Pristimantis achupalla sp. n., a new minute species of direct-developing frog (Amphibia, Anura, Strabomantidae) inhabiting bromeliads of the montane forest of the Amazonian Andes of Puno, Peru

Introduction

The neotropical amphibian genus Pristimantis (Anura, Terraranae, Strabomantidae) is the most speciose genus of terrestrial vertebrates; Pristimantis frogs are often important components of ecological communities in terms of both species composition and individual abundance (Padial, Grant & Frost, 2014). Among approximately 569 currently described species (Frost, 2021), 142 species of Pristimantis are currently known from Peru (Chávez, García-Ayachi & Catenazzi, 2021). New species are described every year, mainly from the Andes (Lehr et al., 2017; Catenazzi & Lehr, 2018).

The remarkable diversity of this terrestrial group may be associated with the success of their reproductive mode. By reproducing through direct development, individuals can be independent of water, and they can colonize new terrestrial niches (Hedges, Duellman & Heinicke, 2008). It is thought that this ability to colonize and establish populations away from water promoted the rapid accumulation of population genetic isolation across the landscape (Elmer, Davila & Lougheed, 2007; Fouquet et al., 2007). Our knowledge of the diversity of this genus is thus still incomplete, and the rate of species discovery remains high throughout the Andes, including in eastern Peru, where several species have recently been described (e.g., Shepack et al., 2016; Chávez & Catenazzi, 2016; Padial et al., 2016; Catenazzi & Lehr, 2018).

Here we describe a new species of Pristimantis found in the bromeliads of a cloud forest remnant in the Cordillera de Carabaya (Fig. 1), in the southern Peruvian department of Puno, along a tributary of the Inambari River. This specimen does not resemble any of the previously described species of Pristimantis. Furthermore, molecular data indicate that the sequence of a fragment of 16S rRNA does not match any known sequence of Pristimantis. The species is most closely related to an undescribed species from cloud forests in Cusco, Peru. On the basis of these lines of evidence, here we describe the species.

Materials & methods

During a rapid survey at a primary cloud forest (2,220 m a.s.l.) near Thiuni village, Ollachea Valley, department of Puno in southern Peru, on 14 August 2017, we collected specimens of Pristimantis frogs by opportunistic inspections in epiphytes bromeliads. The individuals sampled were photographed alive and euthanized with 20% benzocaine. Samples of tissues were extracted from some specimens and preserved in 96% ethanol for molecular analysis. Specimens were fixed in 10% formalin and stored in 70% ethanol. We deposited specimens in the herpetological collection at Centro de Ornitologia y Biodiversidad (CORBIDI) and Herpetology Department, Museo de Biodiversidad del Peru (MUBI). We conducted this research under collection permits (permits #0292-2014-MINAGRI-DGFFS/DGEFFS, #029-2016-SERFOR-DGSPFS) issued by The Dirección General Forestal y de Fauna Silvestre, Ministerio de Agricultura y Riego. The protocol of collection and animal care of our research was approved by the Institutional Animal Care and Use Committee of Florida International University (protocol #18-009).

We wrote the definition (morphological characterization of the new species) following the standard format of Duellman & Lehr (2009). The description follows Lynch & Duellman (1997). We follow Heinicke et al. (2018) for family placement and taxonomy classification. Under a stereomicroscope and using a caliper, we measured the following variables to the nearest 0.1 mm, as described by Duellman & Lehr (2009): snout–vent length (SVL), head length (HL, obliquely from angle of jaw to tip of snout), head width (HW, at level of angle of jaw), eye diameter (ED), tympanum diameter (TD), interorbital distance (IOD), upper eyelid width (EW), internarial distance (IND), and eye–nostril distance (E–N, straight line distance between anterior corner of orbit and posterior margin of external nares), forearm length (FAL), hand length (HAL, distance from proximal edge of palmar tubercle to the tip of Finger III), tibia length (TL), foot length (FL, distance from proximal margin of inner metatarsal tubercle to tip of Toe IV). Fingers and toes are numbered preaxially to postaxially from I–IV and I–V respectively. We compared the lengths of toes III and V by adpressing both toes against Toe IV; lengths of fingers I and II were determined by adpressing the fingers against each other. We used field notes and photographs we took in the field to describe coloration in life. In addition to the type series of the new species, we obtained morphological data from the original descriptions and examined specimens of related congeners (Appendix I) for comparisons of diagnostic characters. See Table 1 for morphometric measurements of the type series.

We used phylogenetic analyses to examine relationships between the new species and other species of Pristimantis. We sequenced a fragment of the 16S rRNA mitochondrial gene. 16S rRNA is the gene most commonly amplified gene for anuran and Terrarana taxonomy (Fouquet et al., 2007; Hedges, Duellman & Heinicke, 2008; Padial & De la Riva, 2009; Vences et al., 2005), and the gene with the largest number of available sequences on Genbank. We used liver tissues from two paratypes of the new species to obtain DNA sequences (accession codes MW724812 and MW724813; Appendix II). Additionally, we obtained DNA sequences from samples of described and undescribed Pristimantis species collected in southern Peru (Cusco and Madre de Dios) (Shepack et al., 2016). Finally, we downloaded sequences from Genbank (Appendix II) of morphologically similar species in the putative P. lacrimosus group (sensu Arteaga, Yáñez-Muñoz & Guayasamin, 2013; Rivera-Correa & Daza, 2016; 2020; Ron et al., 2020), along with representative samples of other species groups of Pristimantis. We followed standard protocols of DNA extraction, amplification, and DNA sequencing (Hedges, Duellman & Heinicke, 2008; Catenazzi & Ttito, 2016). For amplification, we used the primers16Sar (forward: 5′-CGCCTGTTTATCAAAAACAT-3′) and 16Sbr (reverse: 5′-CCGGTCTGAACTCAGATCACGT-3′) (Palumbi et al., 2002) and the following polymerase chain reaction thermocycling conditions (Proflex thermal cycler, Applied Biosystems): one cycle at 96 °C/3 min; 35 cycles at 95 °C/30 s, 55 °C/45 s, 72 °C/1.5 min; and one cycle at 72 °C/7 min. We used Exosap-IT (ThermoFisher) to purify PCR products. MCLAB (South San Francisco, CA, USA) performed sequencing. We used AliView version 1.26 (https://ormbunkar.se/aliview/) to align sequences with the MUSCLE v3.8.31 alignment program (Edgar, 2004) and trimmed sequences to a length of 1623 bp. Our analysis included 86 terminals representing 61 nominal species within Pristimantis and one terminal as outgroup Niceforonia dolops. We inferred the best evolution model using ModelFinder under the BIC criterion (Kalyaanamoorthy et al., 2017). We employed a Maximum Likelihood (ML) as optimality criterion to infer a molecular phylogeny generated using W-IQ-TREE web server (Trifinopoulos et al., 2016) based on the best model inferred TIM2 + F + I + G4. To assess node support, we made 1,000 replicates for the SH-like approximate likelihood ratio test (SH-aLRT, Guindon et al., 2010) and 1,000 ultrafast bootstrap (UFBoot2, Hoang et al., 2018). We considered that nodes with SH-aLRT values ≥80 and ultrafast bootstrap values ≥95 had strong support, also (SH-aLRT ≤ 50% and UFBoot ≤ 70% slightly overestimate the true probability, Minh, Nguyen & Von Haeseler, 2013). We also estimated pairwise, uncorrected genetic distances (p-distances) for 16S rRNA between the new species and the closest related species (i.e., Pristimantis amaguanae, P. bromeliaceus, P. moro, Pristimantis sp. QCAZ 60398, Pristimantis sp. CORBIDI 12183) and the closest geographical species of P. lacrimosus species group (i.e., P. cf. olivaceus, P. pluvialis, and P. pulchridormientes) using the software MEGA 7 (Kumar, Stecher & Tamura, 2016).

Results

Generic placement

We tentatively allocate P. achupalla sp. n. to the putative P. lacrimosus group sensu Arteaga, Yáñez-Muñoz & Guayasamin (2013) due to the presence of rostral tubercle, acuminate snout profile, distinct tympanic membrane, moderately long limbs, Finger I shorter than Finger II, and expanded digital disks. Furthermore, according to our phylogeny (Fig. 2), the new species is nested in a clade that includes the species of the Pristimantis lacrimosus group (clade B, sensu Rivera-Correa & Daza, 2016). The topology for the sampled Pristimantis includes nodes whit moderate to strong support and is similar to topologies of previous phylogenies (Arteaga, Yáñez-Muñoz & Guayasamin, 2013; Padial, Grant & Frost, 2014; Rivera-Correa & Daza, 2016; Rivera-Correa & Daza, 2020; Ron et al., 2020). The new species is the sister taxon of a putative new species with high support, and both are related to recently described species P. amaguanae from Ecuador (see Ron et al., 2020), and the known species P. moro from Panama. Furthermore, these taxa form a small clade that is sister to a small clade composed of P. bromeliaceus and one undescribed species P. sp. QCAZ62940 (Fig. 2). We found substantial genetic distances (uncorrected p-distances of 0.05–0.11, Table 2) between P. achupalla and the most closely related species. Pristimantis achupalla is most closely related to one undescribed Pristimantis, from Yanamayo, Kosñipata Valley in Cordillera Vilcanota approximately 145 km NE of the type locality (paratypes MUBI 17604 05, 16S uncorrected p-distance: 0.05). Other closely related species are P. amaguanae (0.06–0.07) Pastaza, Ecuador; P. moro (0.07–0.09) from Chilibre and Darien, Panama; P. sp. QCAZ 60398 (0.07) Bombuscaro, Zamora Chinchipe, Ecuador; P. bromeliaceus (0.10–0.11) from Morona and Zamora respectively, Ecuador.

Table 2:

Uncorrected p-distances for the fragment of the mitochondrial 16S rRNA gene.

Taxon		1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16
1	P. bromeliaceus QCAZ 16699 ECU
2	P. bromeliaceus QCAZ 62940 ECU	0.04
3	P. sp. QCAZ 60398 ECU	0.10	0.12
4	P. moro AJC1753 PAN	0.09	0.10	0.09
5	P. moro AJC1860 PAN	0.08	0.09	0.07	0.05
6	P. amaguanae QCAZ39274 ECU	0.20	0.20	0.20	0.07	0.07
7	P. sp. CORBIDI12183 PER	0.10	0.10	0.07	0.10	0.08	0.08
8	P. achupalla sp. n. MUBI 17604 PER	0.10	0.11	0.07	0.09	0.07	0.06	0.05
9	P. achupalla sp. n. MUBI 17605 PER	0.11	0.12	0.07	0.09	0.07	0.07	0.05	0.00
10	P. pulchridormientes CORBIDI15566 PER	0.16	0.16	0.09	0.11	0.11	0.08	0.12	0.12	0.14
11	P. pulchridormientes CORBIDI15563 PER	0.16	0.16	0.09	0.11	0.11	0.08	0.12	0.12	0.14	0.00
12	P. pluvialis CORBIDI11862 PER	0.11	0.11	0.10	0.12	0.09	0.08	0.10	0.11	0.11	0.07	0.07
13	P. pluvialis CORBIDI16695 PER	0.17	0.17	0.10	0.12	0.09	0.08	0.10	0.12	0.17	0.11	0.11	0.00
14	P. cf. olivaceus NMP-P6V74067 BOL	0.18	0.17	0.10	0.12	0.10	0.10	0.12	0.12	0.17	0.12	0.12	0.08	0.10
15	P. cf. olivaceus CORBIDI17473 PER	0.15	0.15	0.10	0.11	0.09	0.09	0.11	0.11	0.15	0.10	0.10	0.06	0.10	0.05
16	P. cf. acuminatus CORBIDI17676 PER	0.11	0.10	0.09	0.10	0.09	0.08	0.12	0.11	0.11	0.08	0.08	0.07	0.07	0.04	0.03

DOI: 10.7717/peerj.11878/table-2

Note:

Comparisons between P. achupalla and the closest related species, and other closest geographical species within P. lacrimosus group.

Pristimantis achupalla sp. n.

urn:lsid:zoobank.org:act:D9EFDD3D-F0C3-40D8-BEBF-71372B43D42D; urn:lsid:zoobank.org:pub:88FB4422-449E-45BF-B2E3-0A30D00090B8.

Holotype

CORBIDI 18736, an adult male from near Thiuni, 13.67603 S, 70.46588 W (WGS84), 2,225 m a.s.l., Distrito Ollachea, Provincia Carabaya, Departamento Puno, Peru, collected by A. Catenazzi and A. Ttito on 14 August 2017 (Figs. 1–3).

Paratypes

Three total: two adult males, CORBIDI 18730 and, MUBI 17604, and one juvenile, MUBI 17605 collected at the type locality by A. Catenazzi and A. Ttito on 14 August 2017 (Fig. 4).

Coloration of holotype in preservative

Dorsal surfaces of head brown, dorsal surface of body is cream, with slightly dark brown regions around scapulae (Fig. 4). Interorbital bar as a brown blotch that extends anteriorly and posteriorly as a mask; canthus rostralis dark brown. Upper eyelids dark bluish. Dorsal surfaces of hind limbs with dark transverse bars blurred. Iris dark gray. Throat, chest, and belly pale white to cream; ventral surfaces of thighs the color is pale green; plantar and palmar surfaces and tips of digits pale green, tubercles darker gray.

Variation

The SVL of paratypes (all males) are (in mm): CORBIDI 18737 = 11.7, MUBI 17604 = 10.0, MUBI 17605 = 10.4 (Table 1). A brown to a cream interorbital bar is present in CORBIDI 18737, MUBI 17604 and MUBI 17605 (Figs. 5–6). CORBIDI 18737 and MUBI 17604 possess faint brown barring on hind legs. Dorsal skin is generally rough whit scattered tubercles (CORBIDI 18737 just rough), indicating that skin texture might be a variable trait (Guayasamin et al., 2015).

Etymology

The name of the new species is a Quechua noun, used in apposition, “achupalla” = bromeliads, in reference to the use of bromeliads as its microhabitats.

Distribution, natural history and threats

We found four specimens of Pristimantis achupalla by inspecting epiphytic bromeliads in a relictual cloud forest along the Macusani-San Gabán road (Ollachea Valley), which connects the Peruvian Altiplano to the Amazon rainforest and the interoceanic highway between Peru and Brazil. The relictual montane forest covers a small ridge and is accessible through a maintenance trail for a high-voltage power line (Catenazzi & Ttito, 2018, 2019). We found the holotype and male CORBIDI 18737 in the same bromeliad, a medium-sized bromeliad on a tree branch overhanging the power maintenance trail, along with a third specimen that escaped capture during the inspection. Following the discovery of these individuals at 16 h 20, we haphazardly searched several bromeliads along the trail and around the high-voltage power pole until 17 h 00, and found the two additional specimens (MUBI 17604 and MUBI 17605), as well as a juvenile of Gastrotheca cf. testudinea. On the basis of microhabitats associated with all four specimens, and the flattened body shape of head and body allowing frogs to slide between leaves, we hypothesize that P. achupalla are bromeliad specialists. Searching bromeliads, we surveyed the leaf litter and found Pristimantis platydactylus, and the two recently described species Psychrophrynella glauca and Noblella thiuni (Catenazzi & Ttito, 2018, 2019). We suggest classifying P. achupalla sp. n. as “Data Deficient” according to the IUCN red list categories and criteria (IUCN Standards & Petitions Committee, 2019) based on the limited information on its geographic range and population abundance, and due to the known threats of deforestation, small-scale agriculture, as well as hydropower, road construction, and similar large-scale projects affecting the upper Ollachea Valley.

Discussion

The previously defined Pristimantis lacrimosus species group was non-monophyletic group (Rivera-Correa & Daza, 2016, 2020; Gonzalez-Duran et al., 2017; Ron et al., 2020), but its redefinition by Ron et al. (2020), who included Pristimantis lacrimosus in their matrix, corresponds with clade B, sensu Rivera-Correa & Daza (2016); and includes P. acuminatus, P. amaguanae, P. aureolineatus, P. bromeliaceus, P. crucifer, P. ecuadorensis, P. enigmaticus, P. eremitus, P. galdi, P. jorgevelosai, P. lacrimosus, P. limoncochensis, P. mendax, P. mindo, P. nankints, P. nyctophylax, P. olivaceus, P. omeviridis, P. ornatissimus, P. pluvialis, P. pulchridormientes, P. romeroae, P. schultei, P. subsigillatus, P. tantanti, P. zorro (Ron et al., 2020; Rivera-Correa & Daza, 2020). Currently, the distribution of P. lacrimosus species group comprehends Central America, Colombia, the Guianas, Ecuador, Peru and Bolivia. We associated a priori P. achupalla with the P. lacrimosus group for sharing some synapomorphies proposed by Padial et al., (2104), such as a dorsoventrally compressed head and body, upper eyelids bearing large tubercles, and presence of a rostral papilla. Our phylogenetic analysis suggests with moderate to strong support that P. achupalla is more closely related to one undescribed Pristimantis species from the montane forest of Cusco, and both species are related to species of the P. lacrimosus group sensu Ron et al. (2020). Furthermore, we obtained a topology that is relatively consistent with previous studies (Rivera-Correa & Daza, 2016, 2020; Gonzalez-Duran et al., 2017; Ron et al., 2020). However, some nodes present low support, probably because it is based on only one genetic marker, in addition to the known challenges with alignment of 16S rRNA. Furthermore, existing gaps in the distribution of P. lacrimosus group continue to affect the reconstruction of the evolutionary history of this group (Rivera-Correa & Daza, 2020).

On the other hand, the analysis of genetic distances (uncorrected p-distances) shows that Pristimantis achupalla has moderate genetic distance (p-distance, 0.05–0.06 respectively, Table 2) with Pristimantis sp. from Cusco, and P. amaguanae from Pastaza, Ecuador (Ron et al., 2020). Although there is no fixed threshold for delimiting species, genetic distances among closely related strabomantid species often exceed 0.03, and for 16S, a distance of 0.03 is a reasonable criterion to identify putative new species (Fouquet et al., 2007; Chávez, García-Ayachi & Catenazzi, 2021). Pristimantis achupalla, Pristimantis sp. and P. amaguanae also show low morphological differences and share similar mircrohabitat and presumably ecological niche, but different geographical space. Other closely related species are P. moro and P. bromeliaceus, which are larger than P. achupalla. In contrast, other species such as P. cf. olivaceus, P. pluvialis and P. pulchridormientes, show higher genetic distances between 10% and 17%. Pristimantis achupalla occurs in remnant of montane forest at 2225 m a.s.l. in the Ollachea valley, part of the Cordillera de Carabaya. Recent herpetological surveys in the Cordillera de Carabaya have discovered several new species and allocated species to new genera (see De la Riva et al., 2017; Catenazzi & Ttito, 2018; Catenazzi & Ttito, 2019). Moreover, P. achupalla together with P. olivaceus (Peru and Bolivia) represent the most southern distribution of the P. lacrimosus group. Most suitable areas for terraranas in the Andes of Peru and Bolivia have yet to be explored, and there are good reasons to suspect that many new species will be found (Lehr & Catenazzi, 2009; De la Riva et al., 2017; Catenazzi & Ttito, 2019). Futhermore, the Cordillera de Carabaya appears to host substantial beta diversity of Holadeninae and Strabomantinae, suggesting an intriguing and underappreciated evolutionary history for this groups in southern Peru.

Conclusions

We describe a new species of terrestrial breeding frog in the genus Pristimantis. We justify the generic location-based on morphological similarity and phylogenetic analyses. The new species P. achupalla is nested within the P. lacrimosus group and is closely related to an undescribed species P. sp. from Peru, P. amagunae, P. sp. from Ecuador, and P. bromeliaceus. These species of minute, bromeliad-living frogs form a clade well supported in the phylogeny. Additionally, several meristic traits distinguish P. achupalla from similar species in P. lacrimosus group. We discuss the difficulty of justifying the position of P. achupalla within groups such as P. lacrimosus, but we also confirm the evolutionary unicity of P. achupalla which supports our taxonomic decision to describe it as new species. Our contribution increases the knowledge of the rich diversity of terrestrial breeding frogs found at high elevations on the eastern slopes of the Cordillera de Carabaya.

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