Integrative taxonomy reveals first country record of Hyalinobatrachium mondolfii Señaris and Ayarzagüena 2001, and distribution range extensions for Cochranella nola Harvey 1996, and Rulyrana spiculata Duellman 1976 (Anura: Centrolenidae) in Peru Chávez, Germán Pradel, Renzo Catenazzi, Alessandro Zootaxa 2019 2019-11-04 4691 5 541 560 4TPFH Duellman, 1976 Duellman 1976 [199,418,1178,1205] Amphibia Centrolenidae Rulyrana GBIF Animalia Anura 10 551 Chordata species spiculata    On  28 November 2013, we collected a specimen of  Rulyrana spiculata( CORBIDI13906) at Campamento Hospital, in El Sira Community Reserve( 09°28’43.24”S, 74°46’41.36”W;  791 ma.s.l), Puerto Inca Province, Huanuco Department, Peru.  On  21 March 2014we collected six more specimens ( CORBIDI14406–11) ( Figure 5A–F) at the same locality. On  6 November 2014, we collected a specimen of  Rulyrana( CORBIDI15431) at 3 de Mayo Native Community, in Tingo Maria National Park( 09°25’13.06”S, 75°59’33.36”W;  948 ma.s.l), Leoncio Prado Province, Huanuco Department, Peru, and on  20 November 2015two more specimens ( CORBIDI16518, 16572) were collected by Andy Barbozanear to Agua Nueva Local Community( 09°43’1.39”S, 75°48’58.77”W;  1186 ma.s.l), Huanuco Province, Huanuco Department, Peru.   Thecoloration patterns, overall appearance, and measurements ( Table 4) of the specimens we collected from El Sira, Tingo Maria National Park, and other locations in the Huanuco Department( Fig. 1) resembled those of the typeseries of  Rulyrana spiculatafrom the Kosñipata Valleyof Cusco Department and of specimens that we observed and collected at the typelocality on multiple occasions ( Catenazzi et al.2011, 2013). The main source of variation we observed was the distinctly wider dorsal color pattern range in specimens from Huanuco varying from pale green ( CORBIDI14406, Figure 6A-B) to greenish brown ( CORBIDI14409, Figure 6C-D), in contrast to the green or dark green coloration of typeand topotype specimens ( Duellman 1976; Catenazzi et al.2011, 2013), as well as Bolivian material ( Harvey and Noonan 2005). A trait not included in the original description of  R. spiculata, but later reported for specimens from La Paz Department, Boliviaidentified as  Rulyrana spiculata( Harvey & Noonan 2005), is the presence of a pair of enlarged round glandular tubercles below the venter. Our examination of specimens from El Sira Community Reserve( CORBIDI14406, 14409, Fig. 6) and of CORBIDI19185 from the typelocality ( Fig. 6 E, F) confirms the presence of these tubercles. Additionally, specimens from El Sira Community Reserveseem to have longer heads and larger eyes ( Table 4), but the small sample size prevents us from performing statistical comparisons. Despite these variations, diagnostic characters of the species do not overlap with those of other species of the genus, ensuring field identifications through morphological traits. However, we still compared our morphological identifications with those provided by genetic analyses.   TABLE 4.Measurements and proportions (in percentage) of specimens of  Rulyrana spiculatafrom Peru and Bolivia. Our newly reported specimens correspond to El Sira Community Reserve. NA = Data not available; n= number of individuals.    Character Type series (n=2) Bolivian males (n=2) E1 Sira males (n=7)  SVL 21.4‒21.5 (21.4 ± 0.0) 22.6‒23.1 (22.8 ± 0.3) 19.8‒22.2 (20.9 ± 0.8)  HL 6.8‒7.2 (7.0 ± 0.2) 7.2–7.4 (7.3 ± 0.1) 6.6‒7.8 (7.2 ± 0.4)  HW 7.8‒8.1 (7.9 ± 0.2) 8.4 7.1‒8.2 (7.6 ± 0.4)  IOD NA NA 2.2‒2.8 (2.6 ± 0.2)  EL NA NA 2.7‒2.9 (2.8 ± 0.0)  EW NA NA 2.0‒2.1 (2.0 ± 0.0)  ES NA NA 2.4‒2.5 (2.5 ± 0.0)  FIII NA NA 1.0‒1.1 (1.1 ± 0.0)  FEL NA NA 11.1‒12.5 (11.8 ± 0.5)  TL 12.3‒12.4 (12.3 ± 0.0) 12.7‒13.5 (13.1 ± 0.5) 11.4‒12.7 (12.4 ± 0.8)  FL 9.8‒9.9 (9.8 ± 0.0) 11.1‒13.1 (12.1 ± 1.4) 8.1‒10.0 (9.5 ± 0.6)  HL/HW 86.3‒88.0 (87.1 ± 1.2) 86.2‒87.8 (87.0 ± 1.1) 92.5‒96.5 (94.9 ± 1.0)  HL/SVL 32.0‒33.6 (32.8 ± 1.1) 32.3‒32.4 (32.3 ± 0.0) 33.3‒36.1 (34.5 ± 1.0)  EL/FIII 196.1‒229.9 (213 ± 23.9) 159.2‒167.0 (163.1 ± 5.5) 238.0‒268.9 (262.0 ± 1.5)  EW/IOD 55.6‒58.8 (57.2 ± 2.2) 58.9‒76.3 (67.6 ± 12.3) 73.2‒90.9 (79.9 ± 7.0)  TL/SVL 57.70 56.6‒58.5 (57.5 ± 1.3) 57.6‒62.1 (59.5 ± 1.6)  FL/TL 79.90 87.5‒97.1 (92.3 ± 6.7) 71.5‒79.4 (76.1 ± 3.0)   FIGURE 4.Oscillogram (top) and spectrogram (bottom) of the advertisement call (n = 2; T air= 24ºC) of a male of  Hyalinobatrachium mondolfii(CORBIDI 18211) from Las Piedras Amazon Center (LPAC), Madre de Dios, Peru.   Weobtained DNA sequences from CORBIDI13906, 14409, 144010, 15431 and 16572 from Huanucoand from a male ( CORBIDI19185) from the typelocality. Theuncorrected genetic distances for 16S between the topotype individual and specimens from Huanucosupport our identifications ( Table 5). Allspecimens we sequenced from El Sira(range 0.2–1.29 %), Tingo Maria National Park(1.29%), and the Agua Nueva Community(1.29%) were similar to the sequence we obtained from CORBIDI19185. Furthermore, we confirmed the identification of MHNSM24867 from Vista Alegre, Provincia Satipo, in the Junin Departmentas  R. spiculata( Guayasamin et al.2008), albeit the sequence available ( EU663022) seems to be of low quality due to the presence of indels not seen in any other sequenced specimen of  Rulyrana. Wecan also confirm that  R. spiculataoccurs in Boliviabecause the 16S sequence of specimen CBG806 from Boquerón, La Paz Departmentclosely matches the 16S sequence of CORBIDI19185 from the typelocality (only three nucleotide substitutions within a 550 bp sequence). We found  Rulyrana spiculatain El Sira Community Reserve( CORBIDI13906, 14406–11) at night along streams in the foothill primary forest. We found several males calling between 19:00–21:00 hours from leaves of riparian vegetation at the end of the wet season. We did not find egg masses or females. The fast-flowing stream had clear water, and the riparian vegetation consisted mainly of bushes, tall herbs,  Heliconiasp., and some trees 25–30 meters high. We examined several epiphytic bromeliads, which did not contain amphibians. Sympatric amphibians included  Hyloscirtuscf. phyllognathusalong the same stream, and  Allobatessp.,  Ameerega petersii, and  Pristimantis iiapin the nearby forest. We also recorded species of snakes, which may prey on  R. spiculata, such as  Leptodeira annulataand  Chironius fuscus( Cantor and Pizzatto 2008,  Muscat et al. 2017). Specimens from Tingo Maria National Park ( CORBIDI15431) and Agua Nueva Local Community ( CORBIDI16518, 16572), both in the Huallaga River basin, inhabited secondary montane forest. The males we found were perched on leaves at 1–2 meters above the stream between 19:00-22:00 hours. The riparian vegetation consisted of bushes and tall herbs (  Heliconiasp.), and we also found males of  Hyloscirtuscf. phyllognathusalong the same stream.   FIGURE 5.A) Habitat of  Hyalinobatrachium mondolfiiin the floodplain Amazonian rainforest at Las Piedras Amazon Center (LPAC), Madre de Dios. Peru; B) gelatinous mass containing undeveloped eggs in a clutch in the same locality; C) embryos of   TABLE 5.Uncorrected genetic p-distances estimated from the 16S rRNA mitochondrial fragment between  Rulyrana spiculatafrom El Sira Community Reserve, Tingo Maria National Park, and the type locality (CORBIDI 1985), Peru (all in boldface) and a subset of related taxa of  Rulyrana(see Appendix 3 for full list of specimens considered).      Rulyrana flavopunctata( EU 663009)   Rulyrana susatamai( EU 663024)   Rulyrana tangarana( KF 534370)   Rulyrana saxiscandens( KM 068266)   Rulyrana mcdiarmidi( KM 068279)   Rulyrana saxiscandens( KM 068284)   Rulyrana tangarana( KM068290)   Rulyrana mcdiarmidi( KY 611470)   Rulyrana adiazeta( KY 611471)   Rulyranasp. ( KY611473)    Rulyrana flavopunctata( EU663009)    Rulyrana susatamai( EU663024) 3.77    Rulyrana tangarana( KF534370) 0.38 3.77    Rulyrana saxiscandens( KM068266) 0.56 3.95 0.19    Rulyrana mcdiarmidi( KM068279) 0.19 3.58 0.19 0.38    Rulyrana saxiscandens( KM068284) 0.56 3.96 0.19 0.38 0.38    Rulyrana tangarana( KM068290) 0.56 3.58 0.19 0.38 0.38 0.38    Rulyrana mcdiarmidi( KY611470) 0.19 3.58 0.19 0.38 0.00 0.38 0.38    Rulyrana adiazeta( KY611471) 3.58 0.19 3.58 3.76 3.39 3.77 3.39 3.39    Rulyranasp. ( KY611473) 4.71 3.20 4.71 4.89 4.52 4.90 4.52 4.52 3.01    Rulyranasp. ( KY611474) 4.71 3.20 4.71 4.89 4.52 4.90 4.52 4.52 3.01 0.00    Rulyranacf. spiculata( EU663006) 3.58 3.20 3.20 3.38 3.39 3.39 3.01 3.39 3.01 4.33    Rulyrana spiculata( EU663022) 6.31 6.12 5.93 6.11 6.12 6.12 5.74 6.12 5.93 6.69     Rulyrana spiculata( MN509215)  3.58  3.20  3.20  3.38  3.39  3.39  3.01  3.39  3.01  3.77     Rulyrana spiculata( MN509216)  3.58  3.20  3.20  3.38  3.39  3.39  3.01  3.39  3.01  3.39     Rulyrana spiculata( MN509217)  3.39  3.01  3.01  3.19  3.20  3.20  2.83  3.20  2.83  3.58     Rulyrana spiculata( MN509218)  2.45  2.64  2.07  2.26  2.26  2.26  1.88  2.26  2.45  3.96     Rulyrana spiculata( MN509219)  2.45  2.64  2.07  2.26  2.26  2.26  1.88  2.26  2.45  3.96     Rulyrana spiculata( MN509220)  3.44  3.25  3.06  3.24  3.25  3.25  2.87  3.25  3.06  4.40    Rulyrana mcdiarmidi( KM068279) 0.19 3.58 0.19 0.38 0.00 0.38 0.38 0.00 3.39 4.52    Rulyrana saxiscandens( KF534369) 0.38 3.77 0.00 0.19 0.19 0.19 0.19 0.19 3.58 4.71  TABLE 5. (Continued)      Rulyranasp. ( KY 611474)  Rulyranacf. spiculata( EU 663006)   Rulyrana spiculata( EU 663022)    Rulyrana spiculata( MN 509215)    Rulyrana spiculata( MN 509216)    Rulyrana spiculata( MN 509217)    Rulyrana spiculata( MN 509218)    Rulyrana spiculata( MN 509219)    Rulyrana spiculata( MN 509220)   Rulyrana mcdiarmidi( KM 068279)   Rulyrana saxiscandens( KF 534369)    Rulyrana flavopunctata( EU663009)    Rulyrana susatamai( EU663024)    Rulyrana tangarana( KF534370)    Rulyrana saxiscandens( KM068266)    Rulyrana mcdiarmidi( KM068279)    Rulyrana saxiscandens( KM068284)    Rulyrana tangarana( KM068290)    Rulyrana mcdiarmidi( KY611470)    Rulyrana adiazeta( KY611471)    Rulyranasp. ( KY611473)    Rulyranasp. ( KY611474)    Rulyranacf. spiculata( EU663006) 4.33    Rulyrana spiculata( EU663022) 6.69 4.24     Rulyrana spiculata( MN509215)  3.77 1.32 2.92     Rulyrana spiculata( MN509216)  3.39 1.69 3.30 0.38     Rulyrana spiculata( MN509217)  3.58 1.51 3.11 0.19 0.19     Rulyrana spiculata( MN509218)  3.96 1.69 4.43 1.51 1.51 1.32     Rulyrana spiculata( MN509219)  3.96 1.69 4.43 1.51 1.51 1.32 0.00     Rulyrana spiculata( MN509220)  4.40 0.77 3.92 0.96 1.34 1.15 1.34 1.34    Rulyrana mcdiarmidi( KM068279) 4.52 3.39 6.12 3.39 3.39 3.20 2.26 2.26 3.25    Rulyrana saxiscandens( KF534369) 4.71 3.20 5.93 3.20 3.20 3.01 2.07 2.07 3.06 0.19   H. mondolfiiprior to hatching, same locality as above, showing the characteristic pink coloration.  Our records extend the known distribution of  Rulyrana spiculata~ 195 kmto the northeast ( Fig. 1) and represent the farthest known populations from the typelocality (~ 595 kmby airline). The farthest Bolivian locality is 521 kmby airline south of the typelocality ( Harvey & Noonan 2005). The typelocality is in the upper Madre de Dioswatershed, whereas other Peruvianlocalities to the north, such as the Perené Valley( Cannatella and Duellman 1982) and El Sira Community Reserve(this study) are within the Ucayaliwatershed, and Bolivianlocalities to the south are part of the upper Beniwatershed. Theoccurrence of  R. spiculatain three large watersheds (along> 1000 kmby airline from north to south) suggests the species may be widely distributed in the eastern slopes of the Andesof Peruand northern Bolivia.   FIGURE 6.Dorsolateral and ventral views in life of adult males of  Rulyrana spiculata. CORBIDI 14406 (A, B) and CORBIDI 14409 (C, D) from El Sira Community Reserve, Huanuco, Peru; CORBIDI 19185 (E, F) from Kosñipata Valley, Cusco, Peru, the type locality. The IUCNRed List considered  R. spiculataas endemic to Peruwith a distribution extended from south to central regions of the country and also is listed as Near Threatened ( Rodríguez et al. 2004). We recommend updating the distribution range to include our newly confirmed localities and to add Boliviaas a country of occurrence. In spite of the new records which put in evidence a wider but fragmented occurrence, the species is known to have been locally extirpated at localities where chytrid-driven declines have occurred ( Catenazzi et al. 2011, 2014), thus we recommend further revision of material deposited in collections which could reveal new localities, as well as monitoring of existing populations to confirm species persistence, particularly at sites in the cloud forest known to have being affected by chytrid epizootics. Therefore, we consider at the moment there is not enough data to allow us to update its IUCNred list category. 2848517305 2013-11-28 On & Campamento Hospital Peru Department 791 -9.478678 El Sira Community Reserve 1 -74.77816 Puerto Inca Province 10 551 1 Huanuco 2848517302 2014-03-21 2014-11-06 2014-03-21 On & de Mayo Native Community Peru Department 948 -9.420295 Tingo Maria National Park 1 -75.9926 Leoncio Prado Province 10 551 1 Huanuco 2848517309 2015-11-20 Andy Barboza & Agua Nueva Local Community Peru 1186 -9.717052 Department 1 -75.81633 10 551 1 Huanuco 2848517306 The & El Sira & Huanuco Department Bolivia El Sira Community Reserve Tingo Maria National Park El Sira Community Reserve 10 551 4 La Paz 2848517303 We & Huanuco & The & All & El Sira & Agua Nueva Community & Furthermore & Vista Alegre & Provincia Satipo & Junin Department Bolivia Tingo Maria National Park 12 553 Boqueron We 11 552 2 La Paz 2848517307 Peru Andes Peruvian The 15 556 3 Madre de Dios