 dB threshold.
The advertisement call of
The trill note (type 1) of
A total of 54 individuals of
Of the 56 encounters, 48 frogs were male and 8 female. Males of this species were found calling from vegetation approximately 0.5 to 2 m high. Vocalising males were often within close proximity to one another, positioned on different leaves of the same plant. All but one of the eight females were found in axillary amplexus with males (
The suggested conservation status of this species was assessed using the criteria and guidelines of the IUCN Red List (
Habitat of
Count and frequency of
Date | Transect Location | No. of frogs | Transect Length (m) | Frequency /100 m |
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29/10/2011 | Ankarafa North ‘upstream’ | 3 | 511 | 0.59 |
02/11/2011 | Ankarafa North ‘midstream’ | 5 | 350 | 1.43 |
25/11/2011 | Ankarafa South | 3 | 814 | 0.37 |
26/11/2011 | Ankarafa North ‘downstream’ | 7 | 609 | 1.15 |
15/12/2011 | Ankarafa North ‘upstream’ | 18 | 511 | 3.52 |
29/12/2011 | Ankarafa North ‘upstream’ | 11 | 511 | 2.15 |
05/01/2012 | Ankarafa North ‘midstream’ | 7 | 350 | 2 |
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If suitable habitat is considered to be all areas of Ankarafa Forest (likely an over-estimate) then this area totals less than 5 km2, giving an EOO (extent of occurrence) of less than 100 km2. If plots with a scale of 2 km2 are used to estimate AOO (area of occupancy), then this species occurs within 4 km2 of habitat, resulting in an AOO of less than 10 km2. Therefore the Critically Endangered thresholds for extent of occurrence and area of occupancy are both met (EOO < 100 km2 and AOO < 10 km2) (CR B1+2). The most serious threat to the species is habitat destruction through
The closest relative of
All breeding behaviour of
The frog was only found within intact forest and appears sensitive to anthropogenic disturbance. Intact forest is rare across the peninsula and aside from Anabohazo, where the species was not found, Ankarafa represents the largest area of remaining forest. Isolated populations of
Anthropogenic disturbance within Ankarafa Forest:
Despite its protected status, Ankarafa is experiencing widespread deforestation (
We would like to thank the staff of the AEECL research station in Ankarafa Forest for their help in the field including: Fan, Théophile, Régis, Falisara, Avitsara, Lauricia and Marlene. We would also like to thank Nicola Davies of Bristol Zoological Society for her help with the IUCN Red Listing and the anonymous reviewers for their valuable comments and suggestions towards the manuscript. We are grateful to the Ministère de l’Environnement et des Eaux et Forêts for providing the research permits, and MICET for logistical help. This work was financially supported by the European Association of Zoos and Aquaria (EAZA). A. Crottini was supported by a postdoctoral grant from the Portuguese ‘Fundação para a Ciência e a Tecnologia’ (FCT) (SFRH/BPD/72908/2010) under the Programa Operacional Potencial Humano – Quadro de Referência Estratégico Nacional funds from the European Social Fund and Portuguese Ministério da Educação e Ciência. G. M. Rosa is funded by the Doctoral Programme (SFRH/BD/69194/2010) of the FCT. We acknowledge the project ‘Genomics and Evolutionary Biology’ co-financed by north Portugal Regional Operational Programme 2007/2013 (ON.2 - O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF).
Calls of
Data type: audio file
Explanation note: Calls of