Zoosyst. Evol. 100 (2) 2024, 565-582 | DOI 10.3897/zse.100.119143 vi olaaeesial MoO ee ON ________am__. D> PENSOFT. Nite = NATURKUNDE BERLIN Similar looking sisters: A new sibling species in the Pristimantis danae group from the southwestern Amazon basin (Anura, Strabomantidae) Jorn Kohler!, Frank Glaw*, César Aguilar-Puntriano*, Santiago Castroviejo-Fisher*, Juan C. Chaparro®®, Ignacio De la Riva’, Giussepe Gagliardi-Urrutia®, Roberto Gutiérrez Miguel Vences'', José M. Padial!*1% QO ’ Hessisches Landesmuseum Darmstadt, Friedensplatz 1, 64283 Darmstadt, Germany Zoologische Staatssammlung Miinchen (ZSM-SNSB), Miinchhausenstr. 21, 81247 Miinchen, Germany Universidad Nacional Mayor de San Marcos, Museo de Historia Natural (MUSM), Departamento de Herpetologia, Av. Arenales 1256, Lima 11, Peru Departamento de Zoologia, Facultad de Biologia, Universidad de Sevilla, Av. Reina Mercedes, 41012 Sevilla, Spain Museo de Biodiversidad del Peru, Urbanizacion Mariscal Gamarra A-61, Zona 2, Cusco, Peru Museo de Historia Natural de la Universidad Nacional de San Antonio Abad del Cusco, Paraninfo Universitario (Plaza de Armas s/n), Cusco, Peru Museo Nacional de Ciencias Naturales-CSIC, C/ José Gutiérrez Abascal 2, 28006 Madrid, Spain Direccion de Investigacion en Diversidad Bioldgica Terrestre Amazénica, Instituto de Investigaciones de la Amazonia Peruana (IIAP), Av. Abelardo CON DO BP WDM Quifiones km 2.5, Iquitos, Peru 9 Museo de Historia Natural de la Universidad Nacional de San Agustin de Arequipa (MUSA), Ay. La Pampilla s/n, Cercado, Arequipa, Peru 10 Servicio Nacional de Areas Naturales Protegidas por el Estado (Sernanp), Calle 17 N° 355, San Isidro, Lima, Peru 11 Zoological Institute, Technische Universitat Braunschweig, Mendelssohnstr. 4, 38106 Braunschweig, Germany 12 Departamento de Zoologia, Facultad de Ciencias, Universidad de Granada, Avenida de Fuente Nueva s/n, 18071 Granada, Spain 13 Department of Herpetology, American Museum of Natural History, Central Park West at 79th St, 10024 New York, NY, USA https://zoobank. org/3 DOB 1824-9405-4F 44-A DB9-68 90BOC5C0D3 Corresponding author: Jorn Kohler (joern.koehler@himd.de) Academic editor: Pedro Taucce # Received 19 January 2024 # Accepted 22 April 2024 # Published 16 May 2024 Abstract We describe a new frog species that is the sibling of Pristimantis reichlei. These two sister species inhabit the Amazonian lowlands and adjacent foothills of the Andes, from central Bolivia to central Peru. Pristimantis reichlei occurs from central Bolivia to southern Peru (Alto Purus National Park), while the new species occurs from northern Bolivia (Departamento Pando) to Panguana in central Peru (Departamento Huanuco), at elevations between 220 and 470 m a.s.l. In spite of their morphological crypsis, these siblings occur in syntopy without evidence of interbreeding (in the Alto Purus area) and are recovered as reciprocally monophyletic. Their uncorrected pairwise genetic distances in the 16S rRNA gene range from 9.5—13.5%, and their advertisement calls differ in both qualitative and quantitative traits. Moreover, our study found uncorrected pairwise distances within the new species of up to 5.0% and up to 9.3% within P. reichlei. We therefore cannot rule out the possible existence of hybrids or additional species-level lineages hidden in this complex. Furthermore, we found another potential pair of sibling species composed of P. danae and an unnamed lin- eage, with divergences of 9.4% in the 16S gene, whose in-depth analysis and taxonomic treatment are pending future revision. With the new nominal species, the Pristimantis danae species group now includes 20 species, distributed across the upper Amazon basin and in the eastern Andes, from western Brazil to Bolivia and Peru. Our study, together with an increasing number of other studies, indicates that sibling species are far from being rare among Amazonian amphibians and that species resolution remains low even for groups that have received considerable attention in recent years. Key Words Amphibia, bioacoustics, integrative taxonomy, morphology, molecular genetics, systematics, Bolivia, Brazil, Peru Copyright Kohler, J. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 566 Introduction The genus Pristimantis Jiménez de la Espada, 1870 rep- resents an extremely species-rich and ecologically diverse clade of Neotropical frogs. It 1s considered challenging for researchers with respect to their systematics because of the many genealogically distant species co-occurring in sympatry yet showing highly similar phenotypes, and due to the numerous geographically and phylogenetical- ly distant yet also similar-looking species (e.g., Elmer et al. 2007; Padial and De la Riva 2009; Garcia et al. 2012: Pinto-Sanchez et al. 2012). These patterns of variation and distribution led to the erection of many non-monophylet- ic species groups (see Padial et al. 2014). Although our knowledge of species diversity and relationships remains rudimentary, the application of molecular phylogenetics has improved the systematics of the genus considerably over the last two decades (e.g., Heinicke et al. 2007; Hedg- es et al. 2008; Canedo and Haddad 2012; Pinto-Sanchez et al. 2012; Padial et al. 2014; Paez and Ron 2019; Ron et al. 2020; Sanchez-Nivicela et al. 2021; Arroyo et al. 2022). Among other results, many non-monophyletic species groups and genera have been redefined so as to represent monophyletic groups (Padial et al. 2014; Gonzales-Duran et al. 2017; Paez and Ron 2019; Chavez et al. 2021; Zumel et al. 2021). Still, most nominal Pristimantis species re- main unassigned to any species group, and the affinities of many assigned ones remain uncorroborated, both because of our limited understanding of phenotypic variation and a lack of genetic data (e.g., Padial et al. 2014). Hedges et al. (2008) erected the Pristimantis peruvianus Species group for some species formerly in the P. conspicil- latus and P. unistrigatus groups. However, these authors did not study topotypic material of P peruvianus. Instead, they relied on samples from populations in the Amazon lowlands of SW Peru, which at the time were considered P. peruvianus (Padial and De la Riva 2005) and which later were identified as belonging to a new species, P. reichlei (Padial and De la Riva 2009). Thus, the nominal species group could not be maintained as the species for which it was named belonged to a different clade. Padial et al. (2014) re-allocated P. peruvianus to the P. conspicillatus group and erected the P. danae species group for some spe- cies formerly considered part of the P. peruvianus group (sensu Hedges et al. 2008), which included P danae and P. reichlei as well as others (P. albertus, P. aniptopalmatus, P. cuneirostris, P. ornatus, P. pharangobates, P. rhabdo- laemus, P. sagittulus, P. stictogaster, P. toftae). They found this group to be monophyletic and sister to the P. conspicil- latus group. Further research resulted in new species that were part of this species group (Lehr and von May 2017; Herrera-Alva et al. 2023: Lehr et al. 2017; Venegas et al. 2023), adding six species to it, all but two (P. clarae, P. sim- ilaris), from high elevations of the Pui Pui Protected Forest in central Peru (P. attenboroughi, P. bounides, P. humboldti, P. puipui). In addition, Herrera-Alva et al. (2023) revealed P. scitulus as part of the P. danae species group. zse.pensoft.net Kohler, J. et al.: New sibling species in the Pristimantis danae group The systematics among several populations in south- western Amazonia and along the eastern Andean slopes of Bolivia and Peru, today considered part of the P. con- spicillatus and P. danae species groups, has been histori- cally complex and partly chaotic, with certain populations misidentified at the species level and repeatedly allocated to the wrong groups (see Padial and De la Riva 2005, 2009). The reason for these long-lasting uncertainties was caused by superficial morphological similarities among species as well as by traits shared among species in both species groups. Padial and De la Riva (2009), with the descriptions of P. koehleri (P. conspicillatus group) and P. reichlei (P. danae group), cleared part of the existing cha- os, as both species were previously confused with other taxa of both groups, respectively. They also demonstrated that among Amazonian frogs, there could be many sister and similar-looking unnamed species (i.e., sibling spe- cies; see Bickford et al. 2007) hidden under names that are applied to populations of more than one species. In a recent study on the taxonomy and systematics of species in the P. conspicillatus group, Kohler et al. (2022) included species of the related P. danae group and pro- vided a first indication of the presence of two divergent lineages among populations currently considered part of P. reichlei, as later also demonstrated by Herrera-Alva et al. (2023). In this new study, we follow clues provided by Kohler et al. (2022) and Herrera-Alva et al. (2023), and, based on genetic and bioacoustic evidence, we describe and name a new species of Pristimantis that is morpho- logically most similar and phylogenetically sister to P. re- ichlei, and with which it occurs in sympatry in some areas of the southwestern Amazon basin. Material and methods Fieldwork and voucher specimens Fieldwork was conducted in different areas of the south- western Amazon basin. Specimens were observed and collected during opportunistic searches during the day and night using torchlights and headlamps. Geographic position was recorded using handheld GPS receivers set to WGS84 datum. Collected specimens were euthanized with an overdose of 5% lidocaine or benzocaine gel ap- plied to the ventral surfaces of individuals (McDiarmid 1994). Tissue samples were taken prior to fixation and stored in pure ethanol, while specimens were fixed using 96% ethanol or 4% formalin and subsequently stored in 70% ethanol. Newly collected specimens were deposited in the herpetological collections of the Museo de Histo- ria Natural, Universidad Nacional Mayor de San Marcos (MUSM), Lima, Peru; Zoologische Staatssammlung Munchen (ZSM), Germany; Museo de Biodiversidad del Peru (MUBD, Cusco, Peru; and Carnegie Museum of Natural History (CM), Pittsburgh, USA. KU refers to the University of Kansas, Museum of Natural History, Lawrence, USA; MNCN refers to the Museo Nacional Zoosyst. Evol. 100 (2) 2024, 565-582 de Ciencias Naturales, Madrid, Spain; MCZ refers to the Museum of Comparative Zoology, Harvard University, Cambridge, USA; MNK-A refers to the amphibian col- lection of the Museo Noel Kempff Mercado, Santa Cruz, Bolivia; NMP6V refers to the Department of Zoology, National Museum, Prague, Czech Republic; SMNS re- fers to Staatliches Museum fiir Naturkunde Stuttgart, Germany; ZFMK refers to Zoologisches Forschungsmu- seum A. Koenig, Bonn, Germany; FGZC refers to Frank Glaw field numbers. External morphology Morphometric measurements (in millimeters) were taken by CAP, JCC, JK, and JMP with calipers to the nearest 0.1 mm. Measurements taken and used throughout the text are those used by Kohler et al. (2022): SVL, snout— vent length (distance from tip of snout to posterior end of the body); TL, tibia length (distance from knee to distal end of tibia); HW, head width (at level of angle of jaws); HL, head length (from posterior margin of lower jaw to tip of snout); IOD, interorbital distance (distance between upper eyelids); ED, horizontal eye diameter (length of the visible eye); E—-N, eye—nostril distance (straight line distance between anterior corner of orbit and posterior margin of external nares); TD, tympanum diameter (hor- izontal distance between the peripheral borders of the tympanic annulus); HandL, hand length (from proximal border of outer metacarpal tubercle to tip of third finger); FootL, foot length (from proximal border of inner meta- tarsal tubercle to tip of fourth toe). Fingers and toes are numbered preaxially to postaxially from I-IV and I-V, respectively. The lengths of fingers I and II were deter- mined by adpressing the fingers against each other. For character state definitions, we followed Duellman and Lehr (2009). Descriptions and diagnostic schemes fol- low Padial et al. (2016). Coloration in life was described based on digital photographs. Bioacoustics Vocalizations in the field were recorded using an Ed- irol R-09 digital recorder with built-in microphones (at 44.1 KHz and 16-bit resolution, saved as uncompressed wave format) or a Sony WM-D6C tape recorder with an attached directional microphone Sennheiser Me-80 (for calls of P. reichlei). Recordings were sampled or re-sam- pled at 22.05 kHz and 32-bit resolution and analyzed using the software Cool Edit Pro 2.0. Frequency infor- mation was obtained through Fast Fourier Transforma- tion (FFT; width 1024 points) at the Hanning window function. Spectrograms were obtained with the Blackman window function with 256 bands resolution. Sensitive filtering was applied to remove background sounds, but only to frequencies outside the prevalent bandwidths of calls. Temporal measurements are summarized as a 567 range, with the mean + standard deviation in parenthe- ses. Description, terminology, and methods follow those recommended by Kohler et al. (2017), using the call-cen- tered terminological scheme. Representative sections of the call recordings analyzed were archived in the Zeno- do repository under DOI https://doi.org/10.5281/zeno- do. 10852627. Molecular data Monophyly and sister relationships between the focal lineage and putative new species were tested through phylogenetic analyses of a fragment of the mitochon- drial 16S rRNA gene. As revealed by preliminary data presented by Kohler et al. (2022), the focal lineage (P. cf. reichlei sensu Kohler et al. 2022; hereafter re- ferred to as P. aff. reichlei) is part of the P. danae species group. For taxon sampling, we therefore selected repre- sentative samples of this group as well as of its sister clade, the P. conspicillatus group (Padial et al. 2014). As the focal lineage is morphologically most similar to P. reichlei and P. danae, which are sister species (Padial and De la Riva 2009; Padial et al. 2014), we included all homologous DNA sequences of these species avail- able from GenBank. We rooted our tree with Yunganas- tes, which is sister to Pristimantis (Padial et al. 2009). We extracted DNA using a standard salt extraction pro- tocol and performed polymerase chain reactions (PCR) with primers 16SAr-L (5’-CGCCTGTTTATCAAAAA- CAT-3’) and 16SBr-H (5’-CCGGTCTGAACTCAGAT- CACGT-3’) (Palumbi et al. 1991). PCR products were then directly sequenced on automated DNA sequencers by LGC Genomics (Berlin, Germany). All new DNA sequences were submitted to GenBank (accession num- bers PP621188—PP621210). We used MAFFT (Katoh and Standley 2013) with the G-INS-i algorithm as im- plemented in Concatenator (Vences et al. 2022) to align sequences to reference sequences of other Pristimantis downloaded from GenBank. After removing differences in length resulting from variations in priming regions, we obtained an alignment of 496 base pairs and 75 terminals. We used these data to infer the best model (TIM2+F+I+ R3) under Model Finder (Kalyaanamoorthy et al. 2017), as well as the maximum likelihood tree (log-likelihood: -4805.1412) (Nguyen et al. 2015), using IQ-TREE 2.2.0 (Minh et al. 2020). Branch support was assessed through 500 replicates of standard non-parametric bootstrapping as well as 1000 iterations of the Shimodaira-Hasegawa approximate likelihood ratio test (SH-aLRT) (Shimodai- ra and Hasegawa 1999), both implemented in IQ-TREE. Genetic divergences were quantified as uncorrected pairwise distances (p-distances) on pairwise alignments, not considering gaps using TaxI2 (Vences et al. 2021). A table with all used sequences, GenBank accession numbers, voucher numbers, and locality information is archived in the Zenodo repository under DOI https://do1. org/10.5281/zenodo. 10958854. zse.pensoft.net 568 Results Molecular data The inferred maximum likelihood tree (log-likelihood: -4805.1412; Fig. 1), based on an alignment of 496 nucle- otides of the 16S rRNA gene, recovered the Pristimantis conspicillatus and P. danae species groups as monophy- letic, with higher support for the latter. Our samples of P. reichlei are grouped into two well-supported and highly divergent clades, although the support for their sister rela- tionship is low (bootstrap/SH-aLRT = 45/78). One clade includes all topotypical samples, and we considered it P. re- ichlei sensu stricto. Within this clade of nominal P. reichlei, considerable differentiation is evident among samples. The other clade includes samples from Panguana, central Peru, south-eastern Peru, and northernmost Bolivia. We labeled this clade P. aff. reichlei. The clade containing P. aff. re- ichlei and P. reichlei is sister to P. aff. danae samples from Bolivia, and the clade containing P. aff. reichlei, P. reichlei, and P. aff. danae is sister to nominal P. danae samples from Peru. These four mentioned subclades together are sister to all samples in the P. danae group from high elevations, and these all together are sister to samples of P. cosnipatae + P. toftae and Pristimantis sp. (GenBank accession number KY652655; a sample formerly identified as P. pharango- bates, see Herrera-Alva et al. 2023), although some of the basal nodes received no significant bootstrap support. Genetic divergences between the target lineages were rather high compared to other known sister lineage di- vergences in Pristimantis (e.g., Kohler et al. 2022; Her- rera-Alva et al. 2023). Pristimantis aff. reichlei differed from P. reichlei in uncorrected pairwise distances by 9.5-13.5% (mean 11.4%), and P. aff. danae from Bolivia differed from P. danae from Peru invariably by 9.4% for all available sequences. Pristimantis aff. danae differed from P. reichlei by 11.9-15.2% (mean 12.7%) and from P. aff. reichlei by 7.4-9.1% (mean 8.3%). The respective values for nominal P. danae were 12.3—14.8% (mean 13.2%) and 7.4—9.1% (mean 8.3%). Genetic distances were also high within two of these four main lineages. While no sequence divergence was found among the P. danae samples from Peru (all from the same or nearby localities) and only up to 0.2% among sequences from Bolivian P. aff. danae, uncorrected pairwise distanc- es within P. aff. reichlei amounted up to 5.0% and within P. reichlei up to 9.3% (between samples from the Bolivian type locality Los Guacharos and Santo Domingo de Cara- baya, Peru, but <6% between numerous other populations). See the Zenodo archive (DOI https://doi.org/10.5281/zeno- do. 10958854) for a table with all pairwise distances. Morphology Our external morphological examination of specimens of the focal lineage P. aff. reichlei and comparisons with paratype specimens of P. reichlei from close to its type locality (Provincia Chapare, Departamento Cochabamba, zse.pensoft.net Kohler, J. et al.: New sibling species in the Pristimantis danae group Bolivia; ZFMK 66975, 72564-72565, 72587-72589) re- vealed no diagnostic differences in measurements (Table 1), proportions, or color pattern. The only qualitative mor- phological differences observed are comparatively faint and were: (1) specimens of P. aff. reichlei exhibit a barely recognizable small, flat, round outer metatarsal tubercle in life, which is virtually absent in preservative, whereas in P. reichlei it is distinct, prominent, ovate, and subconical, both in life and preservative (Fig. 4); (2) a difference in the outline of the canthus rostralis when viewed in straight angle from above. In P. aff. reichlei, the canthus rostralis runs almost straight from the anterior corner of the eye to the tip of snout, whereas in P. reich/ei, the canthus rostra- lis is concavely curved around the nostrils, resulting in an indentation that allows to see a larger portion of the nostrils in straight dorsal view (Fig. 5). However, given the limited number of individuals available for direct ex- amination (Table 1), we are not completely confident that the latter is a constant diagnostic character to distinguish among both major lineages across their entire ranges. Bioacoustics The advertisement call of P. aff. reichlei from Panguana is characterized by being a single pulsatile note, simple in structure, and repeated at irregular intervals (see species account below for a detailed call description). Compared to Bolivian calls of P. reichlei from close to its type lo- cality (Kohler 2000; Marquez et al. 2002 [as Eleuthero- dactylus danae}]), the sister taxon of the focal lineage, the following differences are evident: Advertisement calls of P. reichlei are always composed of two pulsed calls (= notes) emitted in conjunction (a two-note call when following the note-centered terminology of Kohler et al. 2017) that differ from each other in duration and domi- nant frequency, with the second call being slightly longer in duration and higher in dominant frequency compared to the first call. Call duration (= note duration) in calls of P. reichlei is longer (49-68 ms) compared to calls of P. aff. reichlei at Panguana (23-47 ms), without overlap, and calls are emitted at a different temporal pattern (see Kohler 2000). Moreover, calls of P. reichlei are distinctly pulsed, with pulses being clearly separated and countable (pulse rate 155—194 pulses/second), versus pulses being largely fused and barely separated from each other but emitted at a distinctly higher rate in P. aff. reichlei (pulse rate ca. 300-375 pulses/second). Despite similar male body sizes (Table 1), the dominant frequency in calls of P. reichlei is significantly lower (1685—2384 Hz) when compared to those of P. aff. reichlei (3289-3628 Hz). Calls of both species are immediately distinguishable in the field, as they sound quite different to the human ear. Although only very few call recordings are available for analysis and respective comparisons among the fo- cal lineages in consideration (calls of two individuals of P. aff reichlei and three individuals of P. reichlei), the qualitative and quantitative differences in advertisement call structure revealed are far beyond those that could Zoosyst. Evol. 100 (2) 2024, 565-582 569 Pristimantis aff. reichlei ON710988 Peru: Huanuco: Panguana 84/90] pristimantis aff, reichlei ON710984 Peru: Hudnuco: Panguana Pristimantis aff. reichlei MUSM 29028 Peru: Hudnuco: Panguana Pristimantis aff. reichlei ON710989 Peru: Hudnuco: Panguana Pristimantis peruvianus JN991461 Peru: Huanuco: Panguana Pristimantis reichlei EU712719 Bolivia: Pando: Bioceanica Pristimantis aff. reichlei MUBI 14816 Peru: Ucayali: Rio Sepahua 64/93| Pristimantis aff. reichiei CM 158600 Peru: Ucayali: Rio Sepahua Pristimantis aff. reichlei CM 158675 Peru: Ucayali: Rio Sepahua Pristimantis aff. reichlei CM 158894 Peru: Ucayali: Rio Yurua P. aff. reichlei iat ; a (=P. asimus sp. nov.) Pristimantis aff. reichlei MUBI 9858 Peru: Cusco: La Convencion, Porotobango Pristimantis reichlei EU192285 Peru: Puno: entre Puerto Leguia y San Gaban 78/80 Pristimantis reichlei MNCN 44255 Peru: Cusco: entre Puerto Leguia y San Gaban Pristimantis reichlei MW075411 Bolivia: Pando: San Antonio Pristimantis reichlei MW075413 Bolivia: Pando: San Antonio Icavalj- Pl whe : ayali: PN Alt iru: Pristimantis reichlei MUBI 8348 Peru: Madre de Dios: Tambopata Pristimantis reichlei MUBI| 8347 Peru: Madre de Dios: Tambopata Pristimantis reichlei MUBI 8392 Peru: Madre de Dios: Tambopata Pristimantis reichlei EU712720 Peru: Cusco: Pantiacolla Pristimantis reichlei MUBI 6925 Peru: Cusco: Quispicanchi 97/98| Pristimantis reichlei MUBI 6974 Peru: Cusco: Quispicanchi —/63 400/100 Pristimantis reichlei EU192286 Bolivia: Cochabamba: Los Guacharos Pristimantis reichlei EU192287 Bolivia: Cochabamba: Los Guacharos P. reichlei 100/91 Pristimantis reichiei EU192288 Peru: Cusco: near Quincemil Pristimantis reichlei MNCN 43249 Peru: Cusco: San Lorenzo a Quincemil 100/100] 98/99), Pristimantis reichlei KY652657 Peru: Cusco: Kosfipata valley 85/68] ) Pristimantis reichlei MUBI 7015 Peru: Cusco: Quispicanchi 86/98! Pristimantis reichlei MUBI 7047 Peru: Cusco: Quispicanchi Pristimantis reichlei MNCN-DNA 20568 Peru: Cusco: Santo Domingo de Carabaya 100/98! Pristimantis reichlei MUBI 5945 Peru: Cusco: Santo Domingo de Carabaya 50/94 Pristimantis danae EU192260 Bolivia: La Paz: Santa Cruz de Valle Ameno Pristimantis danae EU192261 Bolivia: La Paz: trail San José to Apolo 100/100 Pristimantis danae EU192262 Bolivia: La Paz: Huairuro Pristimantis danae EU192263 Bolivia: La Paz: Arroyo Huacataya P. aff. danae Pristimantis danae EU192264 Bolivia: La Paz: Arroyo Huacataya Pristimantis danae EU192265 Bolivia: La Paz: Serrania Bella Vista Pristimantis danae EU192266 Peru: Cusco: Kosfipata valley, Union Pristimantis danae KY652652 Peru: Cusco: Kosfiipata valley Pristimantis danae EU192269 Peru: Cusco: Kosfipata valley Pristimantis danae EU192270 Peru: Cusco: Kosfipata valley P da nae Pristimantis danae EU192272 Peru: Cusco: Kosfipata valley 100/100 Pristimantis danae EU192268 Peru: Cusco: Kosfipata valley 187 Pristimantis danae EU192270 Peru: Cusco: Kosfipata valley 400/007 Pristimantis albertus KY594749 Peru: Junin: Cerro San Pedro —/63 Pristimantis albertus MW075394 Peru: Junin: Pui Pui Forest Pristimantis cf. stictogaster MW075395 Peru: Junin: Pui Pui Forest Pristimantis stictogaster EF493704 Peru: Pasco: NE of Oxapampa Pristimantis ornatus EU186660 Peru: Pasco: Aquimarca gg/gar Pristimantis aniptopalmatus KY006082 Peru: Pasco: Yanachaga-Chemillen 87 Pristimantis aniptopalmatus KY006085 Peru: Pasco: Yanachaga-Chemillen soo) Prstimantis bounides KY962794 Peru: Junin: Runda palices Pristimantis bounides KY962796 Peru: Junin: road Satipo-Toldopampa Pristimantis cf. aniptopalmatus KY006088 Peru: Paco: Yanachaga-Chemillen Pristimantis humboldti KY962798 Peru: Junin: Quebrada Tarhuish {75 oh Pristimantis humbolati KY962799 Peru: Junin: Quebrada Tarhuish 400/95 Pristimantis attenboroughi KY594752 Peru: Junin: Pui Pui Forest 100/99 Pristimantis attenboroughi KY594755 Peru: Junin: Pui Pui Forest 191 Pristimantis puipui KY962800 Peru: Junin: Pui Pui Forest Pristimantis sagittulus EF493705 Peru: Pasco: NE of Oxapampa Pristimantis cosnipatae MW724815 Peru: Cusco: Kosfipata valley, San Pedro 0.06 —/80 Pristimantis toftae JN991439 Peru: Huanuco: Panguana Pristimantis danae 100/100! Pristimantis toftae ZSM 790/2010 Peru: Hudnuco: Panguana Ss pecies g rou p Pristimantis sp. KY652655 Peru: Cusco: Kosfipata valley --16T Pristimantis palodutrai JX267360 Brazil: Bahia: Canavieiras Pristimantis vilarsi KP149278 Colombia: Meta: Finca Los Almendros 85/86 Pristimantis bipunctatus EF493702 Peru: Pasco: E of Oxapampa 99/96 Pristimantis symptosus ON710980 Peru: Huanuco: Cordillera de Carpish 186 84/91 Pristimantis nebulosus ON710981 Peru: Huanuco: Cordillera Azul, Abra La Divisoria Pristimantis cf. skydmainos ON710985 Peru: Hudnuco: Panguana 75185 Pristimantis fenestratus KU495458 Brazil: Amazonas: Manaus 100/100 Pristimantis koehleri EU192278 Bolivia: Santa Cruz: Angostura-Samaipata road Pristimantis samaipatae EU192292 Bolivia: Santa Cruz: Angostura-Samaipata road 56/83 97/93 Pristimantis buccinator KY652650 Peru: Madre de Dios: Los Amigos Biological Station ® . . sas 93/88) Pristimantis conspicillatus EF493529 Ecuador: Sucumbios Pristimantis consp icillatus Pristimantis citriogaster EF493700 Peru: San Martin: 14 km NE Tarapoto species grou p Pristimantis lymani EF493392 Ecuador: Loja: 3.9 km E of Loja 63/89 Yunganastes pluvicanorus EU192247 Bolivia: Cochabamba: Penstock bridge Figure 1. Maximum likelihood phylogenetic tree of selected samples of Pristimantis, representing the P. conspicillatus and P. danae species groups, inferred from an alignment of 496 nucleotides of the mitochondrial 16S rRNA gene. Yunganastes pluvicanorus was used to root the tree. Numbers at nodes are bootstrap values in percent (500 replicates; not shown if <50%) followed by SH-aLRT values (1000 iterations; not shown if <50) as calculated with IQ-Tree. The taxon name is followed by the GenBank accession num- ber, or voucher number for newly produced sequences (bold terminals), and sample locality. Samples highlighted in green indicate the sympatric locality of P. asimus and P. reichlei. Inset photos depict paratopotypes of P. asimus sp. nov. (MUSM 29074) and P. reichlei (ZFMK 66975) in life, respectively. zse.pensoft.net 570 Kohler, J. et al.: New sibling species in the Pristimantis danae group Table 1. Morphometric measurements (in mm) of specimens of Pristimantis asimus sp. nov., P. reichlei, and P. danae, including holotypes and paratypes. BO = Bolivia; PE = Peru; m = male; f= female; subad. = subadult (all other adult specimens); * = paratype; ** = holotype; — = not available. For other abbreviations used, see Material and methods. sex SVL TL HL P. asimus MUSM 41241** PE: Panguana m 30.6 17.9 Tit MUSM 29028* PE: Panguana m 2h 4 17.1 10.6 MUSM 29073* PE: Panguana m 27.7 fA Le MUSM 29074* PE: Panguana m 28.3 17.0 113 ZSM 1986/2008* PE: Panguana m 28.4 1.3 12.7 ZSM 1985/2008* PE: Panguana m 30.6 L7.2 12.8 SMNS 8856* PE: Panguana m 29.8 17.7 11.9 MUBI 12368* PE: La Novia m 30.6 18.5 11.9 MUBI 14816* PE: Rio Sepahua_ fsubad. 26.0 15.4 9.6 SMNS 6386* PE: Panguana f 37.9 22.4 14.7 P. reichlei ZFMK 66975* BO: Chapare m B05 19.8 12.7 ZFMK 72588* BO: Chapare m oe 15.6 LO ZFMK 72589* BO: Chapare m 28.2 18.3 143 ZFMK 72564* BO: Chapare m 28.8 16.9 #31 ZFMK 72565* BO: Chapare m 27.9 16.9 11.4 MNCN 43028* BO: Chapare m 255 15.4 9.6 MNK-A 6620** BO: Chapare f 32.3 20.5 i226 MNCN 43024* BO: Chapare f 3530 21.4 Lead ZFMK 72587* BO: Chapare f 39.1 24.4 15.2 MUBI 8347 PE: Tambopata f 33.4 19.8 12:1 MUBI 8347 PE: Tambopata f 3125 Fee: 12:0 MUBI 12367 PE: La Novia f 30.9 19.2 1225 P. danae KU 162307** PE: Kosnipata m 34.3 16.8 13.6 MCZ 93305* PE: Koshipata m 30.7 17.6 12.0 MCZ 93306* PE: Kosnhipata m 26.0 15.9 10.6 MUSM 13957* PE: Santa Isabel m 24.3 15.7 10.9 HW lOD ED TD E-N HandL_ FootL dle Syl 4.3 ie: Al 7.8 14.3 9.8 Sul 4.3 7 3:3 7H 13.6 9:9 3:6 4.0 2.0 Se: Fil 13-3 10.1 3.5 4.0 2.1 3.6 8.1 14.2 10.4 3.2 Al 1.9 3.5 7.8 14.2 10.9 3.4 3.9 2.0 4.2 7.6 13.9 10.6 CH 3:9 a 3.9 10.0 14.1 let 3.0 4.0 1.9 4.0 8.6 16.9 9.5 2./ 3.0 1.5 eae! 7.2 11.4 1S Pf 4.7 48 25 5/1 10.8 Wher, aa | 3:2 4.0 2.0 3.9 9.0 16.3 9.8 331 4.0 1.7 3.8 8.3 14.0 10.8 O20 4.6 2:2 3:6 Se: 15.4 10.5 3.6 oo) 2.0 4.0 8.6 14.4 9.6 26 3.9 2:3 3:2 8.5 14.5 gal — 3.6 1.6 3.2 — 12.6 11.8 — 3.8 Lal 4.0 — 17.0 12.1 — 4.6 2.0 44 — 17.4 15.0 4.3 5.4 2.6 48 INES! 28 11.4 4.0 4.2 1.8 4.2 8.7 14.8 LTS 3.8 3.4 1.8 ork 8.5 13.5 11.4 3.4 4.0 1.9 4.0 8.5 14.8 12.9 — 5.6 al 4.3 — 16.6 11.4 — 49 1.9 Al — Bera 9.6 — 4.4 Le S. — 13.4 9:9 i) 4.2 1.8 a0 UBS: ees be attributed to intra-specific call variation (see Kohler et al. 2017). Even if more recordings become available in the future, 1t is more than unlikely that an increased sample size would alter the conclusions derived from our bioacoustic analyses by interpreting observed differences in calls as intra-specific variation. On the contrary, call parameters and spectrograms available in the published literature (e.g., Schltiter 1980; Rodriguez 1994; Duell- man 2005) indicate a misidentification of respective populations and corroboration of the call characteristics described for both lineages compared herein (see below). Calls of P. danae from Manu National Park in Peru (https://soundcloud.com/user-416416746/pristiman- tis-danae? in=user-4 1 6416746/sets/cantos-de-nfibios-del- pn-manu) are trill-like calls of approximately 80—120 ms duration, composed of 3—4 widely spaced pulses (pulse rate approximately 30 pulses/second). Calls of P. aff. da- nae from Bolivia are characterized by being composed of two notes, each note containing two distinctly separated pulses only (Padial and De la Riva 2009). In summary, the bioacoustic differences observed among the clades here allocated to P. reichlei, P. danae, P. aff. danae, and the focal lineage P. aff. reichlei provide zse.pensoft.net further evidence for the specific distinctness of respec- tive lineages, particularly in view of the partly sympatric occurrence of P. reichlei and P. aff. reichlei (see below). Consequently, we describe the lineage hitherto referred to as P. aff. reichlei as a new species. Taxonomy Pristimantis asimus sp. nov. https://zoobank.org/298F59D4-1918-4833-8C64-B935FA7E826E Remarks. This species has been previously referred to as Eleutherodactylus peruvianus by Schltter (1980) and Lehr (2002 [partim]); E/eutherodactylus fenestratus by Rodriguez (1994); Eleutherodactylus aff. conspicillatus by Schltiter (2005); Pristimantis cf. danae by Moravec and Aparicio (2005); Pristimantis reichlei by Padial and De la Riva (2009 [partim]), Moravec et al. (2020 [par- tim]), and Herrera-Alva et al. (2023 [partim]); Pristiman- tis peruvianus by Pinto-Sanchez et al. (2012), de Oliveira et al. (2020), and Fouquet et al. (2022); and Pristimantis cf. reichlei by Kohler et al. (2022). Zoosyst. Evol. 100 (2) 2024, 565-582 Type material. Holotype. MUSM 41241 (FGZC 5342, formerly ZSM 177/2017), adult male (Figs 2, 3), from the Area de Conservacion Privada Panguana (9.6166°S, 74.9333°W, 260 m above sea level), lower Rio Yuyapichis, Provincia Puerto Inca, Departamento Huanuco, Peru, col- lected on 29 September 2017 by F. Glaw. GenBank acces- sion number for 16S: ON710989. Paratypes. A total of 16 specimens: ZSM 1985— 1986/2008 (FGZC 3388-3389), two adult males, same locality as holotype, collected on 10 December 2008 by F. Glaw; MUSM 29073-29074 (FGZC 3300, 3273), two adult males, MUSM 29028 (FGZC 3274), an adult S71 male (call voucher), same locality as holotype, collect- ed between 26 November and 2 December 2008 by F. Glaw; FGZC 6334 (to be deposited in ZSM), an adult male, same locality as holotype, collected on 17 Novem- ber 2019 by E. Castillo-Urbina, F. Glaw and J. Kohler; SMNS 6386, an adult female, same locality as holotype, collected on 11 November 1985 by A. Schliiter; SMNS 8856, an adult male, same locality as holotype, collect- ed in 1972 by R. Aussem; MUBI 14816, a subadult fe- male, CM 158675, an adult male, from Campamento 4, between Quebrada Sungaro and Quebrada Esther, Rio Sepahua (11.0801°S, 72.1258°W, 395 m a.s.l.), Distrito Figure 2. Male holotype of Pristimantis asimus sp. nov. (MUSM 41241, FGZC 5342) from Panguana, Departamento Huanuco, Peru, in life (SVL 30.6 mm): a. Dorsolateral view; b. Dorsal view (showing posterior thighs coloration); ¢. Ventral view. zse.pensoft.net 572 Kohler, J. et al.: New sibling species in the Pristimantis danae group Figure 3. Preserved male holotype of Pristimantis asimus sp. nov. (MUSM 41241, FGZC 5342) from Panguana, Departamento Huanuco, Peru: a. Dorsal view; b. Ventral view; c. Palmar surface of the right hand; d. Lateral view of the head. Sepahua, Provincia Atayala, Departamento Ucayali, Peru, collected on 3 and 6 March 2014, respectively, by J. M. Padial, L. A. G. Gagliardi, J. C. Chaparro and R. Guti- érrez; CM 158600, an unsexed adult, from a track across the river from Campamento 2, Rio Sepahua (11.0491°S, 72.4489°W, 408 m a.s.l.), Distrito Sepahua, Provincia Atayala, Departamento Ucayali, Peru, collected on 24 February 2014 by J. M. Padial, L. A. G. Gagliardi, J. C. Chaparro and R. Gutiérrez; CM 158894, an adult male from a track ca. 4 km west of Breu, on the road to Victo- ria (9.5451°S, 72.7933°W, 223 m a.s.l.), Distrito Yurua, Departamento Ucayali, Peru, collected on 12 February 2015, by J. M. Padial, L. A. G. Gagliardi, R. Gutiérrez, O. Rojas and S. Castroviejo-Fisher, MUBI 12368, an adult male, from Puesto de Control y Vigilancia La No- via, Rio La Novia (9.9883°S, 70.7084°W, 262 m a.s.1.), Distrito Purus, Departamento Ucayali, Peru, collected on 25 January 2013 by J. M. Padial, L. A. G. Gagliardi, R. Gutiérrez and S. Castroviejo-Fisher, MUBI 9858, an un- sexed adult from Porotobango (11.4311°S, 73.3471°W, 469 m a.s.l.) Provincia La Convencion, Departamento zse.pensoft.net Cusco, Peru, collected on 25 January 2010 by L. Teja- da; NMP6V 72578/1—2, two adult males (also paratypes of P. reichlei), from Bioceanica (11.1333°S, 69.3666°W, 290 m a.s.1.), Departamento Pando, Bolivia, collected on 25 January 2005 by J. Moravec. Definition. A medium-sized species of the Pristiman- tis danae species group (based on molecular relationships and morphological similarity), with 27.7—30.6 mm SVL in adult males (n = 7), and 37.9 mm SVL in adult fe- males (n= 1), characterized by: (1) skin on dorsum finely shagreened, lacking enlarged tubercles or warts; throat smooth, venter areolate; discoidal fold inconspicuous; dorsolateral folds absent; upper eyelid lacking tubercles and granules; posterior surfaces of thighs smooth; (2) tympanic membrane and annulus distinct, slightly high- er than long, their length less than half of eye diameter; supratympanic fold prominent, curved, slightly covering upper edge of tympanic annulus; (3) head slightly lon- ger than wide; snout subacuminate in dorsal view, blunt- ly rounded in lateral profile; canthus rostralis straight in dorsal view, slightly rounded in profile; (4) cranial crests Zoosyst. Evol. 100 (2) 2024, 565-582 absent; (5) dentigerous process of vomers elongate, oblique, situated posteromedial to choanae; (6) males with vocal slits, single subgular vocal sac; indistinct nup- tial asperities on dorsal surface of thenar tubercle; (7) hands with slender fingers, first finger slightly shorter or about equal in length to second; subarticular tubercles subconical, prominent; supernumerary tubercles absent; palmar tubercle cordiform; thenar tubercle prominent, elongated; terminal discs of inner two fingers enlarged and round, those of external fingers enlarged, truncate, about twice the width of digit proximal to disc; circum- ferential grooves conspicuous, ungual flap not indented; narrow lateral fringes on fingers weakly developed; basal webbing between fingers absent; (8) ulnar tubercles ab- sent; (9) tubercles on heel and tarsus absent, tarsal fold present; (10) inner metatarsal tubercle prominent, ovate; outer metatarsal tubercle small, round, flat, barely recog- nizable in life, virtually absent in preservative; supernu- merary tubercles absent; (11) toes long and slender; later- al fringes narrow, weak; basal toe webbing present; toe V reaching beyond distal level of penultimate subarticular tubercle of toe IV; tips of toes rounded to slightly ovate, enlarged: circumferential grooves conspicuous; (12) in life, dorsal coloration light brown, reddish-brown, or tan, usually with dark brown chevrons and flecks on dorsum; dark brown bars on dorsal surfaces of arms and legs; a pair of black spots dorsolaterally in scapular region; black supratympanic stripe; black canthal stripe; belly creamy white; throat with fine dark mottling in males; posterior surfaces of thighs blackish with yellowish-cream spots and flecking; iris bronze, with black reticulation in life; posterior iris periphery cream to turquoise; bones white; (13) advertisement call consisting of a single pulsatile note of 23-47 ms duration and with a dominant frequen- cy of 3289-3628 Hz, repeated at irregular intervals, con- taining groups of 24 calls repeated in faster succession. Diagnosis. Pristimantis asimus differs from other spe- cies in the Pristimantis danae species group as follows: The new species differs from P. albertus, P. attenbor- oughi, P. bounides, P. clarae, P. cosnipatae, P. humboldti, P. ornatus, P. pharangobates, P. puipui, P. rhabdolaemus, P. sagittulus, P. similaris, P. stictogaster, and P. toftae, at least by the lack of dorsolateral folds (versus presence). Furthermore, P. attenboroughi, P. bounides, P. humboldti, and P. puipui have stout bodies with relatively shorter legs when compared to P. asimus. Pristimantis attenboroughi and P. puipui lack a tympanum (prominent in the new spe- cies), and P. attenboroughi lacks vocal slits in males (pres- ent in the new species). Pristimantis clarae additionally differs from the new species by dorsal and ventral color pattern, advertisement call, and smaller adult male size (12.9-15.6 versus 27.7—30.6 mm). As the new species, P. aniptopalmatus \acks dorsolateral folds (Duellman and Hedges 2005) but differs by its smaller adult male size (16.5—23.2 versus 27.7-30.6 mm), presence of a tubercle on the upper eyelid, and color pattern (Duellman and Lehr 2009). Pristimantis cuneirostris lacks dorsolateral folds but differs from the new species by a long wedge-shaped 53 snout (unique in the P. danae species group; Duellman and Pramuk 1999; Duellman and Lehr 2009), lack of toe webbing (versus basal webbing present), and posterior surfaces of thighs uniformly brown (versus blackish with yellowish-cream blotches and flecks). Pristimantis scitu- /us mainly differs from the new species by the presence of a single conical tubercle on upper eyelid (absent), denti- gerous processes of vomers absent (present), presence of a conical tubercle on heel (absent), and webbing on toes absent (basal webbing present) (Duellman 1978). Morphologically, P. asimus is most similar or even cryptic to P. danae and P. reichlei. However, as a ten- dency, nominal P danae (from Kosfipata valley, Peru) exhibit a more contrasting dorsal color pattern in life (see, e.g., Duellman and Lehr 2009: fig. 150) when com- pared to P. asimus. Moreover, in dorsal view, the snout in P. danae is rounded (versus subacuminate in P. asimus). From P. reichlei, the new species seems to differ by a very small, round, flat outer metatarsal tubercle, barely recog- nizable in life and virtually indistinguishable in preser- vative (versus distinct, ovate, subconical, recognizable in life and in preservative; Fig. 4). Moreover, there are slight differences in the dorsal outline of the canthus rostralis, with a relatively smaller portion of the nostrils being vis- ible when viewed from straight above in P. asimus com- pared to P. reichlei, where nostrils are almost completely visible from above as the canthus rostralis shows a curved indentation around the nostrils (Fig. 5). The new species differs from both P. danae and P. reichlei by substantial differentiation in the 16S gene and differences in the ad- vertisement call (see above). The new species occurs in sympatry with some species of the P. conspicillatus group, which superficially may have a similar appearance. However, these are distin- guishable from P. asimus by molecular phylogenetic rela- tionships, differences in advertisement calls, and most of them by different relative finger lengths, 1.e., the first fin- ger being longer than the second. However, the sympatric P. iiap has the first and second fingers equal in length but differs from the new species at least by exhibiting distinct dorsolateral folds (Padial et al. 2016). Description of the holotype. An adult male, in good state of preservation (Fig. 3), with subgular vocal sac and vocal slits. Head slightly longer than wide (HL/HW = 1.08); snout subacuminate in dorsal view, bluntly round- ed in profile; nostrils oriented posterolaterally; canthus rostralis straight in dorsal view, slightly rounded in pro- file; loreal region slightly concave; lips not flared; upper eyelid without tubercles; cranial crests absent. Supratym- panic fold prominent, long, slightly curved, covering up- permost tympanic annulus; tympanic membrane and an- nulus distinct; tympanic membrane slightly higher than long, its length slightly less than half the eye diameter; one flat round postrictal tubercle. Choanae not concealed by palatal shelf of the maxillary arch when roof of mouth is viewed from below; choanae large, oval, separated by distance equal to five times diameter of a choana; dentig- erous process of vomers present, but barely evident, flat, zse.pensoft.net 574 MUSM 41241 ZSM 1985/2008 ZSM 1986/2008 Pristimantis asimus sp. nov. Kohler, J. et al.: New sibling species in the Pristimantis danae group ZFMK 72564 ZFMK 72588 ZFMK 72589 Pristimantis reichlei Figure 4. Comparative plate showing plantar surfaces of the right feet of preserved type specimens of Pristimantis asimus sp. nov. (all from Panguana, Huanuco, Peru) and P. reichlei (all from Chapare, Cochabamba, Bolivia). Red arrows point to the outer meta- tarsus, showing the virtual absence versus the presence of an outer metatarsal tubercle. Not to scale. Pristimantis asimus sp. nov. holotype (MUSM 41241) Pristimantis reichlei paratopotype (ZFMK 66975) Figure 5. Schematic line drawings of dorsal views of the heads of male Pristimantis asimus sp. nov. and P. reichlei, exemplifying the differing outlines of the canthus rostralis (bold line), resulting in the visibility of different portions of the nostrils (red) in straight dorsal view. Not to scale. elongate, not in contact, oblique, situated posteromedial to choanae, bearing vomerine teeth; tongue removed for tissue sample; vocal sac subgular, vocal slits placed pos- terolaterally. Skin on dorsum finely shagreened, lacking enlarged tubercles or warts; dorsal surfaces of hind limbs finely shagreened, dorsal surfaces of forearms and flanks finely shagreened; skin on throat and chest smooth, that on belly areolate; occipital folds absent; dorsolateral folds absent; discoidal fold indistinct. Arm without ulnar tuber- cles; palmar tubercle cordiform, about double in size to elongate thenar tubercle; supernumerary tubercles absent; subarticular tubercles prominent, subconical; fingers long and slender; finger tips enlarged, rounded on inner two fingers, on two outer fingers truncate, their width about twice the width of digit proximal to disc; circumferential grooves conspicuous, ungual flap not indented on outer fingers; lateral fringes and keels on fingers weak, bare- ly recognizable; basal webbing between fingers absent; relative length of fingers III>IV>H=I; nuptial asperities on dorsal surface of thenar tubercle indistinct. Toes long zse.pensoft.net and slender (FootL 47% of SVL); heel and tarsus lacking tubercles; tarsal fold present; inner metatarsal tubercle ovate, prominent; outer metatarsal tubercle not recogniz- able; supernumerary tubercles absent; subarticular tuber- cles prominent, subconical; narrow lateral fringes on toes present, weakly developed; basal toe webbing present; toe tips enlarged, rounded, their width about 1.5 times the width of toe proximal to disc; circumferential grooves conspicuous; relative length of toes [V>V>II>II>I; toe V reaching slightly beyond distal level of penultimate sub- articular tubercle of toe IV. Tibiotarsal articulation reach- ing distinctly beyond tip of snout when hindlimb flexed parallel to axis of body; heels broadly overlapping when hind limbs flexed perpendicular to axis of body. For mor- phological measurements, see Table 1. In life (Fig. 2), dorsal ground color light brown, with darker brown chevron-shaped marking slightly anterior to sacral region; irregular dark brown U-shaped line in scapular region, bordered posteriorly by orange-brown blotch; irregular indistinct dark brown markings and lines Zoosyst. Evol. 100 (2) 2024, 565-582 575 We Se Figure 6. Paratypes of Pristimantis asimus sp. nov. in life (dorsolateral and ventral views): a. ZSM 1985/2008 (FGZC 3388; SVL ae a 30.6 mm); b. ZSM 1986/2008 (FGZC 3389; SVL 28.4 mm); e. MUSM 29074 (FGZC 3273; SVL 28.3 mm), all from the type local- ity Panguana, Huanuco, Peru; d. CM 158600, from Rio Sepahua, Ucayali, Peru. on dorsum; minute cream flecks, irregular in outline, scattered on dorsum; triangular cream fleck on snout tip; dark brown bars on dorsal surfaces of arms and legs; dark brown interorbital line, partly interrupted, not extending to upper eyelids; a pair of small black spots dorsolateral- ly in scapular region; black supratympanic stripe; broad blackish canthal stripe; lips dark brown to black, irregu- larly barred with cream; flanks light brown with irregular dark brown markings; belly creamy-white; anterior throat grayish-white with scattered fine gray mottling, posteri- or throat yellowish with irregular fine brown mottling; chest pinkish-white; ventral surfaces of thighs and shanks pinkish-gray; posterior surface of thighs blackish with ir- regular yellow-cream blotches and flecks; tarsus, plantar, and palmar surfaces dark brown; iris bronze, with black reticulation, with a dark reddish-brown median streak; posterior iris periphery cream with a turquoise tint; bones white. After six years in preservative (Fig. 3), the general color pattern remains the same as in life. Brown ground coloration turned to grayish-tan, with some pinkish tint, particularly on upper eyelids; brown flecks, bars, and markings on dorsum slightly faded; chest and ventral surfaces of thighs yellowish-cream; belly creamy-white; throat creamy-white with gray mottling. Variation. For variation in morphological measure- ments among type specimens, see Table 1. Females are sig- nificantly larger than males, approximately reaching 25% greater SVL. We observed some limited variation in color and color pattern among the specimens studied. In some individuals, the dorsal ground coloration is yellowish-tan to yellowish-brown in life (e.g., ZSM 1985/2008; Fig. 6a), whereas in most specimens, the dorsal ground color was reddish-brown in life (e.g., ZSM 1986/2008, MUSM 29074, CM 158600; Fig. 6b, c, d), as 1t was in the holotype. Darker dorsal markings might be more or less distinctly outlined with fine cream lines, with some individuals seem- ingly lacking these fine cream lines (Fig. 6a, b). A dark interorbital stripe is present in all specimens but narrow, less conspicuous, and barely extending on upper eyelids in the holotype and some paratypes (Figs 2, 6a, b), whereas a broader, distinct stripe outlined with fine cream lines ex- tends to the upper eyelids in other specimens (Figs 6c, d, 7). Scattered dorsal cream spots and flecking might be pres- ent to different extent or completely absent. The contrast- ing color pattern on the posterior surfaces of thighs (dark brown with yellow-orange flecking) 1s present in all studied specimens, with CM 158600 having some orange color ex- tending to the groin (Fig. 6d). Some males exhibit shades of zse.pensoft.net 576 ; its # ys RU Ren ite Figure 7. Male paratopotype (FGZC 6334) of Pristimantis asi- mus sp. nov. in life at: a. Night coloration in situ; b. Day color- ation the day after collection, demonstrating a limited ability of color change. Note the dark brown, almost black, loreal region and upper lip in the day coloration, resulting in a dark facemask. yellow on the throat in life (Fig. 6). Remarkably, the male paratype MUBI 14816 exhibits a well-marked, W-shaped occipital fold that is lacking in other specimens. Natural history. At Panguana, individuals of this spe- cies have been observed being active at night, perching on small trees and bushes within disturbed primary forest. Schltiter (1980) reported males calling from vegetation mainly at 1-2 m height, with calling activity being most intense at dusk and at dawn, which is confirmed by our own observations. Schltiter (1980) described a vertical calling position with the head down as typical, but we observed calling males in a horizontal position on the up- per side of leaves. Individuals of this species have some limited ability to change color, with nocturnal color being characterized by a light brown loreal region, whereas di- urnal color is characterized by a dark loreal region and upper lip, turning dark brown to almost black (see Fig. 7). The habitat close to the type locality has recently been impacted by illegal gold mining activities, which consti- tute a potential threat to the anuran fauna of Panguana. Vocalization. Advertisement calls of Pristimantis asi- mus, emitted by the male MUSM 29028, were recorded on 29 November 2008, at dusk (18:15 h) at the type lo- cality (ambient temperature not recorded). The calling male was sitting on top of a horizontally oriented leaf at zse.pensoft.net Kohler, J. et al.: New sibling species in the Pristimantis danae group approximately 2.1 m height within the forest. The call con- sists of a single short pulsatile note, repeated at somewhat irregular intervals (see below). In our recording, 4 to 14 calls were emitted in succession and then interrupted by few seconds of silence. Each call (= note) has a clearly pulsatile structure, although ‘pulses’ are largely fused and, in most cases, barely countable. However, in some cases, distinct and thus countable energy peaks (4—10/call) are recognizable in the oscillograms of calls. In these calls, the pulse rate varies between 300 and 375 pulses/second. There is further amplitude modulation recognizable within each call, with maximum call energy being present around the center of each call, rapidly decreasing to a lower lev- el, and further fading towards the call’s end. Calls were usually emitted in slow succession but regularly contained sections of more rapidly repeated calls, usually 2-4 calls emitted at shorter intervals, altering the relatively regular pattern of call repetition (see Fig. 8a). In other words, call repetition rate may change temporarily from ca. 140 calls/ min to ca. 900 calls/min. The character of calls in these rapidly repeated sections did not differ from other calls. It remains unknown whether this increased speed in call succession has a different function (e.g., territorial) or is just part of the ordinary advertisement call. Numerical pa- rameters for 65 analyzed calls of the mentioned male are as follows: call duration (= note duration) 23-47 ms (36.0 + 7.1 ms); inter-call interval in slow calling sections 305— 597 ms (394.6 + 73.6 ms); inter-call interval in rapid call- ing sections 26—37 ms (31.8 + 4.6 ms); dominant frequen- cy 3289-3628 Hz (3518 + 97 Hz); second frequency peak at around 2000 Hz; prevalent bandwidth 1600-5400 Hz. Schltiter (1980) described the call from the same lo- cality (under the name Eleutherodactylus peruvianus). The spectrogram and numerical parameters (call duration ca. 40 ms) provided by him agree with our analysis. The call described by Rodriguez (1994) as that of Eleuthero- dactylus fenestratus from Cocha Cashu, Manu National Park, Madre de Dios, Peru, is also clearly referable to P. asimus. The parameters described by Duellman (2005) for calls of P. fenestratus from Cusco Amazonico clearly differ from those of P. asimus, but the corresponding au- diospectrogram and oscillogram are in disagreement with the numerical parameters provided and may possibly cor- respond to P. asimus. Although difficult to compare ac- cording to another terminology used, the call described for E. fenestratus from Manaus, Brazil, by Zimmerman and Bogart (1984) seems to agree with that of P. asimus. Distribution. As far as known and confirmed by bio- acoustic and/or genetic data, P asimus occurs in lowland rainforests from the southern Departamento Huanuco (Pan- guana, type locality) and eastern Departamento Ucayali (Breu, Rio Yurua) southward across most of the lowlands of southeastern Peru, reaching the border of Machiguen- ga Communal Reserve (Departamento Cusco) and Manu National Park (Departamento Madre de Dios) in the south as well as northernmost Bolivia (Bioceanica, Departamento Pando) to the east (Fig. 9). All known localities are at eleva- tions between 220 and 470 m a.s.l. The new species occurs Zoosyst. Evol. 100 (2) 2024, 565-582 577 Pristimantis asimus sp. nov. (Panguana, Peru) Pristimantis reichlei (Chapare, Bolivia) aq co a Frequency (kHz) ie) Relative amplitude Relative amplitude ) 15 150 225 300 0 15 150 225 300 Time (ms) Time (ms) Figure 8. Audiospectrograms and corresponding oscillograms of the advertisement calls of Pristimantis asimus sp. nov. from the type locality Panguana, Peru (emitted by paratype MUSM 29028) and Pristimantis reichlei from close to its type locality in the Chapare region, Bolivia, at the same temporal and spectral scale (4000 ms and 10 kHz, respectively). Oscillograms at the bottom show respective calls (those highlighted in gray in the oscillograms above) at an expanded time scale of 300 ms. Recording of P. asimus high-pass filtered at 700 Hz and that of P. reichlei band-pass filtered at 900-3600 Hz. 80° W 70° W 10°S 15° S 20°S Figure 9. Schematic map of central-western South America showing the known distribution of species of the Pristimantis danae group referred to in the text, as inferred by molecular genetics and/or bioacoustic data. Broadly overlapping symbols may refer to the same locality. Square symbols refer to the type localities of the respective species. zse.pensoft.net 578 in syntopy with P. reich/ei at least in Alto Purts National Park (La Novia, Departamento Ucayali, Peru) and probably in other places in southeastern Peru and northern Bolivia. The new species most likely also occurs further east in the Brazilian Amazon, as indicated by the call description of Zimmerman and Bogart (1984) from Manaus, but respec- tive records are in need of clarification (see Discussion). Etymology. The specific epithet is a Latinized adjec- tive derived from the Greek Gonpoc (4simos), meaning ‘inconspicuous, nameless’. It refers to the morphologi- cally cryptic nature of the new species and the fact that it has been associated with different species names in the past, missing its status as a separate species to be named. Discussion Morphological crypsis among genetically distant (Le., non-sister) species seems to be uncommon among anuran species (e.g., Castroviejo-Fisher et al. 2017). Striking simi- larity between sister or closely related species (1.e., siblings; Mayr 1942) is nonetheless common and has been recog- nized for a long time (see Winker 2005). Similarity caused by recent shared descent poses difficulties distinguishing among species in complexes with little morphological vari- ation, yet difficulties tend to fade once additional evidence becomes available to clarify the sophisticated issues of the complex (e.g., Scherz et al. 2022). Thus, for example, it is not uncommon that after a study reveals multiple closely related species hidden under a single name, the new spe- cies discovered are more readily distinguishable once the external morphology is examined anew with the results of genetics in mind, or when different dimensions of the phe- notype and the geographic ranges of the species are known better (Saéz and Lozano 2005; Korshunova et al. 2019). Like in the case of the species pair Pristimantis asimus and P. reichlei, it is increasingly common that studies of Neotropical amphibians reveal sister and similar-looking species showing large genetic divergences (Padial and De la Riva 2009; Padial et al. 2009; Hutter and Guayasamin 2015; Ortega-Andrade et al. 2015; Paez and Ron 2019; Trevisan et al. 2020). In many cases, these are allopatric species that, upon closer examination, are readily diag- nosable morphologically, but cases of sympatry/parapatry are not rare either, as in the example reported herein. The challenge with such cases is to ascertain whether such morphologically similar but genetically divergent frogs are, in fact, different species. Vicariance with secondary contact after reproductive isolation is the usual evolution- ary scenario invoked to explain the pattern (Mayr 1942; Wiley and Lieberman 2011). Thus, when mitochondrial DNA (mtDNA) analyses of populations of one putative species reveal the existence of two distinct genetic lin- eages in sympatry, and the mtDNA-divergent individuals are found to also differ at least faintly in other, unlinked characters, the results are interpreted as strong evidence for the existence of similar-looking sibling/cryptic species isolated reproductively. However, as shown by Chan et al. zse.pensoft.net Kohler, J. et al.: New sibling species in the Pristimantis danae group (2020) and others (e.g., Ruane et al. 2014), gene flow and past introgression with another species can lead traditional tree analysis of gene sequences and genetic distance anal- yses to results identical to those produced by speciation through vicariance. Therefore, populations of the same species, some of which may carry part of the mitochon- drial genome of a closely related species, will be inferred as two divergent lineages, even in sympatry. As discussed by Chan et al. (2020), this has serious implications. Many barcoding and integrative analyses relying on 16S or COI and traditional ML or Bayesian inference tree analyses are reporting cases of divergent mtDNA gene lineages in sympatric populations. These are often considered full species that are readily described and named. In order to detect such cases, it is key to rely on additional sources of evidence that may allow us to critically evaluate whether the distinct genetic lineages are part of two distinct spe- cies (‘integrative taxonomy’; see Padial et al. 2010), and in cases where they are undescribed, they can be classified as a confirmed candidate species (Vieites et al. 2009). On the other hand, mitochondrial divergences in situations of sympatry can be among the strongest possible lines of ev- idence for the species status of the lineages involved, as soon as they are fully concordant with differentiation in unlinked nuclear-encoded markers, bioacoustics, or mor- phology, and this concordance is confirmed with substan- tial sample sizes (Miralles et al. 2024). In our case, support for the existence of two species is provided by differenc- es in the respective advertisement calls, which are most likely explained as mechanisms of reproductive isolation (Kohler et al. 2017), and possibly by a few morphological traits. However, it remains desirable to assess both the ge- nealogies and differences in calls with increasing sampling to refine and corroborate the limits of these two species. Within the Pristimantis danae species group, Padial and De la Riva (2009) were unable to identify any qualita- tive morphological character distinguishing P. danae from P. reichlei, and the statistical differences in morphometric measurements were at least ambiguous, thus emphasizing the cryptic nature of both species. With the description of P. asimus, we add another morphologically cryptic sibling species to this complex of frogs. Although we detected two minor morphological differences between topotypes of P. asimus and P. reichlei (virtual absence versus pres- ence of the outer metatarsal tubercle in preservative; dif- ferent outlines of the canthus rostralis in straight dorsal view of the head), variation in these character states has yet to be studied in additional specimens and across the entire species’ ranges. However, P. asimus, P. danae, and P. reichlei clearly differ by pronounced molecular diver- gence (but see above) and advertisement call differences. Given that morphological differences between P. asimus and P. reichlei are yet to be corroborated, identification of the two species can be considered reliable only with the aid of bioacoustic and/or molecular genetic analyses. Con- sequently, several published records referring to P. reichlei should be reviewed. These include lowland records from the Bolivian departments of Beni and La Paz (Padial et al. Zoosyst. Evol. 100 (2) 2024, 565-582 2004; Padial and De la Riva 2009), records from Manu National Park in southeastern Peru (Villacampa-Ortega et al. 2017), from Serrania de Sira, central Peru (Whitworth et al. 2016), and Brazilian records from the state of Acre (Melo-Sampaio and de Souza 2010; Bernarde et al. 2011; Melo-Sampaio 2015). These and other populations (e.g., Manaus, Brazil; Zimmerman and Bogart 1984) should be studied genetically and/or bioacoustically for their correct allocation to one of the two species, and researchers should check for the respective morphological character states. With the description of P. asimus, we accounted for a more adequate taxonomic resolution of frogs formerly sub- sumed under the name P. reichlei (sensu Padial and De la Riva 2009). However, the high genetic diversification within P. reichlei sensu this work, with several deeply differentiated mtDNA lineages (Fig. 1) and uncorrected 16S p-distances of up to 9.3%, argue for further investigation of these pop- ulations, particularly of their calls, as possibly the name P reichlei as used herein still represents a complex of species. As evidenced by our molecular phylogenetic analysis and the mentioned differences in advertisement calls, the situation of P. asimus and P. reichlei is somehow par- alleled by a second species pair, namely P. danae from Peru and P. aff. danae from Bolivia (see Fig. 1). Current- ly, the respective populations from the montane rainfor- ests of southern Peru and western Bolivia are all treated as P. danae (Padial and De la Riva 2009). However, the genetic differentiation, with uncorrected p-distances in 16S of 9.4% between both major lineages and apparently significant differences in advertisement calls (see above), makes it highly improbable that nominal P. danae from the Kosfipata valley in Peru are conspecific with P. aff. danae from the Yungas de La Paz, Bolivia, although data from the area in between these two regions are lacking (Fig. 9). Solving the taxonomic status of Bolivian P. aff. danae was beyond the scope of this study, and a detailed investigation of P. danae remains necessary. Despite the still substantial limitations in geographic and character sampling outlined above, our understanding of species limits in Pristimantis is rapidly growing. It is now customary in phylogenetic studies of Pristimantis to discover the existence of unnamed species overlooked and confounded across the range with their similar-looking nominal sister species or closely related ones (e.g., Hutter and Guayasamin 2015; Ortega-Andrade et al. 2015; Paez and Ron 2019; Trevisan et al. 2020; Castillo-Urbina et al. 2023; Herrera-Alva et al. 2023). This increasing level of species resolution is a fundamental step toward more rigorous inferences of evolutionary scenarios as well as to attain taxonomic stability (Padial and De la Riva 2021). In contrast to the growing taxonomic knowledge in the P. danae group, there is a disproportional lack of knowl- edge about the ecology and natural history of its species, which remains almost completely unknown. Martinez and Rodriguez (2007) reported on egg clutch size and egg numbers in P danae from Peru. Quintero-Mufioz and Aguayo (2022) described observations on parental care (clutch guarding) and reproduction in P. reichlei 579 from Bolivia, where a collected gravid female laid an egg clutch 1n captivity (without male individuals being pres- ent during capture or in the tank), from which half the eggs developed into froglets. This remarkable observation indicates either the presence of internal fertilization or parthenogenesis in this group of frogs. These two poten- tial explanations deserve further study, as it either consti- tutes a rarely documented case in oviparous anurans (e.g., Townsend et al. 1981) or 1s so far undocumented in frogs (natural parthenogenesis). Such observations and poten- tial phenomena argue for a more careful scientific consid- eration of these inconspicuous, brown-colored frogs. Acknowledgements Collecting permits for specimens in MUBI and CM were kindly granted by SERFOR (#192-2015-SERFOR-DGG- SPFFS), those for specimens in MUSM and ZSM by IN- RENA (#124-2008-INRENA-IFFS-DCB), and SERFOR (#007-2014-SERFOR-DGGSPFFS, — #0406-2017-SER- FOR-DGGSPFFS). Additional specimens held at MUBI were collected with permits #024-2017-SERFOR-DGG- SPFFS (SERFOR) and #001-2013-SERNANP-PNAP (Resolucion Jefatural del Area Natural Protegida Parque Nacional Alto Purtis). We would like to express our highest gratitude to staff members of the Alto Purts National Park (SERNANP), especially to its director, Arsenio Calle, for encouraging and facilitating our work in the area, as well as to those who participated in the expedition to the Sepa- hua River: Viviana Ramos, Carlos Ruelas, Cerilo Lopez, Diego Saavedra, Michel Diaz, Abraham Ramirez (all SER- NANP), Jamil Ponce (PRONATURALEZA), and Julio Flores (SERFOR). Special thanks to Maira Duarte Quiro- ga for assisting during fieldwork and for filming activities at PN Alto Purus and to Ernesto Castillo-Urbina for joint fieldwork at Panguana. We thank Juliane Diller, Carlos ‘Moro’ Vasquez. Modena, and Hibrain Vasquez Panduro for their generous support of our work at ACP Panguana. Wolfgang Bohme, Ursula Bott, Morris Flecks, Claudia Koch (ZFMK), Alexander Kupfer (SMNS), and Jifi Mora- vec (NMP6V) provided access to specimens under their care and/or miscellaneous information. This article great- ly benefited from comments and suggestions provided by Valia Herrera-Alva, Santiago Ron, and Pedro Taucce. References Arroyo SB, Targino M, Rueda-Solano LA, Daza JM, Grant T (2022) A new genus of terraranas (Anura: Brachycephaloidea) from northern South America, with a systematic review of Zachiramantis. 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