Systematics of the Ameerega Rubriventris Complex (Anura: Dendrobatidae) with Descriptions of Two New Cryptic Species from the East-Andean Versant of Peru

Zootaxa 4712 (2): 211–235 ISSN 1175-5326 (print edition) https://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2019 Magnolia Press ISSN 1175-5334 (online edition) https://doi.org/10.11646/zootaxa.4712.2.3 http://zoobank.org/urn:lsid:zoobank.org:pub:FC7A2CB9-5541-42AA-A1CA-6EF6BFBA0940

Systematics of the Ameerega rubriventris complex (Anura: Dendrobatidae) with descriptions of two new cryptic species from the East-Andean versant of Peru

JASON L. BROWN1,7, KAREN SIU-TING2,3, RUDOLF VON MAY4, EVAN TWOMEY5, WILSON X. GUILLORY1, MICHAEL S. DEUTSCH1 & GERMÁN CHÁVEZ6 1Department of Zoology, Southern Illinois University, 1125 Lincoln Drive, Carbondale, IL 62901, USA. 2School of Biological Sciences, Queen’s University Belfast, 19 Chlorine Gardens, Belfast, BT9 5DL, UK. 3Dpto. de Herpetología, Museo de Historia Natural, Univ. Nacional Mayor de San Marcos, Av. Arenales 1256, Lima, Peru. 4Biology Program, California State University Channel Islands, 1 University Drive, Camarillo, CA 93012, USA. 5Laboratórío de Sistemática de Vertebrados, Pontifícia Universidade Católica do Río Grande do Sul (PUCRS), Avenida Ipiranga 6681, Porto Alegre, 90619-900, Brazil. 6División de Herpetología—CORBIDI, Lima, Peru. 7Corresponding author. E-mail: [email protected]

Abstract

We describe two new species of poison frog from central and southern Peru that have been referred to as Ameerega picta, A. hahneli, or A. altamazonica throughout the past thirty years. Our phylogenies generated with genomic data provide strong support that the two new species are successive sisters to two described taxa, A. rubriventris and A. altamazonica, and collectively comprise the Ameerega rubriventris complex. The first new taxon, Ameerega panguana sp. nov., can be distinguished from all other Ameerega by its combination of a unique white venter and an advertisement call of 1–2 notes per second. The second new taxon, Ameerega imasmari sp. nov., is the only cryptically colored Ameerega species that is disttributed across the Fitzcarrald Arch in Southern Peru which possesses a ‘peep’ advertisement call consisting of 3–4 notes per second and a dominant frequency of 4.3–4.5 kHz. Within the Ameerega rubriventris complex, we observed differences between species in their ventral coloration, tympanum diameter, and call, which suggest that these taxa are reproductively isolated from each other.

Key words: dendrobatid frogs, poison frogs, UCE, ultraconserved elements

Introduction

Species richness of dendrobatid frogs in the Andean foothills of Peru is unparalleled, particularly in the genus Ameerega (Lötters et al. 2007; Roberts et al. 2006). Of the 30 recognized species in this genus, 16 are endemic to the east-Andean versant and surrounding lowlands of Peru. Intensified sampling in this region over the past two decades, combined with molecular phylogenetic analyses, has revealed many new species of Ameerega, with eight having been described since 2006 (Brown & Twomey 2009; Lötters et al. 2009; Neves et al. 2017; Serrano-Rojas et al. 2017; Twomey & Brown 2008; Vaz-Silva & Maciel 2011). Ameerega hahneli (Boulenger 1884 “1883”) was described from a locality near Yurimaguas, a city on the lower Río Huallaga in Peru. In the past two decades, specimens from many other localities in Peru, Brazil, and Bolivia have been referred to as A. hahneli, all bearing a similar pattern of a black or brown dorsum with white dorsolateral stripes (e.g. Lötters et al. 1997; Lötters et al. 2005; Roberts et al. 2006; Schulte 1999). While these were initially considered a single widespread species, in recent years several taxa bearing such a color pattern have been recognized as cryptic species and split off from A. hahneli sensu lato. The first of these, Ameerega rubriventris, was considered to be a morph of A. hahneli until 1997, when Lötters et al. formally described this species based on its unique ventral coloration and distinctive vocalization. In 2008, Twomey and Brown elevated another morph of A. hahneli to specific status by describing A. altamazonica, the sister species to A. rubriventris, on the basis of its dis- tinct advertisement call and genetic distinctiveness from other described Ameerega species.

Accepted by M. Vences: 16 Oct. 2019; published: 19 Dec. 2019 211 During the last two decades, numerous additional populations of cryptically colored Ameerega with affinities to A. altamazonica and A. rubriventris have been discovered in the east-Andean versant of southern and central Peru (Fig. 1). For example, Roberts et al. (2006) presented a phylogeny with a single individual from southern Peru (Ivochote, Cusco region) that was recovered as sister to A. altamazonica, which is endemic to central Peru (then considered E. hahneli); both lineages are separated by more than 700 km. Siu Ting (2012) presented a series of phylogenies that included Ameerega samples from central and southern Peru (Río Pachitea and Ivochote, respectively) that were sister to A. rubriventris and A. altamazonica. The frogs from the Río Pachitea area possessed a unique bright-white venter and several authors have questioned their taxonomic status (Twomey & Brown 2008; Siu Ting 2012). Here we evaluate the status of these populations, along with samples from additional localities, to species, using phylogenomic, morphological, and acoustic analyses, describing two new species of Ameerega belonging to the rubriventris complex.

Figure 1. Species in the Ameerega rubriventris species complex: A & B) Ameerega altamazonica, C) A. rubriventris dorsal view, D) A. rubriventris ventral view E) Ameerega panguana sp. nov. (photo T. Ostrowski), F) Ameerega imasmari sp. nov. (holotype)

Type materials are deposited in the Museo de Historia Natural San Marcos, Universidad Nacional Mayor de San Marcos, Lima, Peru (MUSM); Centro de Ornitología y Biodiversidad, Lima, Peru (CORBIDI); Museum of Zool- ogy, University of Michigan, Ann Arbor, MI, USA (UMMZ); and the American Museum of Natural History, New York, USA (AMNH). Measurements were made with mechanical calipers and a micrometer to the nearest 0.01 mm, following Myers (1982) and Brown et al. (2006): snout-vent length (SVL), femur length from vent to lateral surface of knee (FL), tibia length from heel to lateral surface of knee (TL), knee-knee distance with both legs extended straight (KK), foot length from proximal edge of metatarsal tubercle to tip of toe IV (FoL), hand length from proximal edge of metacarpal tubercle to tip of longest finger (HaL), head length from most exposed corner of occipitum to tip of snout (HL), head width between tympanum (HW), body width under axillae (BW), upper eyelid width (UEW), interorbital distance (IOD), horizontal tympanum diameter (TD), horizontal eye diameter (ED), distance from outer corner of eye to tympanum (DET), length of finger I from proximal edge of median palmar tubercle to tip of finger disc (L1F), length of finger II from proximal edge of median palmar tubercle to tip of finger disc (L2F), width of disc of finger III (W3D), and width of finger 3 just below disc (W3F). All samples with a SVL > 16mm were considered adults. Information on tadpoles is based on field observations. Acoustic Analyses. Calling males were recorded with a Zoom H1n Handy Recorder (stored as wav files with a sampling rate 48Khz) and a Sennheiser ME 66-K6 shotgun microphone. Dominant frequency, average note length, and average space between notes are consistent among individuals and rarely change with environmental condi- tions such as temperature in Ameerega (French et al. 2019). Therefore, these variables are considered bioacoustic standards as means for species delimitation of anurans (Köhler et al. 2017). Initial isolation of the vocalization from background noise outside call’s frequency range was conducted in Audacity 2.1.3 (https://audacityteam.org/), using the Noise Reduction tool. The reduction filter was set to 40 dB, 6.00 sensitivity, and 3 bands for smoothing. After processing, eight consecutive notes from the center of each recording were then trimmed and exported as a .wav file. Measurements for each note exported were done in Raven Pro 1.5 (Bioacoustics Research Program 2014), measuring dominant frequency (kHz), note length (seconds), and space between notes (seconds), with the following settings: window type = Hann; window size = 1024 samples; 3 dB filter bandwidth = 67.4 Hz; brightness = 50%; contrast = 50%; overlap (locked) = 85%; hop size (temporal resolution = 3.21 ms); DFT size (locked) = 1024 samples (spectral resolution = 46.9 Hz). The mean was then calculated for each variable. Our final data set consisted of a total of 25 male advertisement calls from four species: 11 A. altamazonica, 5 A. rubriventris, 5 A. panguana sp. nov., and 4 A. imasmari sp. nov. Results were analyzed in R 3.5.3 (R core team 2019) using one-way ANOVAs. Tukey’s post-hoc tests were used for pairwise comparisons since sample sizes were unbalanced. Molecular Analyses. Sequence acquisition. We acquired 25 samples from field work, museums, and collabo- rators, including all known species in the Ameerega hahneli group. For each sample, we extracted genomic DNA with the Qiagen DNeasy Blood and Tissue Kit (Valencia, CA, USA) and quantified yield with a Qubit 3 fluorometer (ThermoFisher Scientific). We sent extracted DNA to RAPiD Genomics (Gainesville, FL, USA), who performed sequence capture and Illumina sequencing of ultraconserved elements (UCEs) in the manner of Faircloth et al. (2012). The samples were enriched with the Tetrapods-UCE-5Kv1 set of 5,472 probes, targeting 5,060 UCE loci. Read quality trimming, sequence assembly, and alignment. For our UCE dataset, we performed most bioinfor- matic steps in the software package PHYLUCE v1.5.0. We trimmed raw reads using Illumiprocessor v2.0.6 (Fair- cloth 2013), a Python wrapper for the program Trimmomatic v0.36 (Bolger et al. 2014). We assembled the trimmed reads with Trinity v2.0.6 (Grabherr et al. 2011), implemented in PHYLUCE. We next mapped contigs to UCE loci using PHYLUCE, retaining loci found at least once in any of our samples. In total we retained 2,635 UCE loci. We then performed per-locus alignments with MUSCLE v3.8.31 (Edgar 2004), implemented within PHYLUCE. We filtered for matrix incompleteness by only retaining loci present in 75% or more of taxa. This filtering step reduced our dataset from 2,635 to 2,015 loci. When mitochondria-rich tissues such as muscle or liver are sequenced for UCEs, off-target sequencing reads originating from mitochondrial DNA are frequently generated as byproducts and are often abundant enough to allow for assembly of complete mitochondrial genomes (mitogenomes) (Raposo do Amaral et al. 2015). We assembled mitogenomes from UCE reads using the MITObim pipeline (https://github.com/chrishah/MITObim). First, we used two published poison frog mitogenomes (Hyloxalus subpunctatus and Phyllobates terribilis) (Machado et al. 2018) to serve as initial bait sequences where reads aligning to these sequences were written to new fastq files. This step

Systematics of the Ameerega rubriventris complex Zootaxa 4712 (2) © 2019 Magnolia Press · 213 was done with Hisat2 v2.1.0 (Kim et al. 2015) using the hisat2 command with the --al-conc and --score-min L,0,-1.0 options. Reads were then normalized to 100X depth with BBNorm (Bushnell 2014), interleaved with khmer v2.1.1 (Crusoe et al. 2015), and assembled as mitogenomes using MITObim v1.9 (Hahn et al. 2013) with the Hyloxalus subpunctatus mitogenome used as the seed sequence. We ran this pipeline on the Ameerega samples until we were able to assemble a complete mitogenome for one sample, which was A hahneli MUSM-244981. At this point, we added this mitogenome to the set of bait sequences for hisat2 and used it as a seed sequence for MITObim, and re-ran the whole pipeline for every sample. In this way, we were able to assemble mitogenomes for the Ameerega samples from an A. hahneli reference mitogenome, facilitating both the baiting and assembly steps. Overall, the mitogenome assemblies for each sample ranged from highly fragmentary (e.g., A. altamazonica 0040, 56% complete) to complete (A. hahneli 0766, MUSM-244981; 16,704 bp). Mitogenomes were aligned using MAFFT v7.0 (Katoh & Standley 2013). Genetic distances were calculated in MEGA v10.0.4 (Koichiro et al. 2013) using the Maximum Composite Likelihood method with gamma distributed rates among sites. Phylogenetic analyses. We performed maximum likelihood (ML) analysis on our UCE dataset as well as species tree methods consistent with the multispecies coalescent. Our ML analyses were performed in IQ-TREE v1.5.5 (Nguyen et al. 2014), using a general time-reversible (GTR) model and assessing support with 10,000 ultrafast bootstrap replicates (Minh et al. 2013). We performed the analysis on a concatenated, unpartitioned alignment of all UCE loci. For the species tree analysis, we used ASTRAL-III v5.6.1 (Zhang et al. 2018) to summarize per-locus gene trees made in IQ-TREE using the GTR substitution model and 1,000 ultrafast bootstrap replicates. Addition- ally, we used IQ-TREE’s -czb option to collapse near-zero branch lengths in the gene trees into polytomies, an approach recommended by Persons et al. (2016) to avoid downstream bias in the ASTRAL-III analysis. We used a mapping file in conjunction with the -a option in ASTRAL-III to assign each sample to a species. ASTRAL-III then collapsed each species into a single tip. For the mitochondrial genomic data, we performed an ML analysis, again using IQ-TREE. We used the Mod- elFinder Plus option in IQ-TREE (-m MFP) to select the best nucleotide substitution model for the data (Kaly- aanamoorthy et al. 2017). The TIM2+I+G4 model was selected on the basis of BIC. We used 100,000 ultrafast bootstrap replicates to assess node support. A third ML analysis was performed on 16s mtDNA that included additional samples, increasing population and phylogeographic sampling (53 terminals vs. 25 terminals in our UCE/mtDNA genome datasets). The phylogenetic analyses of 16s mtDNA matched the parameters used for the mtDNA genomic data. However, we additionally used the topology recovered from the mtDNA genome data as a constraint tree in our 16s mtDNA phylogenetic inference. This required the resulting 16s mtDNA tree to match the tree topology output for the mtDNA genome data and es- sentially placed taxa with only 16s mtDNA data into our mtDNA genome phylogeny.

Results

Phylogenetic results. Our phylogenetic results were consistent between methods and datasets with respect to the placement of the two putative undescribed species. We consistently recovered Ameerega altamazonica and rubriventris as sister species, with A. panguana sp. nov. as sister to this clade, and A. imasmari sp. nov. as sister in turn to the clade including the former three species. We recovered this topology using both species tree (Fig. 2) and maximum likelihood methods (Figs. 2 and 3) in both nuclear UCE and mitogenomic data (Figs. 2 and 3). We never recovered the two undescribed species as sister taxa, thus precluding their description as a single species. Describ- ing them as a single monophyletic species would necessitate lumping them together with A. altamazonica and A. rubriventris. Additionally, our results are consistent with those of another study that analyzed phylogenomic UCE data from all Ameerega species (Guillory et al. 2020) and found the same relationships between the four frogs mentioned above (note that A. panguana sp. nov. and A. imasmari sp. nov. are referred to as A. sp. “Panguana” and A. sp. “Ivochote” in that study). Guillory et al. (2020) recovered the same relationships with much broader taxon sampling, alleviating any concerns that adding taxa to our dataset would alter the topology.

Figure 2. Phylogenetic relationships of A. rubriventris complex and outgroups. A) Species tree of focal taxa using UCE genome-scale molecular markers. B) Maximum-likelihood phylogeny based on 16s mtDNA and a backbone constraint tree from mtDNA genome data (Fig 3). Art by WXG. *Depicts taxa with mtDNA genome data, whose relationships were fixed in our constraint tree. Support values were not included, as they are misleading due to the use of a constraint tree. See Fig. 3 for more rigorous phylogeny. ǂ =holotype, α =paratype

Systematics of the Ameerega rubriventris complex Zootaxa 4712 (2) © 2019 Magnolia Press · 215 Figure 3. Mirror phylogeny of A. rubriventris complex and outgroups. Left. Maximum-likelihood phylogeny reconstructed from UCE genome-scale molecular markers. Right. Maximum-likelihood phylogeny reconstructed from mtDNA genome data (this tree was used a backbone constraint in Fig 2).

Ameerega panguana sp. nov. Panguana poison frog

Phyllobates pictus (non Bibron): Silverstone 1976 p. 40–41, pattern 5 (partim), Texas Natural History Collections 36469, Río Pachitea near Bosque Nacional de Iparia, Nevati (Kansas University 144351–60, Los Angeles County Museum 92413). Dendrobates pictus (non Bibron): Myers, Daly & Malkin 1978, p. 332 (by implication). Ameerega picta (Schlüter 2005) (by implication). Ameerega altamazonica: Twomey & Brown 2008. p. 56–62 (by implication). Ameerega sp. “Panguana”: Siu Ting 2012. p. 20–22 (by implication); Guillory et al. 2020 p. 3–10.

216 · Zootaxa 4712 (2) © 2019 Magnolia Press BROWN ET AL. Holotype. MUSM 26846 (field number KST263), an adult male collected by K. Siu Ting, S. Flechas and A. Craw- ford in Huánuco region, Peru, 4.1 km (via direct line) S of Puerto Inca along Río Pachitea at the Panguana Biologi- cal Research Station, 240 m elevation, 9° 36′ 49.3′′ S, 74° 56′ 7.8′′ W, 20 December 2007 (Fig 4).

Figure 4. A–C) Holotype of Ameerega panguana in life (MUSM 26846), SVL=17.3mm. D–E) Holotype of Ameerega imasmari in life (CORBIDI 20575), SVL= 19.9 mm, see Fig. 1 for additional image.

Paratopotypes. MUSM 26884 (field number KST301), collected by K. Siu Ting on 20 January 2008 (Supp. Fig. 1E). MUSM 24685–24687 collected between 10–16 November 2003 by W. Hödl, A. Amézquita, K. Siu Ting and A. Lima. Paratypes. All from Huánuco region, Peru: MUSM 26963–26964 (field numbers JLB07-608 and 610), 11 km E of Puerto Inca along a trail to Cordillera El Sira, 298 m above sea-level, collected 25 April 2007 by E. Twomey and J.L. Brown, 9° 26′ 50.86′′ S, 74° 48′ 1.91′′ W. MUSM 26985 (field numbers JLB07-786), 4.5 km S of Puerto Sira along a trail to Cordillera El Sira, collected 3 September 2011 by J. L. Brown. 9° 18′ 44.78′′ S, 74° 51′ 5.83′′ W. AMNH 95663–95666 (field numbers CWM-13104-13107), “Río Llullapichis, nr. Llullapichis (town) on Río Pachitea”, C. Toft and R.L. Dressler, February 1975 (Supp. Fig 3A). Etymology. The specific epithet is in honor of the Koepcke family: parents, Drs. Hans-Wilhelm and Maria Koepcke, and their daughter, Dr. Juliane Diller (born as Koepcke). Hans-Wilhelm and Maria founded the Panguana Biological Research Station in 1968, which was named after the undulated tinamou (Crypturellus undulates), an inconspicuous, partridge-sized bird. The Koepcke family developed the Panguana Biological Research Station into a paragon for land conservation and biodiversity preservation. Under Juliane’s guidance, the station continues to serve as a cornerstone for biologists working in the Amazon Rainforest of central Peru and has become the country’s oldest biological station. Hans-Wilhelm, Maria, and Juliane also made substantial scientific contributions to the knowledge of Peruvian ornithology, botany, and mammalogy, respectively. On December 24th 1971, Maria and Juliane were passengers on LANSA flight 508. Midflight, the plane was struck by lighting and broke into several pieces, which crashed into the dense rainforests of the upper Pachitea drainage, ca. 120 km south-west of Pucallpa. Despite suffering a broken collar bone, a deep forearm cut, and a severe concussion, Juliane courageously made her way downriver towards the village of Tournavista—a trip that spanned 11 days. She was the sole survivor. The core of the distribution of Ameerega panguana contains the Panguana Biological Research Station, the site of the plane crash, and Juliane’s trek to Río Pachitea. Definition. A small species of cryptically-colored frog assigned to Ameerega due to the presence of maxillary and premaxillary teeth, the first finger being longer than the second, and basal webbing occurring between toes II–IV. The species can be characterized by the following combination of characters: (1) mean SVL of adult males 19.0 mm (range 16.9–19.7 mm), mean SVL of adult females 19.8 mm (range 16.6–22.1 mm) (Table 1); (2) dorsal skin granular, especially on the back and the dorsal surfaces of the legs, dorsal surfaces of forelimbs lightly granular, flanks and venter non-granular; (3) finger III expanded in adults; (4) absence of lateral fringes and basal webbing on fingers; (5) maxillary and premaxillary teeth present; (6) fingers and toes discs expanded, larger in toes II, III, and IV; (7) basal webbing between toes II–III and III–IV; (8) finger I longer than finger II; (9) toe III length reaches or surpasses the middle of the central sub-articular tubercle of toe IV; (10) metatarsal fold absent; (11) tympanum

Systematics of the Ameerega rubriventris complex Zootaxa 4712 (2) © 2019 Magnolia Press · 217 conspicuous and small; (12) in life, base dorsal coloration of the head, back, and limbs are dark brown mottled in subtle to conspicuous ovoid blotches variable in size and coloration ranging from gray to slate-blue to light olive green to chartreuse yellow; dorsal blotches often merge or are clustered on dorsum; in some individuals the dorsal blotches are mostly absent; (13) oblique lateral stripe absent; (14) usually with white dorsolateral stripes extending from loreal region to groin; (15) white labial stripe present, starting behind nares and ending at forelimb; (16) flanks black to dark brown, most individuals have bright white or cream, occasionally slate-blue, spotting or marbling extending from venter; (17) venter smooth, bright white to bluish-cream, with sparse black reticulation; (18) yellow to red spots present posterodorsally at the insertions of the forelimbs and hindlimbs, and on medial face of tibia; (19) limbs light to dark brown on dorsal surfaces, ventral surfaces of forelimbs are light blue distally, yellow proximally; underside of head pigmented as the venter but often darker (i.e. more black marbling), especially in males. (20) iris dark brown with golden ring around pupil; (21) in preservative, all lighter pigments and flash colors fade to white or gray; (22) advertisement call is a short ‘peep’ repeated 1.1–1.9 times per s for several min; each note is short (0.23 s, mean), with the dominant frequency from 4.9 to 5.5 kHz; (23) presence of exotrophic tadpoles carried to small terrestrial pools, where they are deposited in groups. Diagnosis. Many Ameerega species possess white dorsolateral stripes and brown dorsal coloration, but only A. hahneli sensu stricto can have a similar white venter. However, A. hahneli has an advertisement call consisting of 6–10 notes per second for several minutes (versus 1–2 notes per second in A. panguana; Figs. 5 & 6), the venter is typical finely marbled (coarsely reticulated in A. panguana), the dark pigmentation on flanks is broad with venter coloration barely visible from profile (dark pigmentation narrow, ca. width, and venter coloration extends midway up obliques and is distinctly visible in profile in A. panguana). The second similar species is Ameerega imasmari sp. nov. (described below), in which northern populations are the most similar, but can have golden dorsolateral stripes (white in A. panguana) an advertisement call consisting of 3–4 notes per second with a dominant frequency 4.3–4.5 kHz (versus 1–2 notes per second with a dominant frequency 4.9–5.5 kHz in A. panguana; Table 3, Figs. 5 & 6). Other species similar in appearance to A. panguana include A. boliviana, A. ignipedis, A. simulans, A. petersi, A. pulchripecta (all of which have yellowish or green dorsolateral stripes and blue venters), A. picta sensu stricto (can possess a white venter, but has yellow dorsolateral stripes), A. shihuemoy (pink dorsolateral stripe and a blue venter), A. rubriventris (reddish-orange venter) and A. altamazonica (blue venter instead of a white venter in A. panguana). Ameerega panguana is also similar in appearance to Allobates femoralis, however, it lacks an oblique lateral stripe (present in A. femoralis) and finger I is longer than or finger II (finger II is longer than or finger I in A. femoralis). Measurements of holotype (in mm). SVL 17.3; FoL 7.6; TL 7.9; KK 15.6; FL 6.1; HaL 3.5; HL 4.1; HW 4.5; BW 4.0; UEW 2.9; IOD 2.4; IND 2.3; TD 2.4; ED 2.4; DET 0.5; L1F 2.8; L2F 2.4; W3D 0.5; W3F 0.4. For paratypes see Table 1. Description of adults. Little to no sexual dimorphism is apparent except for males being slightly smaller, pos- sessing vocal slits and a subgular vocal sac. Tongue gray, ovoid, attaching anteriorly. Head widest at jaw articulations, slightly narrower than body in most individuals (head width at tympanum 71.3–126.0% of body width at axillae); head width 21.9–31.6% of SVL. Snout sloping laterally; bluntly rounded dorsally; truncate ventrally. Nares situated and directed posterolaterally to the tip of snout; nares visible from front and below but not from above. Canthus rostralis sloped, slightly rounded; loreal region nearly vertical and slightly concave. Interorbital distance nearly same width of superior upper eyelid. Eye large and prominent, with a maximum diameter of 14.9–17.3% of the snout vent length; pupil rounded and horizontally elliptical. Tympanum circular, partially concealed posterodorsally, lacking tympanic annulus; tympanum width 21.7–40.0% of eye diameter. Supratympanic fold absent. Hands relatively small, length 18.9–29.3% of SVL. Relative length of appressed fingers: III > IV > II ≈ I. Discs moderately expanded, disc on finger III 1.3–1.7 times width of finger below disc. A large, circular outer metacarpal tubercle on median base of palm; a smaller inner metacarpal tubercle on base of finger I; one well developed and prominent subarticular tubercle on fingers I and II, two on fingers III and IV. Hind limbs relatively small, femur 37.1–50.8% of SVL, tibia 40.7–55.1% of SVL. Relative lengths of appressed toes IV > III > V > II > I; first toe short, barely reaching bottom of subarticular tubercle on base of second toe, with unexpanded disc; toes II and III with barely expanded discs (much smaller than finger discs), and toe IV and V with discs expanded (disc 1.3–1.5 times broader than adjacent phalanx). Inner and a smaller outer metatarsal tubercle present, somewhat protuberant with rounded surfaces. One slightly protuberant subarticular tubercle present on toes I and II, two on toes III, IV, and V. Hands and feet lacking supernumerary tubercles, lateral fringes, and webbing. No toe fringes.

218 · Zootaxa 4712 (2) © 2019 Magnolia Press BROWN ET AL. Figure 5. Advertisement calls of: A) Ameerega hahneli from Panguana Biological Research Station at 25°C. B) A. rubriventris from type locality, Boqueron de Padre Abad, Peru at 24°C. C) A. altamazonica from type locality, near Tarapoto, Peru at 23°C. D) A. panguana from type locality at 24°C. E) A. imasmari from type locality at 23°C. Solid gray line depicts 5 kHz.

Systematics of the Ameerega rubriventris complex Zootaxa 4712 (2) © 2019 Magnolia Press · 219 Vocalization. The advertisement call for A. panguana (Table 1, Figs. 5 & 6) is a short ‘peep’ repeated 1.1–1.9 times per second for several minutes. Each note is short (mean 0.23 s), with the dominant frequency from 4.9–5.5 kHz. This single-note advertisement call is given most frequently in the afternoon and evening as males chorus in small groups. We also note a second call type in A. panguana of two to three notes in quick succession (within 10 ms of each other), repeated once every 3-90 seconds. The latter call appears to function as an aggressive or territorial call and is most frequently heard in the early morning and early evening (Schlüter 1980). Distribution and Natural History. Ameerega panguana is distributed throughout the east-Andean versant, Cordillera Sira, and surrounding lowlands of central Peru at elevations of ca. 200–400 m (Fig. 7). This species appears to be widely distributed throughout the region of Huánuco in lower elevations of the Río Pachitea drainage and foothills of Cordillera Sira, extending to both the western and eastern versant. This species is common and can be locally abundant in disturbed forested habitats (locally known as “Purmas”) that surround, or are nearby, small streams, which collectively provide refugia from the midday heat and habitat for reproduction. Ameerega panguana is less commonly found in secondary and old-growth forests. Male A. panguana were frequently observed calling on leaves 0.5-1m above ground. Populations of A. panguana observed at the Panguana Biological Research Station from December to February in 2003 and 2007 were most active between 05:00 to 09:00 and 16:00 to 18:00 (K. Siu- Ting, pers. obs.). This species commonly co-occurs with three other Ameerega species: A. trivittata, A. petersi, and A. hahneli sensu stricto. Ameerega hahneli sensu stricto is more easily detected than A. panguana at most surveyed sites (Schlüter 1980). Conservation status. Following the IUCN Red List criteria 3.1 (IUCN 2012), we suggest A. panguana be listed as Vulnerable (VU) under the following criteria: (1) we estimate its extent of occurrence at 9,370 km2 (as pictured in Fig. 7), and part of this range lies in a protected forest (San Matías San Carlos) and two communal re- serves (El Sira and Yánesha) and a private concession area (ACP Panguana); (2) it occurs in undisturbed habitat and disturbed areas; (3) population sizes are unknown, but assumed to be large given the abundant forested habitats and tentative large range; (4) populations do not appear to be declining; and (5) demand for the pet trade is presumed to be moderate to low.

Ameerega imasmari sp. nov. Riddle poison frog

Phyllobates pictus (non Bibron): Silverstone 1976 p. 40–41, pattern 5 (partim), trail between Satipo and Puerto Ocopa (Museum of Comparative Zoology, Havard, 24433). Dendrobates pictus (non Bibron): Myers, Daly & Malkin 1978, p. 332 (by implication). Epipedobates hahneli: Roberts et al. 2006. Sample “P1” (by implication). Ameerega sp. “Ivochote”: Siu Ting 2012, p. 17–22.; Guillory et al. 2020 p. 3–10.

Holotype. CORBIDI 20575 (field number JLB-WS-033), an adult male collected by Jason L. Brown, Wilson X. Guillory, and Brian Widmer in the region of Pasco, 1.5 km E of Satipo town in small patch of secondary forest adjacent to Río Satipo, Peru, 605 m elevation, 11° 14′ 14.892 S, 74° 37′ 3.576 W, 20 June 2018 (Figs. 1 & 4). Paratypes. UMMZ 2449821–244984 and MUSM-H 35670–35673 collected by Joanna Larson and Consuelo Alarcón, in the Cusco region, Peru, Villa Carmen Biological Station and Reserve, Pillcopata, Peru, 519 m elevation, 12° 53′ 45.01 S, 71° 24′ 21.69 W, collected between 16 January 2016 and 20 February 2016 (Supp. Figs 2E–F, 4A–E) Etymology. The species name is formed as an adjective, derived from the Quechua word ‘imasmari’ which means ‘riddle’. The epithet is in reference to cryptic color pattern of this species, which is similar to that of Ameere- ga hahneli sensu stricto and A. picta sensu stricto, two species that co-occur with A. imasmari. During the last century, the field identification of A. hahneli, A. picta and other similar species has challenged biologists; with the description of A. imasmari sp. nov. there is another choice to distinguish from. The Quechua language is spoken by the Incan people indigenous to Andean Peru and the adjacent lower elevation areas where A. imasmari is known to occur. Definition. A small species of cryptically-colored frog assigned to Ameerega due to the presence of maxillary and premaxillary teeth, the first finger being longer than the second, and basal webbing occurring between toes II- IV. The species can be characterized by the following combination of characters: (1) mean SVL of adult males 18.6

220 · Zootaxa 4712 (2) © 2019 Magnolia Press BROWN ET AL. mm (range 18.3–19.0 mm), mean SVL of adult females 20.7 mm (range 19.8–21.8 mm) (Table 1); (2) dorsal skin granular, especially on the back and the dorsal surfaces of the legs, dorsal surfaces of forelimbs lightly granular, flanks and venter non-granular; (3) finger III expanded in adults; (4) absence of lateral fringes and basal webbing on fingers; (5) maxillary and premaxillary teeth present; (6) fingers and toes discs expanded, larger in toes II, III, and IV; (7) basal webbing between toes II–III and III–IV; (8) finger I longer than finger II; (9) toe III length reaches or surpasses the middle of the central sub-articular tubercle of toe IV; (10) metatarsal fold absent; (11) tympanum conspicuous and small; (12) in life, base dorsal coloration of the head, back, and limbs are dark brown and finely mottled in brown; flanks black to dark brown; (13) oblique lateral stripe absent; (14) usually with white or cream dorsolateral stripes extending from loreal region to groin; (15) labial stripes present that match dorsolateral stripe coloration, starting behind nares and ending at forelimbs; (16) flanks black to dark brown, most individuals have bright white or cream, occasionally slate-blue, spotting or marbling extending from venter; (17) venter smooth, golden to light blue, with dense black marbling; (18) yellow to red spots present posterodorsally at the insertions of the forelimbs and hindlimbs, and on medial face of tibia; (19) limbs light to dark brown on dorsal surfaces, ventral surfaces of forelimbs are light blue distally, yellow proximally. Underside of head pigmented as the venter but often darker (i.e. more black marbling), especially in males; (20) iris dark brown with golden ring around pupil; (21) in preservative, all lighter pigments and flash colors fade to white or gray; (22) advertisement call is a short ‘peep’ repeated 2.1–2.6 times per second for several minutes; each note is short (mean of 0.17 s), with the dominant frequency from 4300–4500 Hz; (23) presence of exotrophic tadpoles carried to small terrestrial pools, where they are deposited in groups. Diagnosis. Though they are not sister species (Fig. 2), southern populations of A. imasmari and northern populations of A. altamazonica are phenotypically similar and confidently distinguishing one from another may not be possible in the absence of genetic data (Figs. 2 & 3). In A. hahneli sensu stricto, the width of each dorsolateral stripe decreases to a very fine stripe on the snout, occasionally disappearing (versus being wide and remaining the same width around the entire snout in A. imasmari), and its advertisement call consists of 6–10 notes per second for several minutes (versus 2–3 notes per second in A. imasmari). Another similar species is Ameerega panguana sp. nov., with some individuals morphologically similar, but with white dorsolateral stripes and venter coloration (versus golden in Satipo populations of A. imasmari), and with an advertisement call consisting of 1–2 notes per second with a dominant frequency of 4.9–5.5 kHz (versus 2-3 notes per second with a dominant frequency of 4.3–4.5 kHz in A. imasmari, Table 3). Other species similar in appearance to A. imasmari include A. boliviana, A. picta sensu stricto, A. petersi, A. simulans, A. ignipedis, A. pulchripecta (all of which have yellowish or green dorsolateral stripes and blue venters), A. shihuemoy (pink dorsolateral stripes), and A. rubriventris (reddish-orange venter instead of a blue or golden venter in A. imasmari). Measurements of holotype (in mm). SVL 19.9; FL 9.8; TL 10.9; KK 19.5; FoL 9.4; HaL 6.0; HL 7.2; HW 6.8; BW 6.4; UEW 2.2; IOD 2.4; IND 2.5; TD 1.5; ED 2.4; DET 0.5; L1F 4.1; L2F 4.0; W3D 0.6; W3F 0.4. For paratypes see Table 2. Description of adults. Little to no sexual dimorphism is apparent except for males being slightly smaller, pos- sessing vocal slits and a subgular vocal sac. Teeth absent; tongue gray, ovoid, attaching anteriorly. Head widest at jaw articulations, slightly wider than body in most individuals (head width at tympanum 85.0–120.0% of body width at axillae); head width 26.8–32.8 % of SVL. Snout sloping laterally; bluntly rounded dorsally; truncate ventrally. Na- res situated and directed posterolaterally to the tip of snout; nares visible from front and below but not from above. Canthus rostralis sloped, slightly rounded; loreal region nearly vertical and slightly concave. Interorbital distance nearly same width of superior upper eyelid. Eye large and prominent, with a maximum diameter of 7.9–8.9% of the snout vent length; pupil rounded and horizontally elliptical. Tympanum circular, partially concealed posterodorsally, lacking tympanic annulus; tympanum width 47.8–62.5% of eye diameter. Supratympanic fold absent. Hands relatively small, length 23.7–29.6% of SVL. Relative length of appressed fingers: III > IV > II ≈ I. Discs moderately expanded, disc on finger III 1.4–2 times width of finger below disc. A large, circular outer metacarpal tubercle on median base of palm; a smaller inner metacarpal tubercle on base of finger I; one well developed and prominent subarticular tubercle on fingers I and II, two on fingers III and IV. Hind limbs relatively small, femur 42.9–49.7% of SVL, tibia 42.9–53.0% of SVL. Relative lengths of appressed toes IV > III > V > II > I; first toe short, barely reaching bottom of subarticular tubercle on base of second toe, with unexpanded disc; toes II and III with barely expanded discs (much smaller than finger discs), and toes IV and V with discs expanded (disc 1.3–1.5 times broader than adjacent phalanx). Inner and a smaller outer metatarsal

Systematics of the Ameerega rubriventris complex Zootaxa 4712 (2) © 2019 Magnolia Press · 221 tubercle present, somewhat protuberant with rounded surfaces. One slightly protuberant subarticular tubercle present on toes I and II, two on toes III, IV, and V. Hands and feet lacking supernumerary tubercles, lateral fringes, and webbing. No toe fringes. Vocalization. The advertisement call for A. imasmari (Figs. 5 & 6) is a short ‘peep’ repeated 2.1–2.6 times per second for several minutes. Each note is short (mean of 0.17 s), with the dominant frequency from 4.3–4.5 kHz. This single-note advertisement call is given most frequently in the afternoon and evening as males chorus in small groups. We also observed a second call type in A. imasmari consisting of three to four notes, occasionally two notes in quick succession (separated by a mean of 13 ms, of silence), repeated once every 2-30 seconds. The latter call appears to function as an aggressive or territorial call and is most frequently heard in the early morning and early evening (Schlüter 1980).

Figure 6. Advertisement calls of the A. rubriventris complex. The plot in center depicts the 2D call space occupied by each species with the x-axis representing advertisement call dominant frequency and y-axis representing individual note length (in seconds). Density plots extend from each axis and depict the relative frequency of each species’ values. Letters depict significant differences among taxa (ANOVA: dominant frequency, df=3, F= 22.55, p<0.001; note length, df=3, F= 8.55, p<0.001). Black lines in density plots on each axis depict median, upper-, and lower-quartiles for each species. The isopleths in center plot depict a 2D kernel density estimation of each species’ call space. White dots represent values for A. altamazonica.

222 · Zootaxa 4712 (2) © 2019 Magnolia Press BROWN ET AL. Distribution and Natural History. Ameerega imasmari is distributed throughout the east-Andean versant and surrounding lowlands of southern Peru, across the Fitzcarrald Arch, at elevations of ca. 200-400 m (Fig 7). This species appears to be sparsely distributed throughout regions Cuzco, Junín, and Pasco, and may extend into Madre de Dios and Ucayali. This species is locally abundant around Satipo (Pasco) and Villa Carmen (Cusco). Elsewhere it appears to be rare, or more likely undetected. This species is common and can be locally abundant in disturbed forested habitats (Purmas) nearby permanent water sources, which collectively provide refugia from the midday heat and habitat for reproduction. Ameerega imasmari does not appear to be commonly found in secondary and old-growth forests. This species commonly co-occurs with six other Ameerega species: A. trivittata, A. shihuemoy, A. simulans, A. picta, A. macero sensu stricto and A. hahneli sensu stricto.

Figure 7. Known distribution of species in the A. rubriventris complex. Each distribution was created by applying a 20 km buffer to a minimum convex polygon surrounding the known localities of each species. An asterisk indicates samples con- firmed with genetic data. Gray dots represent frogs collected as either A. hahneli and A. picta over the past decades; in much of southern Peru it is impossible to positively ascribe these localities to either species based on observation alone. Thus, we have pooled them here to represent the joint distribution of these two similar looking species.

Systematics of the Ameerega rubriventris complex Zootaxa 4712 (2) © 2019 Magnolia Press · 223 19.0 (16.6–22.1) 8.1 (6.9–10) 9.0 (7.3–10.6) 16.8 (15.0–19.1) 8.0 (6.1–10.1) 4.7 (3.2–6.3) 5.1 (3.7–6.3) 5.3 (3.4–6.3) 5.4 (3.0–8.7) 2.9 (1.2–3.4) 2.6 (1.9–3.0) 2.3 (1.9–2.8) 0.9 (0.5–1.1) 2.6 (1.9–3.0) 0.5 (0.2–0.9) 3.0 (2.8–3.5) 2.9 (2.4–3.5) 0.6 (0.4–0.7) 0.4 (0.3–0.4) 5M 6F, AMNH 95666 18.9 7.9 9.1 17.3 8.2 4.8 5.0 6.3 5.0 3.0 2.6 2.3 1.0 2.7 0.5 3.1 3.1 0.5 0.4 F AMNH 95665 21.3 10.0 10.6 19.1 10.1 5.9 4.8 6.2 8.7 3.4 2.9 2.6 1.1 3.0 0.5 3.5 3.5 0.6 0.4 F AMNH 95664 21.5 8.6 10.3 18.4 9.9 6.3 6.1 6.3 6.6 3.4 3.0 2.6 1.1 3.0 0.5 3.5 3.5 0.6 0.4 F 18.6 7.4 9.2 16.2 7.8 4.8 5.8 5.8 5.2 3.0 2.6 2.2 1.0 2.6 0.5 3.0 3.0 0.5 0.4 F 17.1 7.6 7.9 15.6 6.1 3.5 4.1 4.5 4.0 2.9 2.4 2.3 0.9 2.4 0.5 2.8 2.4 0.5 0.4 M 17.3 7.0 7.6 15.5 6.6 3.9 4.6 3.7 3.7 3.0 1.9 1.9 1.0 2.5 0.3 2.8 2.8 0.4 0.4 M MUSM 24687 16.9 6.9 7.7 15.7 7.1 3.2 3.7 3.4 3.0 2.8 2.2 1.9 1.0 2.6 0.2 2.8 2.7 0.5 0.4 M sp. nov.

MUSM 24686 16.6 7.1 7.3 15.0 6.6 3.4 4.1 4.2 3.9 2.8 2.3 1.9 1.0 2.5 0.3 3.0 2.8 0.5 0.4 F Ameerega panguana Ameerega MUSM 24685 18.7 9.5 10.3 17.3 8.4 5.2 5.9 5.9 4.8 1.2 2.8 2.1 0.5 2.3 0.4 2.8 2.8 0.5 0.3 M MUSM 26964 22.1 8.2 9.0 17.2 8.7 5.1 6.3 6.3 7.5 3.3 2.8 2.8 0.8 2.7 0.8 2.8 2.7 0.6 0.4 F MUSM 26963 19.7 9.0 9.7 18.1 8.3 5.3 5.8 6.1 6.5 3.4 2.9 2.3 0.8 2.6 0.9 2.9 2.7 0.6 0.4 M Measurements of the type series 1. a bl e KK HW IOD IND TD ED DET W3D W3F SEX SV L F L T L Fo L Ha L H L B W U EW L 1F L 2F T

224 · Zootaxa 4712 (2) © 2019 Magnolia Press BROWN ET AL. 19.9 (18.3–21.9) 9.3 (9.0–9.8) 9.8 (9.2–10.5) 18.3 (16.6–19.2) 8.7 (8.2–9.4) 5.3 (5.0–6.0) 7.5 (6.9–8.2) 6.2 (5.1–6.9) 5.8 (5.0–6.3) 1.7 (1.5–1.8) 2.3 (2.1–2.6) 2.6 (2.3–2.8) 1.4 (1.1–1.6) 2.5 (2.3–2.7) 0.5 (0.5–0.7) 4.1 (3.4–4.4) 3.9 (3.2–4.2) 0.6 (0.5–0.7) 0.4 (0.3–0.5) 4M 5F, M U SM-H-35673 18.3 9.1 9.4 17.3 8.2 5.0 6.9 6.0 5.0 1.5 2.1 2.3 1.3 2.4 0.5 3.4 3.2 0.6 0.4 M M U SM-H-35672 19.0 9.0 9.5 18.1 8.4 5.2 7.4 5.1 6.0 1.6 2.3 2.4 1.3 2.3 0.5 4.1 3.8 0.7 0.5 M SM-H-35671 U M 18.4 9.0 9.2 16.6 8.6 5.1 7.1 5.8 5.3 1.6 2.2 2.3 1.1 2.3 0.7 3.7 3.6 0.6 0.4 M M U SM-H-35670 19.8 9.8 10.5 19.2 8.7 5.3 7.3 6.2 5.7 1.6 2.3 2.6 1.5 2.4 0.6 4.0 3.9 0.5 0.3 F sp. nov. U MMZ 244984 20.3 9.2 10.2 19.0 9.2 5.3 7.3 6.5 6.3 1.6 2.3 2.7 1.5 2.6 0.6 4.4 4.2 0.7 0.5 F U MMZ 244983 21.2 9.4 9.8 19.2 8.7 5.5 7.9 6.9 6.3 1.8 2.6 2.8 1.5 2.7 0.5 4.3 4.0 0.6 0.3 F Ameerega imasmari Ameerega 244982 20.3 9.7 10.2 18.7 9.4 6.0 8.2 6.6 5.7 1.8 2.3 2.7 1.2 2.3 0.7 4.4 4.1 0.6 0.3 F U MMZ 244981 21.9 9.4 9.4 18.4 8.9 5.2 8.1 6.8 6.3 1.8 2.6 2.7 1.6 2.6 0.5 4.2 3.9 0.5 0.3 F U MMZ CO RB IDI 20575 19.9 9.8 10.9 19.5 9.4 6.0 7.2 6.8 6.4 2.2 2.4 2.5 1.5 2.4 0.5 4.1 4.0 0.6 0.4 M Measurements of the type series 2. a bl e SV L F L T L Fo L Ha L H L B W U EW L 1F L 2F KK HW IOD IND TD ED DET W3D W3F SEX T

Systematics of the Ameerega rubriventris complex Zootaxa 4712 (2) © 2019 Magnolia Press · 225 Conservation status. Following the IUCN Red List criteria 3.1 (IUCN, 2012), we suggest A. imasmari be listed as Least Concern (LC) or Data Deficient (DD) under the following criteria: (1) we estimate its extent of occurrence at 41,690 km2 (as pictured in Fig. 7), and part of this range lies within two national parks (Otishi and Manu), a protected forest (Pui Pui), a natural sanctuary (Megantoni) and a communal reserve (Asháninka); (2) it occurs undisturbed habitat and disturbed areas; (3) population sizes are unknown, but assumed to be large given the abun- dance of forested habitats and tentative large range; (4) populations do not appear to be declining; and (5) demand for the pet trade is presumed to be low.

Table 3. Main call parameters between species. Mean (minimum-maximum, standard deviation) A. altamazonica (11) 0.22 (0.17–0.31, 0.04) 0.39 (0.26–0.65, 0.11) 4463 (4221-–4823, 181) A. imasmari (5) 0.18 (0.15–0.2, 0.02) 0.24 (0.19–0.29, 0.03) 4388 (4313–4500, 92) A. panguana (4) 0.23 (0.2-–0.25, 0.02) 0.45 (0.28–0.63, 0.13) 5223 (4910–5513, 219) A. rubriventris (5) 0.29 (0.26–0.31, 0.02) 0.31 (0.15–0.44, 0.11) 4863 (4700–5082, 150)

Discussion

Several authors suspected the existence of cryptic species that appear to be individuals of A. panguana and A. imasmari. Schlüter (1980) published spectrographs of A. panguana advertisement calls (as Phyllobates pictus) from the upper Río Pachitea drainage, which had notes repeated at a rate of twice per second and that were frequency modulated. Morales (1992) also presented call data for several species of poison frogs from the Río Pachitea region of central Peru. These studies led Haddad & Martins (1994) and De la Riva et al. (1996) to suspect the existence of two cryptic species in this region that resembled A. picta. Silverstone (1976) assigned specimens of Phyllobates pictus to ‘Pattern 5’ that occurred near Satipo, Navati, and Río Pachitea. These appear to be A. imasmari (Satipo and Navati) and A. panguana (Río Pachitea). Roberts et al. (2006) published a sequence of A. imasmari from Ivochote, which was later included in the phylogenies of Twomey & Brown (2008) and Siu Ting (2012). Siu Ting (2012) presented the first phylogenetic results with all four members of the A. rubriventris species complex, whose 16s topology mostly matches our results. Our phylogenies were based on several data types (nuclear genomic data, mtDNA genomes, 16s mtDNA) and several methods (species tree versus gene tree, likelihood and coalescent), and recovered consistent topologies for specific relationships in the A. rubriventris complex (Figs 2 & 3). Ameerega panguana and A. imasmari display high genetic divergence from all described species (greater than 6.8% and 7.2% divergence in genomic mtDNA, Tables 4 & 5). Divergence time estimations suggest that A. imasmari diverged from other members of the A. rubriventris complex approximately 3.9 million years ago. The other three taxa (A. altamazonica, A. rubriventris, and A. panguana) diverged more than 2.2 million years ago (Guillory et al. 2020). Thus, despite the close geographic proximity of the distributions of A. altamazonica, A. rubriventris and A. panguana, there is little evidence of past or recent gene flow among these species.

Table 4. Mean inter-specific genetic distances of mtDNA genome altamazonica rubriventris imasmari panguana hahneli ignipedis rubriventrisntris 0.0518 imasmari 0.0842 0.0835 panguana 0.0691 0.0677 0.0723 hahneli 0.1136 0.1137 0.1160 0.1064 ignipedis 0.1309 0.1315 0.1338 0.1229 0.1437 pongoensis 0.1218 0.1252 0.1222 0.1135 0.1313 0.0938

We observed phenotypic differences between species in their ventral coloration, tympanum diameter, and call differences, which suggest that these taxa are reproductively isolated from each other. All four members of the A. rubriventris complex possess a ‘peep’ call, but each species’ call is unique in terms of combination of the peep note- length, the rate of calling, and the dominant frequency of each ‘peep’. Ameerega imasmari and A. altamazonica have the shortest average call note length of 0.18 s (0.15–0.20) and 0.22 s (0.17–0.31) respectively, whereas the note

226 · Zootaxa 4712 (2) © 2019 Magnolia Press BROWN ET AL. ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** Genbank BSAPE290-12 Genbank BSAPE292-12 Genbank BSAPE291-12 Genbank BSAPE293-12 Genbank BSAPE286-12 ...... continued on the next page mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA genome & nDNA: UCE mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 0040 0181 0196 0194 0816 0020 0384 0298 0219 0692 0807 0805 0809 0044 0766 0767 0768 0769 0458 0506 0215 HS006 HS010 HS007 HS011 HS002 Tarapoto, San Martin, Peru Tarapoto, Chazuta, San Martin, Peru Sisa, San Martin, Peru Chumia, San Martin, Peru Andreas, San Martin, Peru N. San San Franciso, Martin, Peru Bocaron, Huánuco, Peru Bocaron, Huánuco, Peru Bocaron, Huánuco, Peru Puerto Sira, Huánuco, Peru Satipo, Junín, Peru Satipo, Junín, Peru Satipo, Junín, Peru San Martin de Pangoa, Junín, Peru EBVC, Cusco, Peru EBVC, Cusco, Peru Amigos, Madre de Dios, Peru Los Amigos, Madre de Dios, Peru Los Puño, Loreto, Peru Sargento Contamana, Loreto, Peru Huallaga Canyon, Loreto, Peru PBRS, Huánuco, Peru PBRS, Huánuco, Peru PBRS, Huánuco, Peru PBRS, Huánuco, Peru PBRS, Huánuco, Peru altamazonica altamazonica altamazonica altamazonica altamazonica altamazonica rubriventris rubriventris rubriventris panguana imasmari imasmari imasmari imasmari imasmari imasmari hahneli hahneli hahneli ignipedis pongoensis panguana panguana panguana panguana panguana PBRS=Panguana Biological Research Station, EBVC= Villa Carmen Biological Station, **https://doi.org/10.6084/m9.figshare.9983006.v1. Villa PBRS=Panguana Biological Research Station, EBVC= 5. a bl e Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega T

Systematics of the Ameerega rubriventris complex Zootaxa 4712 (2) © 2019 Magnolia Press · 227 Genbank BSAPE287-12 Genbank BSAPE294-12 Genbank BSAPE291-09 Genbank BSAPE289-12 Genbank DQ523038 Genbank EU523038 Genbank BSAPE299-12 Genbank BSAPE303-12 Genbank BSAPE298-12 Genbank BSAPE304-12 Genbank BSAPE295-12 Genbank BSAPE297-12 Genbank BSAPE302-12 Genbank BSAPE296-12 Genbank BSAPE301-12 Genbank BSAPE532-12 Genbank BSAPE319-12 Genbank BSAPE218-12 Genbank BSAPE388-12 Genbank BSAPE318-12 Genbank BSAPE320-12 Genbank BSAPE233-12 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA 16s mtDNA HS003 HS013 MUSM 26985 (KST263) HS005 KS1 KS2 HF007 HF013 HF006 HF014 HF003 HF005 HF012 HF004 HF011 CORBIDI6125 KST663 KST485 CORBIDI5291 KST557 KST629 KST500 PBRS, Huánuco, Peru PBRS, Huánuco, Peru PBRS, Huánuco, Peru PBRS, Huánuco, Peru Ivochote, Cuzco, Peru Ivochote, Cuzco, Peru Ivochote, Cuzco, Peru PBRS, Huánuco, Peru PBRS, Huánuco, Peru PBRS, Huánuco, Peru PBRS, Huánuco, Peru PBRS, Huánuco, Peru PBRS, Huánuco, Peru PBRS, Huánuco, Peru PBRS, Huánuco, Peru Loreto, Peru PBRS, Huánuco, Peru PBRS, Huánuco, Peru Madre de Dios, Peru PBRS, Huánuco, Peru PBRS, Huánuco, Peru PBRS, Huánuco, Peru panguana panguana panguana panguana imasmari imasmari hahneli hahneli hahneli hahneli hahneli hahneli hahneli hahneli hahneli hahneli hahneli hahneli hahneli hahneli hahneli hahneli BL E 5. (Continued) TA Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega Ameerega

228 · Zootaxa 4712 (2) © 2019 Magnolia Press BROWN ET AL. length is almost doubled in A. rubriventris, on average 0.29 s (0.26–0.31), while Ameerega panguana possesses an intermediate ‘peep’ note-length of 0.23 s (0.2–0.25). The species also diverge in the dominant frequencies of their calls, and both A. imasmari and A. altamazonica have significantly lower dominant frequency calls, 4.4 (4.3–4.5) kHz and 4.5 (4.2–4.8) kHz, respectively; whereas A. rubriventris is intermediate, 4.9 (4.7–5.1) kHz, and A. panguana has the highest average dominant frequency, 5.2 (4.9–5.5) kHz. These differences in dominant frequency are also consistent with changes in tympanum size; adult A. imasmari and A. altamazonica possess larger tympana; 1.4 (1.1– 1.6) mm, n=7; 1.3 (1.1–1.4) mm, n=6, respectively) when compared to A. panguana and A. rubriventris (0.9 (0.5–1.1) mm, n=11; 1.0 (0.9–1.1) mm, n=4, respectively). Lastly, despite A. imasmari and A. altamazonica having similar note lengths and dominant frequencies, the two species call at different rates, with A. imasmari calling 2.3 (2.1–2.6) times per second and A. altamazonica calling 1.7 (1.3–1.9) times per second. The latter calling rate is similar to that observed in A. panguana and A. rubriventris, 1.6 (1.1–1.9) and 1.5 (1.3–1.9) times per second, respectively. Future studies with increased sample sizes, temperature data, and social context from across the distributions of each species and call mate- choice experiments are needed to clarify the full extent of the observed call divergence and its effect on reproductive isolation in this group. However, our preliminary data and genomic analyses support the idea that the observed call differences may function as prezygotic reproductive barriers. Lastly, the ventral coloration within the A. rubriventris complex is quite variable between species ranging from red (in A. rubriventris), blue (in A. altamazonica and southern populations of A. imasmari), cream (in A. imasmari in the north nearby Satipo), to white (A. panguana). It remains unclear if ventral coloration diversity is driven by sexual selection, via stochastic processes (i.e. drift), or both. One of the most interesting phylogenetic results of this study was the variable placement of A. ignipedis. In our mtDNA genome tree, A. ignipedis is the sister taxon to A. pongoensis, a species with a similar phenotype (Fig 3), whereas in our UCE nDNA tree, A. ignipedis is the sister taxon to the A. rubriventris complex (Fig 3). One potential explanation for these dramatic phylogenetic incongruencies is from historic hybridization between the ancestor of the A. rubriventris complex and the ancestor of A. pongoensis, which may have resulted in the speciation of A. ignipedis. This adds to the number of putative historic hybridization events observed in this genus (Brown & Twomey, 2009; French et al. 2019).

Acknowledgements

Special thanks to Mark Pepper, Manuel Sanchez Rodriguez, Joanna Larson, Consuelo Alarcón, Thomas Ostrowski, Thorsten Mahn, Walter Hödl, Adolfo Amézquita, Albertina Lima, Andrew Crawford, Roy Santa-Cruz, Valia Her- rera, and Sandra Flechas for assistance in the field, for collecting associated materials, or for sharing photographs and observations of these species. KS would like to thank Juliane Diller for allowing fieldwork in Panguana Re- search Station and Moro Modena and family for their kind hospitality. We thank César Aguilar (MUSM), Greg Schneider (UMMZ), Alison Davis Rabosky (UMMZ), and Dan Rabosky (UMMZ) for allowing access to specimens under their care. Collection of Ameerega specimens from Los Amigos was funded by a fellowship from the David and Lucile Packard Foundation (to D. Rabosky). Permits were issued by the Servicio Nacional Forestal y de Fauna Silvestre (R.D.G. 120-2012-AG-DGFFS-DGEFFS, R.D.G. 029-2016-SERFOR-DGGSPFFS, R.D.G. 405- 2016-SERFOR-DGGSPFFS, R.D.G. 116-2017-SERFOR-DGGSPFFS, N 002765-AG-INRENA, N 061-2003-IN- RENA-IFFS-DCB, N 050-2006-INRENA-IFFS-DCB, N 067-2007-INRENA-IFFS-DCB, N 083-2017-SERFOR/ DGGSPFFS, N 134-2007-INRENA-IFFS-DCB), Contrato de Acceso Marco a Recursos Genéticos (002-2013-AG- DGFFS-DGEFFS, 359-2013-MINAGRI-DGFFS-DGEFFS), and the administration of Manu National Park (06- 2013-SERNANP-PNM-JEF).

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Systematics of the Ameerega rubriventris complex Zootaxa 4712 (2) © 2019 Magnolia Press · 231 Supplemental Figure 1. Ameerega hahneli and A. panguana from Huánuco region, Peru. A–C) Ameerega hahneli from Panguana Biological Research Station, KST633, KST485, KST234 respectively. D) Ameerega panguana from Panguana Biological Research Station, KST292. E) Comparison of Ameerega panguana (left, KST301) and A. hahneli from Panguana Biological Research Station (right, KST303). F–G) Ameerega panguana observed near a small stream near La Amistad, Río Pichis (Photos: T. Ostrowski). H) Ameerega panguana from San Matías San Carlos (Photos: Roy Santa Cruz). I) Ameerega panguana from secondary forest 2 km SE of Puerto Sira in the foothills of the Cordillera Sira.

232 · Zootaxa 4712 (2) © 2019 Magnolia Press BROWN ET AL. Supplemental Figure 2. A–B) Larvae of Ameerega panguana from La Amistad, Río Pichis, Huánuco region (Photos:T. Ostrowski). C) Ameerega panguana from Iparia (the east versant of Cordillera El Sira), Huánuco region. D) Ameerega imasmari from near Satipo, Junín region, Peru. E–G) Ameerega imasmari from Villa Carmen Biological Station and Reserve near Pill- copata, Cuzco region, Peru. (Photos: Joanna Larson) H) Habitat of A. imasmari from type locality nearby Satipo. I) A. hahneli sensu stricto with mottled dorsal patterning guarding embryos, CICRA Station, Madre de Dios (Photos: Valia Herrera).

Systematics of the Ameerega rubriventris complex Zootaxa 4712 (2) © 2019 Magnolia Press · 233 Supplemental Figure 3. A) Specimens from type series of Amereega panguana: AMNH 95664, 95665, 95666, 95663 (from left to right). B) Specimens from type series of Ameerega imasmari: UMMZ 244982, C) UMMZ 244986, D) UMMZ 244987, E) UMMZ 244989.

234 · Zootaxa 4712 (2) © 2019 Magnolia Press BROWN ET AL. Supplemental Figure 4. Specimens from type series of Ameerega imasmari. A) MUSM-H-35672, B) MUSM-H- 35670, C) MUSM-H-35671, D) MUSM-H-35667, E) UMMZ 244981, F) CORBIDI 20575 (holotype).