Research Article |
Corresponding author: Jörn Köhler ( joern.koehler@hlmd.de ) Academic editor: Uwe Fritz
© 2023 Ernesto Castillo-Urbina, Miguel Vences, César Aguilar-Puntriano, Frank Glaw, Jörn Köhler.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Castillo-Urbina E, Vences M, Aguilar-Puntriano C, Glaw F, Köhler J (2023) Contributing to the taxonomic inventory of green-colored rain frogs: A new species of the Pristimantis lacrimosus group (Anura: Strabomantidae) from the southern Cordillera Azul, central Peru. Vertebrate Zoology 73: 1047-1061. https://doi.org/10.3897/vz.73.e109309
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Abstract
We studied the taxonomic status of a population of Pristimantis from the southern Cordillera Azul, Departamento Huánuco, central Peru. A phylogenetic analysis based on the mitochondrial 16S rRNA gene revealed that it represents a lineage within the Pristimantis lacrimosus species group, being the closest relative of a species of uncertain taxonomic status from a lowland rainforest in central Peru (Panguana), and P. pulchridormientes from the Tingo Maria National Park. However, the focal lineage is divergent from all nominal species in the P. lacrimosus group for which respective data are available by >7.9% uncorrected pairwise distance in the 16S rRNA gene fragment. An integrative taxonomic approach, including morphological and bioacoustic analyses, provided multiple lines of evidence for the focal specimens belonging to an unnamed evolutionary lineage at the species level that we describe and name herein. The systematics of Peruvian populations associated with the P. lacrimosus group are discussed, particularly highlighting problematic taxa with uncertain taxonomic status and unknown relationships. We point to scientific challenges and actions needed to achieve a better taxonomic resolution of this species-rich clade of frogs.
Amphibia, bioacoustics, integrative taxonomy, molecular genetics, morphology, systematics
Within the species-rich Neotropical anuran genus Pristimantis, 17 species groups are currently recognized (
As demonstrated by the most recent progresses in understanding the phylogenetic relationships and taxonomic identity of various populations, the systematics of the P. lacrimosus group are far from being clarified. There are several documented cases of misidentifications and confusion among species, as the result of either the presence of intra-specific morphological variation, and/or inter-specific morphological crypsis (e.g.,
Summarizing the current knowledge, we can expect 16 species of the Pristimantis lacrimosus group to occur in Peru: P. achupalla, P. aureolineatus, P. acuminatus, P. deyi, P. galdi, P. lacrimosus, P. mendax, P. olivaceus, P. padiali, P. pardalinus, P. pluvialis, P. pseudoacuminatus, P. royi, P. schultei, P. tantanti, and P. pulchridormientes (
The Pristimantis lacrimosus group has been demonstrated to exhibit great diversity in the montane forests of the Andean slopes (e.g.,
Fieldwork was conducted in November 2019 in different areas of the departments Huánuco and Ucayali in central Peru (see also
Collected specimens were euthanized using an overdose of 5% lidocaine gel applied on ventral surfaces of the individuals (
Morphometric measurements (in millimetres) were taken by the first author with a digital caliper to the nearest 0.1 mm. Measurements taken and used throughout the text are:
SVL, snout–vent length;
TL, tibia length;
HW, head width (at level of angle of jaws);
HL, head length (from posterior margin of lower jaw to tip of snout);
IOD, interorbital distance;
ED, horizontal eye diameter;
E–N, eye–nostril distance (straight line distance between anterior corner of orbit and posterior margin of external nares);
IND, inter-narial distance;
TD, horizontal tympanum diameter;
HandL, hand length (from proximal border of outer metacarpal tubercle to tip of third finger);
FootL, foot length (from proximal border of inner metatarsal tubercle to tip of fourth toe).
Fingers and toes are numbered preaxially to postaxially from I–IV and I–V, respectively. Lengths of fingers I and II were determined by adpressing the fingers against each other. For character state definitions, we followed
Vocalizations in the field were recorded using an Olympus LS-05 digital recorder with built-in microphones at 44.1 KHz and 16-bit resolution and saved as uncompressed WAVE format. Recordings were re-sampled at 22.05 kHz and 32-bit resolution and computer-analyzed using the software Cool Edit Pro 2.0. We obtained frequency information through Fast Fourier Transformation (FFT; width 1024 points) at Hanning window function. Spectrograms were produced with Blackman window function at 256 bands resolution. Sensitive filtering was applied to remove background sounds, only to frequencies outside the prevalent bandwidths of calls. Temporal measurements are provided as range with mean ± standard deviation in parentheses. Analysis, description and terminology follow the recommendations of
Our genetic analysis aimed at identifying lineage divergence among focal lineages of Pristimantis. For representative taxon sampling, we largely followed an approach as described by
We sequenced a DNA fragment of the mitochondrial 16S rRNA gene (16S) from tissue samples of newly collected specimens. DNA was extracted using a standard salt extraction protocol, Polymerase Chain Reaction (PCR) carried out with primers 16SAr-L (5’-CGCCTGTTTATCAAAAACAT-3’) and 16SBr-H (5’-CCGGTCTGAACTCAGATCACGT-3’) (Palumbi et al. 1991), and the PCR products then directly sequenced on automated DNA sequencers by LGC Genomics (Berlin, Germany). All new DNA sequences were submitted to GenBank (accession numbers OR725032‒OR725036).
To estimate evolutionary lineage divergence within the P. lacrimosus species group we used a Maximum Likelihood (ML) phylogenetic approach. Sequences were exported to AliView v1.26 (
Our morphological examination of the focal specimens from the southern Cordillera Azul revealed a combination of qualitative traits being unique within the P. lacrimosus species group. The color pattern with a yellow belly in life, smooth dorsal skin lacking tubercles, coarsely areolate skin on venter, absence of a rostral papilla, absence of dorsolateral folds, presence of a discoidal fold, presence of a tympanum and tympanic annulus, and an almost truncate snout in lateral profile distinguish the focal samples from all nominal species allocated to the P. lacrimosus group (see
Known advertisement calls of species in the P. lacrimosus group are often rather similar in general character (e.g.,
Our Maximum Likelihood tree (Fig.
Maximum Likelihood phylogenetic tree of selected samples of Pristimantis, representing the P. lacrimosus species group and related taxa, inferred from an alignment of 1637 nucleotides of the mitochondrial 16S rRNA gene. Three samples of Yunganastes were used to root the tree (not shown for better graphical presentation). Numbers at nodes are bootstrap values in percent (2000 pseudoreplicates; not shown if <50%), followed by SH-aLRT values (1000 replicates; not shown if <50) as calculated with IQ-TREE. The taxon name is followed by the GenBank accession number, or voucher number for newly produced sequences (bold terminals), and the sample locality. Inset photo depicts the holotype of P. loeslein sp. nov. (
Uncorrected pairwise distances of the focal samples from central Peru when compared to the closest relatives in the same major clade are as follows: 4.7% to Pristimantis sp. from Panguana; 7.9% to P. pulchridormientes; 11.2% to P. aureolineatus; 10.6–10.7% to P. pluvialis; 10.2% to P. cf. olivaceus from northern Bolivia; 10.9–11.4% to P. zorro; and 9.4–14.4% to P. lacrimosus.
In summary, our analyses provided different lines of evidence that the focal samples represent a divergent evolutionary lineage. The molecular genetic data reconstruct it (along with the unnamed Panguana population) sister to P. pulchridormientes, but it differs from this species by a high genetic distance, longer note duration in advertisement calls, and various morphological characters (see Diagnosis below). We therefore formally describe and name this lineage in the following as new species.
The species is named after the German family Löslein (from Erlangen and Munich) in recognition of supporting taxonomic research and species conservation in Peru through the BIOPAT initiative. The name is treated as a noun in apposition.
A species assigned to the genus Pristimantis and in particular to the P. lacrimosus species group based on molecular phylogenetic relationships and overall morphological similarities. Pristimantis loeslein sp. nov. is characterized by the following combination of characters: (1) Skin on dorsum smooth; skin on flanks with small scattered low subconical tubercles; skin of venter coarsely areolate; discoidal fold present, weakly defined; dorsolateral folds absent; (2) tympanic membrane and tympanic annulus present, round, its length about 2/5 of eye diameter; its upper edge slightly concealed by inconspicuous supratympanic fold; (3) snout acuminate in dorsal view, slightly rounded to almost truncate in profile, lacking distinct rostral papilla; (4) interorbital region flat, broader than upper eyelid; upper eyelid with 1–3 distinct small subconical tubercles; cranial crests absent; (5) dentigerous processes of vomers low, widely separated, posteromedial to choanae; (6) adult males with prominent subgular vocal sac and vocal slits; adult males with inconspicuous nuptial pads on the proximal dorsal part on thumb; (7) first finger shorter than second; all fingers long, discs broadly expanded, rounded to truncate in outline, with circumferential grooves; all fingers bearing a hyperdistal tubercle; fingers with narrow, weakly defined lateral fringes; fingers without webbing; (8) few low ulnar tubercles present or absent; knee and heel tubercles absent; (9) tarsal fold not evident; tarsus bearing row of few elongated low tubercles; (10) inner metatarsal tubercle prominent, oval, approximately 4× the size of round conical outer metatarsal tubercle; few low supernumerary plantar tubercles; (11) all toes with hyperdistal tubercles; toes with narrow lateral fringes; traces of basal webbing present between toes; discs expanded, rounded to truncate in outline, slightly smaller than those on fingers, bearing circumferential grooves; toe V distinctly longer than toe III; (12) in life, predominantly yellowish green to olive-green dorsal coloration, with darker markings; throat, chest and venter yellow; iris copper to bronze with black reticulation; (13) SVL of adult males 20.5–23.3 mm (n = 4); (14) advertisement call consisting of a single tonal note with 98–110 ms duration, repeated in long call series.
Pristimantis loeslein is most closely related to the nominal species P. aureolineatus, P. lacrimosus, P. pluvialis, P. pulchridormientes, and P. zorro (see Fig.
Species without analyzed 16S rRNA sequence data allocated to the P. lacrimosus group (sensu
Although currently not associated with the P. lacrimosus species group, P. rhodostichus shares several characters with other species in the group (see
Adult male (SVL 21.0 mm) in good state of preservation (Fig.
Morphological measurements (in mm) of the male type specimens of Pristimantis loeslein sp. nov. For abbreviations see Materials and Methods.
Holotype | Paratype | Paratype | Paratype | |
|
|
|
|
|
SVL | 21.0 | 20.5 | 22.3 | 23.3 |
TL | 10.7 | 9.5 | 10.9 | 10.9 |
HL | 8.0 | 7.6 | 9.5 | 8.2 |
HW | 7.8 | 7.5 | 8.2 | 8.0 |
ED | 2.7 | 2.7 | 2.8 | 3.0 |
TD | 1.1 | 1.1 | 1.2 | 1.3 |
IOD | 2.9 | 3.0 | 3.2 | 3.0 |
IND | 1.8 | 1.7 | 2.0 | 2.2 |
E‒N | 2.3 | 2.0 | 2.5 | 2.5 |
HandL | 6.4 | 6.4 | 6.4 | 6.5 |
FootL | 9.4 | 8.6 | 9.4 | 9.5 |
In life (Fig.
After two years in preservative (Fig.
For variation in measurements among the four males of the type series, see Table
The type locality is an area of evergreen montane rainforest of moderate height, growing on moderate to steeply sloped terrain (Fig.
Calls were recorded on 8 November 2019, around 20:00 h, at the type locality, Abra la Divisoria, southern Cordillera Azul, 1650 m a.s.l., Departamento Huánuco, Peru (air temperature estimated by subjective feel app. 18°C). The recorded individuals could not be observed calling, but searching at the spot of sound emission revealed individuals of P. loeslein, and other individuals of P. loeslein were seen emitting the same call, leaving little doubt that recorded calls actually correspond to this species. The advertisement call consists of a single tonal note of short duration, repeated in long call series at rather long and irregular inter-call intervals (Fig.
Audiospectrogram and corresponding oscillogram at 4000 ms time scale showing three advertisement calls of Pristimantis loeslein sp. nov. recorded on 8 November 2019 at Abra La Divisoria, 1650 m a.s.l., Departamento Huánuco, Peru. Below an oscillogram showing one single call (the center call from oscillogram above) at 400 ms time scale. Recording band-pass filtered at 1500–6000 Hz.
Known so far only from the type locality in the southern Cordillera Azul (Fig.
Map of central Peru with adjacent regions in northern and southern Peru, showing the known distribution of seven nominal and two candidate species of the Pristimantis lacrimosus species group as discussed in the text. In most cases, symbols refer to the respective type localities. Black lines within Peru do refer to the political borders of respective departments.
As apparent from most recent publications (e.g.,
The identity and relationships of Pristimantis mendax, with its type locality in southern Peru (Colonia Pistipata, SE of Huyro, 1820 m a.s.l., Departamento Cuzco) and paratypes from different low and high elevation localities in the Departamentos Ayacucho, Cuzco, and Huánuco (
Living individual (FGZC 6300) of a second unidentified species of the Pristimantis lacrimosus group occurring at Panguana, Departamento Huánuco, Peru, 260 m a.s.l. (no sequence data available). Morphologically, this species is apparently most similar to P. royi, described from a nearby locality (
Another taxonomic uncertainty concerns Pristimantis olivaceus, with its type locality situated at the lower Andean slopes of central Bolivia (Chapare region, Departamento Cochabamba), that has also been recorded from southern Peru (Pakitza, PN Manu, Departamento Madre de Dios) in the original description (
In addition to these difficulties, the presumed loss of the type specimens of P. royi (
These cases, together with the fact that other nominal species lacking phylogenetic information may actually be part of the P. lacrimosus group (e.g., P. rhodostichus), exemplify the challenging taxonomy of these frogs in Peru and highlight the need for future integrative taxonomic studies, including topotypic samples.
The closest nominal relative of the new species described herein, Pristimantis loeslein, was revealed by our analysis to be P. pulchridormientes occurring at Tingo Maria National Park (uncorrected p-distance 7.9%). Notably, these two species share a similar habitat, altitudinal distribution, and occur in close geographical proximity, with their respective type localities being only around 28 km airline distance apart (see
We are indebted to the Servicio Nacional Forestal y de Fauna Silvestre (SERFOR) for dealing with our research proposals and issuing research and collection permits (RGD 071-2020-MINAGRI-SERFOR-DGGSPFFS and D000067-2021-MINAGRI-SERFOR-DGGSPFFS). We thank Juan C. Chaparro for information on populations from southern Peru. William E. Duellman and Linda Trueb provided access to the collection under their care, and José M. Padial shared his photos of several type specimens of Pristimantis. Juan M. Guayasamin, Edgar Lehr and an anonymous reviewer provided comments that improved the manuscript. Financial support was provided to the first author by the BIOPAT initiative (www.biopat.de).