Rediscovery, redescription and identity of Pristimantis nebulosus (Henle, 1992), and description of a new terrestrial-breeding frog from montane rainforests of central Peru (Anura, Strabomantidae) Author Koehler, Joern Hessisches Landesmuseum Darmstadt, Friedensplatz 1, 64283 Darmstadt, Germany joern.koehler@hlmd.de Author Castillo-Urbina, Ernesto https://orcid.org/0000-0002-8293-758X Universidad Nacional Mayor de San Marcos, Museo de Historia Natural, Departamento de Herpetologia, Av. Arenales 1256, Lima 11, Peru Author Aguilar-Puntriano, Cesar https://orcid.org/0000-0001-6372-7926 Universidad Nacional Mayor de San Marcos, Museo de Historia Natural, Departamento de Herpetologia, Av. Arenales 1256, Lima 11, Peru Author Vences, Miguel https://orcid.org/0000-0003-0747-0817 Zoological Institute, Technische Universitaet Braunschweig, Mendelssohnstr. 4, 38106 Braunschweig, Germany Author Glaw, Frank https://orcid.org/0000-0003-4072-8111 Zoologische Staatssammlung Muenchen (ZSM-SNSB), Muenchhausenstr. 21, 81247 Muenchen, Germany text Zoosystematics and Evolution 2022 2022-06-24 98 2 213 232 http://dx.doi.org/10.3897/zse.98.84963 journal article http://dx.doi.org/10.3897/zse.98.84963 1860-0743-2-213 959396406B7F41E9825CA53024113F54 86D36A45524C5B3CBF9BB1209ADE1633 Pristimantis nebulosus (Henle, 1992) Relationships. The Maximum Likelihood tree, based on an alignment of 584 bp of the 16S gene, is insufficient to establish a reliable hypothesis of phylogenetic relationships among the Pristimantis samples included (Fig. 4 ). The P. conspicillatus species group is recovered paraphyletic with respect to the sample of P. gaigeae from Panama, a member of the P. conspicillatus group sensu Hedges et al. (2008) and Padial et al. (2014) . Together with species representing the P. lacrimosus , P. ridens and P. danae species groups, P. gaigeae is placed sister to the remaining species of the P. conspicillatus group. Within the P. conspicillatus group, most relationships are not resolved and basal nodes did not receive significant support. However, the three specimens of the focal P. nebulosus are recovered with high support (bootstrap value 98%) as members of a clade containing nominal P. bipunctatus and an undescribed candidate species, with P. nebulosus placed sister to nominal P. bipunctatus (without significant support; 36%). The clade containing P. nebulosus , P. bipunctatus , and the candidate species are recovered sister (76%) to a clade containing two samples of P. skydmainos (a close relationship of P. bipunctatus and P. skydmainos has already been revealed by Padial et al. 2014 ). Our P. nebulosus specimens had uncorrected p-distances in the studied 16S gene fragment of 5.4-6.2% to P. bipunctatus (with the greatest distance of 6.2% referring to the P. bipunctatus holotype KU291638) and 4.1% to the mentioned candidate species (Table 2 ). P-distances to other species in the P. conspicillatus species group range from 5.8-17.8%. Table 2. Estimates of uncorrected p-distances in a 491 bp fragment of the mitochondrial 16S gene among selected samples of the Pristimantis conspicillatus species group, calculated with MEGA7 (all ambiguous positions were removed for each sequence pair).

-	FGZC 6253	FGZC 6254	FGZC 6281	FGZC 6207	KU 291638	MUSM 31179	MUSM 31120
P. nebulosus FGZC 6253 (Abra La Divisoria)	-	-	-	-	-	-	-
P. nebulosus FGZC 6254 (Abra La Divisoria)	0.000	-
P. nebulosus FGZC 6281 (Abra La Divisoria)	0.000	0.000	-
P. symptosus FGZC 6207 (Cordillera de Carpish)	0.041	0.041	0.041	-
P. bipunctatus KU 291638 (E of Oxapampa)	0.062	0.062	0.062	0.041	-
P. bipunctatus MUSM 31179 (Pui Pui Forest)	0.054	0.054	0.054	0.033	0.037	-
P. bipunctatus MUSM 31120 (Yanachaga Chemillen )	0.058	0.058	0.058	0.039	0.006	0.031	-
P. bipunctatus NMP6V/75062 (Pui Pui Forest)	0.054	0.054	0.054	0.033	0.037	0.000	0.031

Figure 4. Maximum Likelihood tree inferred from a 584 bp fragment of the mitochondrial 16S gene of Pristimantis species. Values at nodes are bootstrap proportions in percent (values below 49% not shown). Newly obtained sequences for this study are in red font. For other samples used, species names are followed by GenBank accession number and locality. Yunganastes pluvicanorus was used as outgroup (not shown for graphic reasons only). Inset photos depict Pristimantis bipunctatus (MUSM 31120), P. nebulosus (MUSM 40299, FGZC 6254) and P. symptosus sp. nov. (MUSM 40256, FGZC 6207) in life. Diagnosis. Based on the holotype and the newly collected material, we provide the following updated diagnosis for Pristimantis nebulosus (Henle, 1992): a medium-sized species of the Pristimantis conspicillatus species group (based on molecular relationships and morphological similarity), with 27.7-29.1 mm SVL in adult males (n = 3), characterised by: (1) skin on dorsum shagreen, with few scattered small tubercles; flanks shagreen; throat, chest and venter weakly areolate, smooth only in the middle and anterior part; posterior surfaces of limbs smooth; discoidal fold conspicuous; dorsolateral folds present, but low, partly interrupted; dorsal folds absent; two prominent postrictal conical tubercles present; upper eyelid lacking tubercles and granules; (2) tympanic membrane and annulus distinct, nearly round, their length slightly less than half of eye diameter; supratympanic fold long, prominent, curved, surrounding upper tympanum; (3) head slightly longer than wide; snout subacuminate in dorsal view, rounded in lateral profile; canthus rostralis straight in dorsal view, sharp in profile; (4) cranial crests absent; (5) dentigerous process of vomers large, triangular in outline, oblique, situated posteromedial to choanae; (6) males with vocal slits, single subgular vocal sac, and nuptial asperities on dorsal surface of thenar tubercle; (7) hands with long and slender fingers, first finger only very slightly longer than second; subarticular tubercles subconical, prominent; supernumerary tubercles round, smaller than subarticular tubercles; palmar tubercle bifid; thenar tubercle elongated; terminal discs of inner two fingers enlarged and round, those of external fingers enlarged, ovate to triangular, about twice the width of digit proximal to disc; circumferential grooves conspicuous, ungual flap slightly indented; lateral fringes and keels on fingers absent; basal webbing between fingers II and III; (8) ulnar tubercles absent; (9) tubercles on heel and tarsus absent, tarsal fold present, narrow and oblique; (10) inner metatarsal tubercle ovate, prominent; outer metatarsal tubercle round, flat, inconspicuous; supernumerary tubercles absent; (11) toes long and slender (FootL 54-56% SVL); narrow lateral fringes weakly expressed, basal toe webbing present; toe V reaching mid-level of penultimate subarticular tubercle of toe IV; toe V slightly longer than toe III; tips of toes rounded to slightly truncate, enlarged; circumferential grooves evident, ungual flap not indented; (12) in life, dorsal colouration overall brown of different shades (sometimes with orange to reddish tint), with dark brown chevrons or irregular flecks on dorsum, outlined with cream; dark brown bars on dorsal surfaces of arms and legs, outlined with cream; brown interorbital bar, at least anteriorly outlined with cream; a pair of black occipital spots: broad black supratympanic stripe; greyish-black canthal stripe; lips dark brown, barred with cream; tip of snout with triangular cream-coloured fleck; belly yellowish-cream; throat yellow or yellowish-cream, mottled with grey spots; ventral surfaces of thighs and shanks orange brown to reddish-brown; posterior surface of thighs reddish-brown with small light spots; iris bronze, with some dark brown reticulation and a median reddish-brown streak; posterior iris periphery turquoise; bones white; (13) advertisement call consisting of a single pulsed note of 206-382 ms duration with a pulse rate of 156-174 pulses/second, emitted in short series (see below). Comparison. Among the now 41 recognised species in the P. conspicillatus species group ( Padial et al. 2014 , 2016 ; de Oliveira et al. 2017 , 2020 ; Taucce et al. 2020 ), 12 species, including P. nebulosus , exhibit dorsolateral folds ( Duellman and Lehr 2009 ; Padial et al. 2016 ). Among these, P. avicuporum mainly differs by the presence of an interocular dermal fold, fingers bearing lateral fringes, and an upper eyelid with numerous tubercles ( Duellman and Pramuk 1999 ; Duellman and Lehr 2009 ); P. buccinator mainly differs by continuous and prominent dorsolateral folds, an X-shaped or V-shaped dorsal fold and presence of an interorbital dermal ridge ( Rodriguez 1994 ); P. condor mainly differs by larger male size (SVL 32.1-39.5 mm), finger I distinctly longer than finger II, lack of basal webbing between fingers II and III, and lack of basal webbing between toes ( Lynch and Duellman 1980 ); P. conspicillatus mainly differs by finger I being distinctly longer than finger II, fingers with lateral keels, lack of basal webbing between fingers II and III, and posterior surfaces of thighs with orange spots in life ( Duellman and Lehr 2009 ); P. malkini mainly differs by slightly larger male size (SVL 30.4-37.2 mm), finger I distinctly longer than finger II, and more extensive webbing between toes ( Lynch 1980 ; Duellman and Lehr 2009 ); P. meridionalis mainly differs by finger I being shorter than finger II, dentigerous processes of vomers small and round, and fingers bearing lateral fringes ( Duellman and Lehr 2009 ); P. peruvianus mainly differs by prominent and continuous dorsolateral folds, finger I distinctly longer than finger II, fingers with lateral fringes, and lack of basal webbing between toes ( Koehler 2000 ; Padial and De la Riva 2009 ); P. skydmainos mainly differs by more prominent and continuous dorsolateral folds, finger I being shorter than finger II, and a black mid-dorsal tubercle ( Flores and Rodriguez 1997 ; Padial and De la Riva 2005 ). Superficially, P. nebulosus is similar to P. iiap , with which it shares similar adult male size, inconspicuous and partly interrupted dorsolateral folds, finger I only very slightly longer or equal in length to finger II, presence of postrictal tubercles, and similar colour pattern ( Padial et al. 2016 ). However, P. iiap mainly differs by the lack of basal webbing between fingers II and III (present in P. nebulosus ), lack of basal toe webbing (present), upper eyelid with small granules (absent), a copper-coloured iris in life (bronze), and advertisement call ( Padial et al. 2016 ; see below). The closely related species P. bipunctatus also shares many characters with P. nebulosus , such as similar male size, dorsolateral folds present but inconspicuous, finger I about equal in length to finger II, upper eyelid lacking tubercles or granules, and similar colour pattern. However, it differs from P. nebulosus by the lack of basal webbing between fingers II and III (present), bluntly rounded snout in dorsal view (subacuminate), truncate in profile (rounded), basal webbing between toes IV and V (basal webbing between all toes) ( Duellman and Hedges 2005 ; Duellman and Lehr 2009 ), and considerable divergence in the mitochondrial 16S gene. According to Duellman and Lehr (2009) , P. adiastolus is morphologically cryptic to P. bipunctatus . However, P. adiastolus mainly differs from P. nebulosus by more prominent and continuous dorsolateral folds, lack of basal webbing between fingers, and finger I slightly shorter than finger II ( Duellman and Hedges 2007 ). Individuals of the recently described species P. pictus , P. pluvian and P. moa from Amazonian Brazil may sometimes exhibit weakly developed dorsolateral folds, but all differ from P. nebulosus by smooth mid-venter (versus areolate) and advertisement call ( de Oliveira et al. 2020 ). Natural history. Pristimantis nebulosus occurs in an area of evergreen montane rainforest of moderate height. Forest grows at moderate to steeply sloped terrain (Fig. 5 ). See also Myers and Daly (1979) for a detailed description of the area. The area has been heavily altered by different kinds of plantations (e.g. tea). Individuals were found in disturbed habitat along a narrow dirt road. Males were calling from a bushy area during light rain at night, one individual called from near the ground, another one was sitting on a leaf about 30 cm above the ground. The species was found in syntopy with Pristimantis sp. ( Pristimantis aff. divnae ), P. sp. ( Pristimantis lacrimosus group), Rhinella sp. ( margaritifera group), Boana lanciformis , Dendropsophus aperomeus , Scinax garbei , S. sp. ( Scinax aff. ruber ) and Adenomera sp. Figure 5. View of the forest at the type locality of Pristimatis nebulosus at Abra La Divisoria, southern Cordillera Azul, Departamento Huanuco , Peru, at 1650 m a.s.l. Photo taken on 8 November 2019. Figure 6. On top, audiospectrogram and corresponding oscillogram of one advertisement call of a topotypic Pristimantis nebulosus from Abra la Divisoria, Cordillera Azul, 1650 m, Peru, at 1000 ms time scale. Below, an oscillogram at 8000 ms time scale showing one complete call series consisting of 7 calls of the same individual. Recording band-pass filtered at 450-9600 Hz. Vocalisation. Calls were recorded on 8 November 2019, around 20:00 h, at Abra la Divisoria, Departamento Huanuco , 1650 m a.s.l. (air temperature estimated 18 °C). The recorded individual could not be observed calling, but searching at the spot of sound emission revealed an individual of P. nebulosus , leaving little doubt that recorded calls actually correspond to this species. The advertisement call consists of a single regularly pulsed note, repeated at a rather regular succession in short call series (Fig. 6 ). Call series consist of seven calls (n = 2), with the initial call being the longest in duration and separated from the remaining calls of the series by a longer inter-call interval with about two times the duration of other inter-call intervals. Pulses within calls (= notes) are broadly fused, but fairly countable in the oscillogram. Apart from the pulse structure, there is amplitude modulation within each call, with the greatest amplitude occurring at the beginning of the call, then decreasing to about half the initial amplitude at about one third of the call's duration and rapidly decreasing to zero at the call's end, giving the call a bell-shaped appearance in the oscillogram. Numerical parameters of 14 analysed calls (two call series) of one male are as follows: call duration (= note duration) 206-382 ms (254.1 +/- 40.3 ms); inter-call interval within call series 507-1730 ms (796.1 +/- 348.8 ms); pulses per call 33-37 (35.4 +/- 1.5); pulses per second 156-174 (162.8 +/- 7.0); dominant frequency 3186-3359 Hz (3230 +/- 57 Hz); prevalent bandwidth 1500-6600 Hz, but bands of very low call energy recognisable up to 9600 Hz. Apart from the dominant frequency band, additional frequency bands, apparently induced by the pulse rate (not harmonics; see Koehler et al. 2017 ), occur at approximately 1400-1900, 4400-5000, 6000-6600, and 7900-8400 Hz, respectively. Within regular call series, calls were repeated at a rate of 54-72 calls/minute. Duration of call series was 7.13 and 6.05 seconds, respectively. Distribution. So far, P. nebulosus is only known from its type locality in the southern Cordillera Azul, at the border of the Departamentos Huanuco and Ucayali. However, it is very likely that the species occupies a wider range, at least along the same elevational corridor within the Cordillera Azul. Possibly, there are additional records of this species represented by unidentified or misidentified specimens in scientific collections.