An integrative taxonomic revision and redefinition of Gephyromantis (Laurentomantis) malagasius based on archival DNA analysis reveals four new mantellid frog species from Madagascar Author Vences, Miguel https://orcid.org/0000-0003-0747-0817 Zoologisches Institut, Technische Universitaet Braunschweig, Mendelssohnstr. 4, 38106 Braunschweig, Germany m.vences@tu-bs.de Author Koehler, Joern Hessisches Landesmuseum Darmstadt, Friedensplatz 1, 64283 Darmstadt, Germany Author Crottini, Angelica https://orcid.org/0000-0002-8505-3050 CIBIO, Centro de Investigacao em Biodiversidade e Recursos Geneticos, InBIO Laboratorio Associado, Campus de Vairao, Universidade do Porto, 4485 - 661 Vairao, Portugal & Departamento de Biologia, Faculdade de Ciencias, Universidade do Porto, 4099 - 002 Porto, Portugal & BIOPOLIS Program in Genomics, Biodiversity and Land Planning, CIBIO, Campus de Vairao, 4485 - 661 Vairao, Portugal Author Hofreiter, Michael Institut fuer Biochemie und Biologie, Universitaet Potsdam, Karl-Liebknecht-Str. 24 - 25, 14476 Potsdam, Germany Author Hutter, Carl R. Museum of Natural Sciences and Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA Author du Preez, Louis https://orcid.org/0000-0002-3332-6053 Unit for Environmental Sciences and Development ,, North-West University, Potchefstroom campus, Private Bag X 6001, Potchefstroom 2520, South Africa & South African Institute for Aquatic Biodiversity, Private Bag 1015, Makhanda, 6140, South Africa Author Preick, Michaela Institut fuer Biochemie und Biologie, Universitaet Potsdam, Karl-Liebknecht-Str. 24 - 25, 14476 Potsdam, Germany Author Rakotoarison, Andolalao Mention Zoologie et Biodiversite Animale, Universite d'Antananarivo, BP 906, 101 Antananarivo, Madagascar Author Rancilhac, Lois Zoologisches Institut, Technische Universitaet Braunschweig, Mendelssohnstr. 4, 38106 Braunschweig, Germany Author Raselimanana, Achille P. Mention Zoologie et Biodiversite Animale, Universite d'Antananarivo, BP 906, 101 Antananarivo, Madagascar & Association Vahatra, Lot V A 38 LBA Ter Ambohidempona Tsiadana, BP 3972, 101 Antananarivo, Madagascar Author Rosa, Goncalo M. https://orcid.org/0000-0002-8658-8436 Institute of Zoology, Zoological Society of London, London NW 1 4 RY, UK & Centre for Ecology, Evolution and Environmental Changes (cE 3 c), Faculdade de Ciencias, Universidade de Lisboa, 1749 - 016 Lisboa, Portugal Author Scherz, Mark D. Natural History Museum of Denmark, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark Author Glaw, Frank https://orcid.org/0000-0003-4072-8111 Zoologische Staatssammlung Muenchen (ZSM-SNSB), Muenchhausenstr. 21, 81247 Muenchen, Germany text Vertebrate Zoology 2022 2022-05-26 72 271 309 http://dx.doi.org/10.3897/vz.72.e78830 journal article http://dx.doi.org/10.3897/vz.72.e78830 2625-8498-72-271 229EBA83732F477C9B2212222131274C 4D682458C96F5B899212B96F789A9403 Gephyromantis fiharimpe sp. nov. (lineage B) Holotype. ZSM 164/2016 (field number FGZC 5181), adult male, from Mandraka ( 18.9122°S , 047.9144°E , 1235 m a.s.l.), Analamanga Region, Northern Central East of Madagascar, collected on 5 January 2016 by F. Glaw, D. Proetzel , and L. Randriamanana. Paratypes. MRSN A6436 (field number PBZT/RJS 1983), adult male, from Vevembe (Site A: camp foret ), Atsimo Atsinanana Region, Southern Central East of Madagascar, collected on 26 October 2007 by J.E. Randrianirina and J. Randriantsoa; UADBA 20646 (FGMV 2002.530), unsexed, ZMA 19421 (FGMV 2002.415), probable female, and ZSM 746/2003 (FGMV 2002.531), adult male, from Ranomafana National Park, Vatovavy-Fitovinany Region, south central eastern Madagascar, all collected 22-24 January 2003 by F. Glaw, M. Puente, L. Raharivololoniaina, M. Teschke ( nee Thomas), D.R. Vieites; ZFMK 57434, ZFMK 59876, two adult males, from Andasibe, Alaotra-Mangoro Region, Eastern Madagascar, collected between 1-4 January 1994 by F. Glaw and M. Vences; ZFMK 60039, adult male, from Andasibe, Alaotra-Mangoro Region, Eastern Madagascar, collected on 1 February 1995 by F. Glaw. NMBE 233/96, adult male, from Ambohitantely, Analamanga Region, central Madagascar, collected by D. Vallan. The ZFMK and NMBE specimens are included as paratypes despite the lack of associated DNA sequences because they bear a tibial gland and originate from sites where the presence of lineage B was ascertained by genetic data (Fig. 1 ). KU 340759 (CRH 511), UADBA-CRH 486, KU 340863 (CRH 746), collected at Ranomafana National Park by C. R. Hutter and S. Lambert; KU 340736 (CRH 470) collected at Vohidrazana, Alaotra-Mangoro Region, Eastern Madagascar, by C. R. Hutter and S. Lambert; UADBA uncatalogued (APR 7651) collected at Ambohitantely Special Reserve (Jardin botanique), 1560 m a.s.l., Analamanga Region, central Madagascar, by A.P. Raselimanana in 2007. UADBA uncatalogued (APR 8659), male, collected at Ambatovy-Analamay Forest ( 18.7989°S , 048.3242°E , 1100 m a.s.l.), Alaotra-Mangoro Region, eastern Madagascar, by A.P. Raselimanana in 2009. UADBA uncatalogued (APR 8448), collected at Maromizaha Forest ( 18.9757°S , 048.4583°E , 1000 m a.s.l.), Alaotra-Mangoro Region, eastern Madagascar, by A.P. Raselimanana in 2008. UADBA uncatalogued (APR 12214) collected at NAP Anjozorobe-Sahabe ( 18.4208°S , 047.9438°E , 1305 m a.s.l.), Analamanga Region, central Madagascar, by A.P. Raselimanana in 2016. Referred specimen. ZFMK 57435, female, from Ankeniheny, Alaotra-Mangoro Region, Eastern Madagascar, collected on 19 February 1994 by F. Glaw, N. Rabibisoa and O. Ramilison. This female specimen with tibial gland ( Vences et al. 2002 ) is not included in the paratype series because no genetic data are available, neither for the specimen nor for the general locality Ankeniheny. Etymology. The species epithet is derived from the Malagasy words fihary (gland) and fe (leg) which written together become fiharim-pe according to Malagasy grammar. The name makes reference to the tibial gland of the species, and is used as a noun in apposition. Diagnosis. A member of the subfamily Mantellinae based on the presence of intercalary elements between terminal and subterminal phalanges of fingers and toes (verified externally), and on the absence of nuptial pads and presence of femoral glands in males. Assigned to the subgenus Gephyromantis Laurentomantis in the genus Gephyromantis based on the strongly tubercular dorsal skin, absence of foot webbing, completely connected lateral metatarsalia, and molecular phylogenetic relationships. The new species differs from all nominal species of the subgenus Gephyromantis Laurentomantis as follows: From G. horridus by smaller body size (male SVL 22.0-24.0 mm vs. 33.5 mm), less expressed tubercles and ridges on the dorsum, and absence of a dorsal pattern of two blackish transverse patches (vs. presence); from G. ranjomavo by the absence of light brown to orange-brown color covering limbs dorsally (vs. presence), less expressed tubercles and ridges on the dorsum, and possibly slightly smaller body size (male SVL 22.0-24.0 mm vs. 23.5-28.1 mm); from G. striatus by absence of a vertebral stripe posteriorly on dorsum (vs. presence) and a more strongly tubercular dorsal skin; from G. malagasius (as redefined herein) by absence of a distinct bluish gray pattern on a dark venter (vs. presence), and less expressed tubercles and ridges on the dorsum; from G. marokoroko by a somewhat less tubercular dorsal skin, presence of red color ventrally on limbs (vs. absence), absence of orange spots and vermiculations on dorsum (vs. presence), and absence of gray to whitish color on vocal sac (vs. presence). Furthermore, differing from all the aforementioned species by the presence of light reddish color in the inguinal region and ventrally on limbs and posterior belly in life (vs. absence), and from all species except for G. ranjomavo by the presence of a tibial gland (vs. absence), and by a substantial genetic divergence (>6% uncorrected pairwise distance in the 16S gene). According to the molecular phylogeny, G. fiharimpe is closely related to G. matsilo described above, and may be its sister species. It differs from G. matsilo by presence of a tibial gland (vs. absence), shorter note duration in advertisement calls (2-12 ms vs. 13-21 ms), a less tubercular dorsal skin, and an uncorrected genetic distance in the 16S gene of 4.0-5.7%. For a distinction from the other new species described in the following (lineages C and D), see the diagnoses in the respective species accounts below. Description of the holotype. Adult male in good state of preservation (Fig. 18 ), tongue removed as tissue sample for molecular analysis. SVL 23.4 mm, for other measurements see Table 1 . Body slender; head longer than wide, as wide as body; snout rounded in dorsal view, subacuminate in lateral view; nostrils directed laterally, slightly protuberant, much nearer to tip of snout than to eye; canthus rostralis rather indistinct, concave; loreal region slightly concave; tympanum distinct, rounded, 53% of eye diameter; supratympanic fold not recognizable, in its place two large tubercles; tongue removed and thus not available for examination; vomerine teeth weakly recognizable, but present in two minuscule aggregations posteromedially to choanae; choanae rounded; maxillary teeth present. Dermal fold along the lower jaws (the inflatable parts of the vocal sac) weakly expressed. Arms slender, subarticular tubercles single; poorly developed outer and inner metacarpal tubercles recognizable; fingers without webbing; relative length of fingers 1 <2 <4 <3, second finger distinctly shorter than fourth finger; finger discs distinctly enlarged; nuptial pads absent. Hindlimbs slender; tibiotarsal articulation reaching snout tip when hindlimb is adpressed along body; lateral metatarsalia connected; inner metatarsal tubercle distinct, outer metatarsal tubercle small but recognizable; webbing between fingers and toes absent; relative toe length 1 <2 <5 <3 <4. Third toe only slightly longer than fifth toe. Toe discs enlarged. Skin on upper surface granular, with rather indistinct ridges and many smaller irregularly distributed tubercles on head, eyes, and dorsum. Ventral skin smooth on throat, chest and limbs, slightly granular on posterior belly. Femoral glands well delimited and distinctly recognizable from external view, apparently with two large gland granules in external view. Tibial glands distinct, covering about two thirds of the shank. After five years in preservative, dorsal coloration of head and body uniformly dark brown, with darker crossbands on hind- and forelimbs. Posterodorsal surface of thigh largely pigmentless whitish/cream: this area in life was presumably reddish. Ventrally, throat, chest and anterior belly uniformly blackish brown, posteror belly fading into gray-cream. Ventral side of hindlimbs pigmentless cream, probably corresponding to reddish color in life. Variation. A tibial gland is visible in all examined adult specimens, as well as in additional photographed individuals (UADBA-CRH 119, UADBA-CRH 486, UADBA-CRH 510, KU 340759 [CRH 511], KU 340736 [CRH 470]). The specimen ZMA 19421 from Ranomafana is probably a female (sex cannot be unambiguously determined due to removal of inner organs and part of the skin for chromosome analysis); this specimen also appears to have a tibial gland, which however cannot be recognized with full reliability. However, a second female (ZFMK 57435), which is assigned to this species tentatively (due to the lack of genetic data), has distinct tibial glands. The two females are larger than the males (25.7-25.8 vs. 22.0-24.0 mm SVL). For morphometric measurements of ZFMK and NMBE paratypes not included in Table 1 , see Vences et al. (2002) . Call. The advertisement call (Fig. 21 ) was recorded on 18 February 1994 at Ankeniheny (air temperature 23.5°C; Vences et al. 2006 : CD2, track 28). It consists of a multinote call of variable duration, emitted in series at regular intervals. Slight amplitude modulation is recognizable within calls, with notes at the beginning and the end of the call having lower call energy. Notes are very short and appear to consist of a single pulse each. They are repeated at irregular intervals within calls. Numerical call parameters of 12 analyzed calls are as follows (range followed by mean +/- standard deviation in parentheses): call duration 609-935 ms (798.8 +/- 110.8 ms); note duration 7-9 ms (7.8 +/- 0.8 ms); number of notes per call 11-19 (15.5 +/- 2.4); note repetition rate within calls 14.9-25.6 notes/second (18.3 +/- 3.9 notes/second); call repetition rate within call series approximately 24-25 calls/minute; dominant frequency 3638-3723 Hz (3676 +/- 23 Hz); prevalent bandwidth 2500-5200 Hz. Figure 20. Specimens of Gephyromantis fiharimpe sp. nov. (lineage B) in life, in dorsolateral and ventral views. A , B Adult male, probably from An'Ala , photographed 1996. C - E Adult male from Andasibe, photographed 1994. F , G Adult male from Ranomafana, photographed 2003. Individuals not reliably attributable to a voucher specimen. Not to scale. Figure 21. Audiospectrogram and corresponding oscillogram of one advertisement call of Gephyromantis fiharimpe sp. nov. recorded on 18 February 1994 at Ankeniheny. The oscillogram below shows a 100 ms section of the call figured above, showing two notes and their repective amplitude structure. Recording band-pass filtered at 1500-8000 Hz. Additional calls recorded on 1 January 1994 at Andasibe (temperature unknown) generally agree in characteristics with those reported from Ankeniheny, including evident variation in note repetition rate within calls. The main differences compared to calls from Ankeniheny are longer call duration and higher number of notes per call. Numerical call parameters of 7 analyzed calls are as follows (range followed by mean +/- standard deviation in parentheses): call duration 840-1503 ms (1179.3 +/- 232.7 ms); note duration 5-12 ms (7.6 +/- 2.2 ms); number of notes per call 26-43 (36.9 +/- 7.3); note repetition rate within calls 18.2-37.4 notes/second (25.5 +/- 6.5 notes/second); call repetition rate within call series approximately 25-27 calls/minute; dominant frequency 3649-4078 Hz (3796 +/- 157 Hz); prevalent bandwidth 2500-4800 Hz. Calls recorded on 12 January 2015 at Vohidrazana (air temperature 18.5°C; call voucher KU 340736 [CRH 470]) also agree with those from Andasibe in all general characters, being only slightly longer in duration on average and exhibiting slightly shorter note duration. Irregular note repetition rate within calls is evident. Numerical call parameters of 12 analyzed calls are as follows (range followed by mean +/- standard deviation in parentheses): call duration 950-1764 ms (1405.6 +/- 290.4 ms); note duration 2-7 ms (4.3 +/- 1.7 ms); number of notes per call 26-52 (40.7 +/- 11.5); note repetition rate within calls 18.8-54.1 notes/second (32.1 +/- 13.1 notes/second); call repetition rate within call series approximately 21-24 calls/minute; dominant frequency 3726-3913 Hz (3781 +/- 68 Hz); prevalent bandwidth 2300-5000 Hz. Calls recorded on 13 February 2015 at Ambatolahy, Ranomafana National Park (air temperature 17.6°C; call voucher KU 340759 [CRH 511]), based on genetic data assignable to clade B are very similar to the calls described for clade B from Andasibe, Ankeniheny, and Vohidrazana, but differ from these by a regular note repetition rate within calls and pronounced amplitude modulation within calls, with call energy continuously increasing from the beginning, reaching its maximum at the middle of the call, then continuously decreasing towards its end. Numerical call parameters of 14 analyzed calls are as follows (range followed by mean +/- standard deviation in parentheses): call duration 916-1162 ms (1073.9 +/- 86.0 ms); note duration 4-7 ms (6.2 +/- 0.9 ms); number of notes per call 27-34 (30.6 +/- 2.3); note repetition rate within calls 25.6-28.0 notes/second (26.8 +/- 1.0 notes/second); call repetition rate within call series approximately 20-23 calls/minute; dominant frequency 3305-3736 Hz (3523 +/- 145 Hz); prevalent bandwidth 2100-5000 Hz. Distribution and natural history. Based on genetically verified records, the distribution area includes in a south-north direction the localities (1) Vevembe, (2) Ranomafana, (3) Mandraka, (4) Maromizaha, (5) Andasibe, (6) Ambatovy, (7) Vohidrazana, (8) Anjozorobe, and (9) Ambohitantely. Probably the species is also present at Ankeniheny. The known elevational range of the species spans from 580 m a.s.l. (Vevembe) to approximately 1500 m a.s.l. (Ambohitantely). The species apparently is restricted to rather intact rainforest habitat. Males call at night from perch heights of 5-50 cm in the low understory vegetation, not concentrated around water bodies.