A New Genus of Terrestrial-Breeding Frogs (Holoadeninae, Strabomantidae, Terrarana) from Southern Peru

: We propose to erect a new genus of terrestrial-breeding frogs of the Terrarana clade to accommodate three species from the Province La Convenci ó n, Department of Cusco, Peru previously assigned to Bryophryne : B. ﬂammiventris , B. gymnotis , and B. mancoinca . We examined types and specimens of most species, reviewed morphological and bioacoustic characteristics, and performed molecular analyses on the largest phylogeny of Bryophryne species to date. We performed phylogenetic analysis of a dataset of concatenated sequences from fragments of the 16S rRNA and 12S rRNA genes, the protein-coding gene cytochrome c oxidase subunit I (COI), the nuclear protein-coding gene recombination-activating protein 1 (RAG1), and the tyrosinase precursor (Tyr). The three species are immediately distinguishable from all other species of Bryophryne by the presence of a tympanic membrane and annulus, and by males having median subgular vocal sacs and emitting advertisement calls. Our molecular phylogeny conﬁrms that the three species belong to a new, distinct clade, which we name Qosqophryne , and that they are reciprocally monophyletic with species of Microkayla . These two genera ( Qosqophryne and Microkayla ) are more closely related to species of Noblella and Psychrophrynella than to species of Bryophryne . Although there are no known morphological synapomorphies for either Microkayla or Qosqophryne , the high endemism of their species, and the disjoint geographic distribution of the two genera, with a gap region of ~310 km by airline where both genera are absent, provide further support for Qosqophryne having long diverged from Microkayla . The exploration of high elevation moss and leaf litter habitats in the tropical Andes will contribute to increase knowledge of the diversity and phylogenetic relationships within Terrarana.


Introduction
Terrestrial-breeding frogs of the high Andes display an impressive degree of evolutionary convergence [1][2][3][4]. Such convergence is associated with life in the cloud forest and high-Andean grassland. Frogs in many genera of Terrarana have evolved strikingly similar body forms [4,5], typically a small, compact body with very short legs and feet, short arms and hands, loss of toe pads and discs, head wider than long, small eyes directed anterolaterally, and, in many groups, reduction or loss of tympanic structure and function [3]. The high similarity of body forms has delayed obtaining a

Materials and Methods
We are familiar with most described species of Bryophryne, which we have seen in the field or inspected in collections. We provide a complete list of examined specimens in Appendix 1. We used the literature (i.e., original species descriptions) for species whose specimens we could not examine. We have described the advertisement calls of B. gymnotis and B. mancoinca [14,17], and have heard and provided a short description of the call of B. flammiventris [15]. We refer readers to the original publications for details on recording methods.
We combined DNA sequences available from GenBank with sequences from newly collected tissues to generate molecular phylogenies of Bryophryne and closely related Holoadeninae taxa (Table 1). We considered sequences for a fragment of the 16S rRNA gene (16S), a fragment of the 12S rRNA gene (12S), the protein-coding gene cytochrome c oxidase subunit I (COI), the nuclear protein-coding gene recombination-activating protein 1 (RAG1), and the tyrosinase precursor (Tyr). All taxa selected for our comparisons belong to the subfamily Holoadeninae [1,23,24].

Materials and Methods
We are familiar with most described species of Bryophryne, which we have seen in the field or inspected in collections. We provide a complete list of examined specimens in Appendix A. We used the literature (i.e., original species descriptions) for species whose specimens we could not examine. We have described the advertisement calls of B. gymnotis and B. mancoinca [14,17], and have heard and provided a short description of the call of B. flammiventris [15]. We refer readers to the original publications for details on recording methods.
We combined DNA sequences available from GenBank with sequences from newly collected tissues to generate molecular phylogenies of Bryophryne and closely related Holoadeninae taxa (Table 1). We considered sequences for a fragment of the 16S rRNA gene (16S), a fragment of the 12S rRNA gene (12S), the protein-coding gene cytochrome c oxidase subunit I (COI), the nuclear protein-coding gene recombination-activating protein 1 (RAG1), and the tyrosinase precursor (Tyr). All taxa selected for our comparisons belong to the subfamily Holoadeninae [1,23,24].

Laboratory Work
We followed protocols of extraction, amplification, and sequencing of DNA previously used for terrestrial-breeding frogs [1,20,22]. For the focal taxa (the three species members of the new genus), we extracted DNA from tissue samples obtained from six specimens collected in the field (two specimens per species). We also obtained DNA sequences from seven specimens in five other species of Bryophryne, and two specimens representing two species in other genera (Noblella and Psychrophrynella), and the remaining sequences are legacy data from GenBank.
We extracted DNA from liver tissue preserved in 70% ethanol by using a commercial extraction kit (IBI Scientific, Dubuque, IA, USA). We used selected primers (Table 2) to amplify DNA from each gene using the polymerase chain reaction (PCR) [22,32]. We obtained sequence data by running purified PCR products in an ABI 3730 Sequence Analyzer (Applied Biosystems), except sequences of B. mancoinca and B. phuyuhampatu, which we shipped to MCLAB (San Francisco, CA) for sequencing. We deposited all new sequences in GenBank (Table 1). We provide updated names of 86 terminals included in the analysis for 314 GenBank sequences.

Molecular Phylogenetic Analyses
We inferred the phylogenetic relationships among taxa through analysis of concatenated DNA sequences of the five gene fragments (16S, 12S, COI, RAG1, Tyr). We used Niceforonia dolops to root the tree. We aligned sequences with Geneious R6, v. 6.1.8 (Biomatters 2013), using the built-in Geneious Aligner program. We then used PartitionFinder, v. 1.1.1 [36] to select the best partitioning scheme and substitution model for each gene using the Bayesian information criterion (BIC). The best partitioning scheme included the following six subsets (best fitting substitution models are in parentheses): partition subset 1 includes 12S and 16S sequences (GTR + I + G), partition 2 is the first codon position of COI (SYM + G), partition 3 is the second codon position of COI (F81), partition 4 is the third codon position of COI (HKY + G), partition 5 includes the first and second codon positions of RAG together with the first and second codon positions of Tyr (HKY + I + G), and partition 6 includes the third codon position of RAG together with the third codon position of Tyr (K80 + G).
We used MrBayes, v. 3.2.0 [37] to infer a molecular phylogeny for the 106 terminals and 2632 bp concatenated partitioned dataset (16S, 12S, COI, RAG1, Tyr). We performed an MCMC Bayesian analysis that included two simultaneous runs of 10 million generations, sampled once every 1000 generations. Each run had one "cold" chain and three heated chains, and the burn-in was set to discard 25% samples from the cold chain. Upon completion of the MCMC Bayesian analysis, the average standard deviation of split frequencies was 0.003916. We used Tracer version 1.5 [38] to examine the effective sample sizes (ESS), to verify convergence, and to verify that the runs reached stationarity. The observed effective sample sizes were satisfactory for all parameters (ESS > 200). Lastly, we used FigTree v. 1.4.2 [39] to visualize the majority-rule consensus tree and assess node support (based on posterior probability values). The electronic version of this article in portable document format will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) and the associated information can be viewed through any standard web browser at http://zoobank.org/urn:lsid:zoobank.org:pub:0B8FFBEE-96AA-46E1-BA6F-541DC9FA73BF.

Results
We recovered a phylogenetic tree ( Figure 2) that was largely congruent with previous analyses [2,24]. However, our tree recovered three species of Bryophryne not previously included in phylogenetic analyses (B. gymnotis, B. flammiventris, and B. mancoinca) as a clade that is sister to the clade containing all species of Microkayla. Thus, species of Microkayla, instead of other species of Byrophryne, share the most common shared ancestor with B. gymnotis, B. flammiventris, and B. mancoinca. The presence of large, external tympanic membrane and annulus, and males with a median subgular vocal sac and production of vocalizations, immediately distinguishes the newly recognized genus from all other species of Bryophryne. At least four species of Bryophryne were described as having small, barely visible (under the skin surface) tympanic membranes and annuli (B. bustamantei, B. quellokunka, B. tocra, B. wilakunka), but their external appearance does not look that different from the other species of Bryophryne known to lack a visible tympanic membrane [2,14,18]. One of these species, B. bustamantei was described as producing a short whistle, but there is no recording of the call nor voucher associated with a call [18]. The distribution range of B. bustamantei overlaps with that of B. gymnotis in the cloud forest near Abra Málaga [14,18,40], and thus it is possible that the call of B. gymnotis was erroneously associated with males of B. bustamantei. There also seems to be some problems identifying specimens of this species, as shown by our phylogeny where specimens identified as B. bustamantei by one of us do not group with sequences from one of the paratypes of B. bustamantei (MHNC 6019). Finally, our inferred phylogeny suggests that there are at least seven additional putative new species of Bryophryne, Noblella, and Psychrophrynella (Figure 2), and confirms previous findings of cryptic species diversity particularly in leaf litter, cloud forest frogs in the Noblella/Psychrophrynella clade [22]. These putative new species, similarly to most known species of high-elevation Holoadeninae [4], are highly endemic and known from single localities (or, around those localities, from within a narrow elevational range in the same valley, [22]). Of special interest among the putative new species, Psychrophrynella MUSM 27619 is the first specimen of the Noblella/Psychrophrynella lineage known from the Vilcabamba range. We propose to erect the new genus Qosqophryne gen. n. to accommodate Bryophryne gymnotis, B. flammiventris, and B. mancoinca. Several lines of evidence support the idea that Qosqophryne is distinct from its sister genus Microkayla. The molecular phylogeny indicates there is a degree of divergence comparable to that observed between other genera of strabomantid frogs (Figure 2). Our molecular analyses show strong support for the divergence of Microkayla and Qosqophryne gen. n. The lack of geographic overlap between the two genera, with a gap region of~320 km by airline where both genera are absent, further supports this divergence by preventing recent gene flow among species of both genera ( Figure 3). Furthermore, several glaciated peaks, including the massive Ausangate mountains and associates peaks of the Cordillera de Vilcanota, are interspersed along this gap region of 320 km.  Similarly to recent phylogenies [28,41], we found that Noblella is not monophyletic: the species from southern Peru along with species of Psychrophrynella form a clade that is sister taxon to Microkayla + Qosqophryne, whereas the species of Noblella from northern Peru and Ecuador are closely related to "Eleutherodactylus bilineatus" and Barycholos (Figure 2). Because the type species N. peruviana occurs in southern Peru, and the most similar species sequenced to date N. thiuni is part of the Noblella/Psychrophrynella clade [28], our findings support the hypothesis that Noblella occurs only in southern Peru and northern Bolivia, and that species from northern Peru and Ecuador belong to a different genus [28,41]. Furthermore, our tree suggests that species of Noblella and Psychrophrynella belong to the same lineage, as supported by the respective type genera sharing several morphological traits [2,5,20,28,42]. Therefore, the two possibilities are that some species of Noblella have been misidentified as Psychrophrynella (and vice versa), or that Psychrophrynella is a junior synonym of Noblella. We will not be able to resolve the taxonomic uncertainty associated with Noblella and Psychrophrynella until we obtain DNA sequences from the respective type species N. peruviana and P. bagrecito [2,19,20,28].
There are no known morphological synapomorphies for Qosqophryne, but the three known species share the following traits (Table 3): (1) males with median subgular vocal sac produce whistle-like tonal calls composed of 1-4 short notes; (2) tongue ovate; (3) skin on venter smooth to weakly areolate (in Q. flammiventris); (4) inner tarsal fold absent. Four other genera of Holadeninae occur south of the Apurimac canyon, a proposed biogeographic barrier for high-elevation terrestrial breeding frogs [13][14][15]. Bryophryne differs from Qosqophryne in lacking an externally visible tympanum, and having males without vocal sac and not emitting vocalizations [2,12,16]. Oreobates have head about the same width as body, smooth venter, subarticular and supernumerary tubercles large, conical or subconical, projecting, and range in snout-vent length from 20-63 mm [1,5]. Noblella and Psychrophrynella have smooth venter, elongated tongue, two prominent metatarsal tubercles, and in most species facial masks and/or a tarsal fold-like, sigmoid tubercle [2,19,20,28]. Qosqophryne is most similar to its sister genus Microkayla. Putative synapomorphies of Microkayla are a rounded tongue, areolate belly, and absence of prominent metatarsal tubercles [2]. It is presumed that all species of Microkayla vocalize, and known calls consist of a simple, short whistle-like tonal note [2,4]. Qosqophryne differs from most Microkayla in having (except for Q. flammiventris) fingers and toes with lateral fringes (absent in Microkayla), and having (except Q. flammiventris) dentigerous processes of vomers (absent in Microkayla). Future examination of osteological characters, for example through computed tomography, might help identify such characters, and resolve the condition of the tympanic apparatus in the three genera Bryophryne, Microkayla and Qosqophryne. Etymology. The name refers to the city of Cusco, using the spelling Qosqo which more closely reflects the name in Quechua. Qosqo is used in apposition with phryne, from the greek for "frog". Thus, the name for the new genus alludes to the geographic distribution of the three known species in the Peruvian Department of Cusco.
Distribution, natural history, and conservation. The three species of Qosqophryne occur within a region of~150 km 2 in the upper montane forests and grasslands of the Cordilleras de Urubamba and Cordillera de Vilcabamba, Provincia La Convención, Department Cusco, Peru. These frogs inhabit cloud forests, elfin forests, montane scrub and humid grasslands (puna) from 3270 to 3800 m a.s.l. Similar to other regions in the high Andes, these habitats and their amphibian communities are threatened by pasture burning, climate change and associated expansion of agricultural activities, deforestation, and the fungal disease chytridiomycosis [43,44]. Although chytridiomycosis has caused the collapse of montane frog communities at several sites in Departamento Cusco [45,46], terrestrial-breeding frogs have generally declined the least, and several species challenged in experimental infection trials appears to resist or tolerate infection [47]. Protection of natural habitats will benefit conservation of these frogs. Two of the three species occur within naturally protected areas: Q. gymnotis within the Área de Conservación Privada Abra Málaga, and Q. mancoinca within Machu Picchu Historic Sanctuary.
Remarks. The new genus is distinguished from all species of Bryophryne by the presence of tympanum and tympanic annulus, and median subgular vocal sacs in males. Furthermore, males of all three species of Qosqophryne are known to emit advertisement calls (unknown in all species of Bryophryne, except possibly for B. bustamantei). We have described the advertisement calls of Q. gymnotis and Q. mancoinca [14,17]. One of us (LM) has recorded the advertisement call of a male Q. flammiventris (MUBI 13365) at the type locality, and this call is composed of 3-4 short notes (~15-35 ms duration) at dominant frequency~3000 Hz. Females of Q. gymnotis attend clutches of 14-16 eggs [39], but unattended clutches of up to 19 eggs have also been found [14].
The new genus Qosqophryne is supported by our molecular phylogeny, the most complete to date covering three mitochondrial and two nuclear gene fragments, as well as most described species of Bryophryne and Microkayla. Despite the absence of known synapomorphies for the sister clades Microkayla and Qosqophryne, we are confident that our proposed arrangement reflects the evolutionary history of these organisms, and yet still takes into consideration taxonomic stability [48]. There is strong support (bootstrap probabilities) at the node where Microkayla and Qosqophryne diverge, and the relative branch lengths leading to their respective living species is similar, or in some cases exceed the branch lengths separating other genera within Terrarana (e.g., Euparkerella and Holoaden, or Barycholos and the "northern clade" of Noblella).

Discussion
Our study integrating molecular, acoustic and morphological information justifies the erection of the new genus of strabomantid frog Qosqophryne. The molecular phylogeny we inferred, the most complete phylogeny to date in terms of terminal sampling for genera of Holoadeninae [2,24], provides strong support for this new genus forming a sister clade to Microkayla. Furthermore, our phylogeny confirms taxonomic uncertainty regarding the genera Noblella and Psychrophrynella [2,19,20], suggests the presence of several undescribed species of Noblella and Psychrophrynella, and generalizes the idea of high species endemism in high elevation Andean strabomantids [2,4,[19][20][21][22]49].
Morphological synapomorphies for the new genus Qosqophryne have not been recognized, and there does not appear to be a unique combination of meristic traits to distinguish all species of Microkayla from species of Qosqophryne. However, there are some characteristics that help distinguish the two genera. Some of the traits present in Qosqophryne but absent in Microkayla are fingers and toes with lateral fringes, venter smooth (areolate in Microkayla), and presence of dentigerous processes of vomers (but absent in Q. flammiventris). The structure of the advertisement call, when known, appears to be similar in both genera, i.e., a whistle-like call, but composed of a single note in Microkayla vs. 2-4 notes in Qosqophryne (except for Q. gymnotis). There is limited information on parental care, but it appears that females attend clutches in Q. gymnotis [39], whereas males attend clutches in M. illimani and M. teqta [50,51]. Similarly to Qosqophryne, females attend clutches in B. cophites [52], B. hanssaueri and B. nubilosus (Catenazzi, pers. obs.). However, we lack natural history information from most species of strabomantid frogs, and thus any generalization on parental care is premature.
In support of our proposed new genus, there is a wide gap, both in terms of airline distance and the highly dissected topography, in the distribution range of species of Microkayla and Qosqophryne. These are all highly endemic, terrestrial-breeding frogs most likely characterized by extreme low vagility, as suggested by their patchy distribution in cloud forests and grasslands. All species of Microkayla occur from extreme southern Peru (Department Puno) to the western limits of department Santa Cruz in central Bolivia (Serranía Siberia), whereas the three species of Qosqophryne occur in the Vilcabamba mountain range in the Peruvian Department of Cusco. The gap of 320 km by airline between the southernmost locality of Qosqophryne (Q. gymnotis; −13.07558, −72.38201) and the northernmost locality of Microkayla (M. boettgeri) overlaps with the distribution range of Bryophryne. At the northern limit, B. abramalagae and B. bustamantei are marginally sympatric with Q. gymnotis, whereas at the southern limit, B. wilakunka (Ayapata, Puno, −13.85294, −70.31450) occurs~80 km NW of the type locality of M. boettgeri (Phara, Puno, −14.16247, −69.66250). Although many species in these genera of Holoadeninae are likely "micro-endemic", researchers have seldom invested much effort in documenting the distribution ranges of most species, and it is possible that some of these species occur more widely than presently known. Therefore, currently five genera of Holoadeninae occur in the tropical Andes south of the Apurimac canyon in Cusco, Puno and northern Bolivia: Bryophryne, Psychrophrynella and Qosqophryne in the Vilcabamba mountain range; Bryophryne, Noblella and Psychrophrynella in the Vilcanota range; Bryophryne, Microkayala, Noblella and Psychrophrynella in the Carabaya range, and Microkayala south of the Apolobamba range.