﻿A new Bolitoglossa (Amphibia, Caudata, Plethodontidae) from the Cordillera Oriental of Colombia

﻿Abstract A new salamander species of the genus Bolitoglossa is here described from the cloud forests of the western slopes of the Cordillera Oriental of Colombia, in the Cundinamarca department. The most salient characters of this new species are its numerous maxillary and vomerine teeth, its moderate webbing on hands and feet, its short and robust tail, and its chromatic variation. Based on molecular analyses this new species is assigned to the adspersa species group and its status established as the sister species of B.adspersa, with which it was previously confused. Lastly, the distribution, natural history, and conservation status of the new species are discussed.


Specimen collection and fieldwork
The holotype and most of the paratypes were collected within vereda Roble Hueco, Bojacá municipality, Cundinamarca department, Colombia (4.6963,, 2630 m a.s.l.; Fig. 1). Specimens were captured by hand using free searches over two 10-day field trips, kept in plastic bags until weighed and photographed, and then euthanized by applying 2% lidocaine gel. Tissue samples were obtained from the tail or liver of individuals and preserved in absolute ethanol. Specimens were then fixed in 10% formaldehyde and then stored in 70% ethanol. All specimens were deposited at Colección de Anfibios, Instituto de Recursos Biológicos Alexander von Humboldt, Villa de Leyva, Boyacá, Colombia (IAvH).

Morphology and taxonomy
Measurements and counts of morphological characters were taken using a Neiko digital caliper rounded to the nearest 0.1 mm under a Leica Stemi 2000 stereoscope, using the diagrams in Bingham et al. (2018) as a model. The following morphological traits were analyzed for all specimens: snout-vent length (SVL); head length (HL); head width (HW); tail length (TL); maxillary teeth (MT); vomerine teeth (VT); and additional measurements only for the description of the holotype: interorbital distance (IOD); eye diameter (EYD); snout length (SNL). Color descriptions are based on field notes and photographs of preserved specimens using the color catalogue of Köhler (2012). Format of diagnosis and description follows Meza-Joya et al. (2017). Species comparisons were made following Brame and Wake (1963) or their original descriptions for those species described after 1963, as well as by examining Bolitoglossa specimens housed at Colección de Anfibios, Instituto de Recursos Biológicos Alexander von Humboldt, Villa de Leyva, Boyacá, Colombia (IAvH), Laboratorio de Anfibios, Instituto de Ciencias Naturales, Universidad Nacional de Colombia, Bogotá D.C., Colombia (ICN), Colección de Anfibios, Museo La Salle, Universidad de La Salle, Bogotá D.C., Colombia (MLS) and Colección Herpetológica of Universidad Industrial de Santander, Bucaramanga, Colombia (UIS).

Molecular and phylogenetic analyses
We extracted whole genomic DNA from liver or muscle tissue of specimens of Bolitoglossa preliminary identified as B. adspersa, using the DNeasy Blood & Tissue Kit (Qiagen, #69506). Extracted DNA samples were amplified by PCR for the partial non-coding 16S rRNA (16S ≈ 517 bp) and the protein-coding cytochrome b (cyt b ≈ 742 bp) mitochondrial genes, using the primers 16Sar-L and 16Sbr-H (Palumbi et al. 1991) and MVZ15 and MVZ16 (Moritz et al. 1992), respectively. Amplification protocols (reaction mix and thermocycler programs) are as described in Meza-Joya et al. (2017). The amplicons were cleaned and then sequenced at Macrogen Inc. (Seoul, Korea) by capillary electrophoresis using an ABI3730 genetic analyzer. The partial sequences obtained were visualized, cleaned, and assembled with Geneious v. 9.1.6 (Kearse et al. 2012); we only used sequences with a quality score higher than 90%. DNA sequences were deposited in GenBank (Appendix 1).
Homologous sequences from other Bolitoglossa in the adspersa group were downloaded from GenBank and compared with our molecular data. Representatives from other species groups within Eladinea (epimela, schizodactyla, and subpalmata) were used as outgroups (Appendix I). We also included partial sequences of the nuclear proteincoding recombination activating gene 1 (RAG1 ≈ 792 bp), a relatively well-sampled gene fragment, for other species of Bolitoglossa. We performed multiple alignments in MAFFT v. 7.304 (Katoh and Standley 2013) using the G-INS-i algorithm. Phylogenetic analyses were performed on the concatenated dataset. We inferred the best-fit partition scheme and the best-fit evolution models with PartitionFinder v. 2.1.1 (Lanfear et al. 2017) under the Bayesian Information Criterion (BIC). For this, we performed an exhaustive search of all possible partitioning schemes on our dataset, placing the 16S gene in a separate partition whereas protein-coding genes were further partitioned by codon position. We performed a maximum likelihood (ML) phylogenetic analysis on IQ-TREE 2.0 (Nguyen et al. 2015), running 10,000 ultrafast bootstrap pseudoreplicates for internal node support (Hoang et al. 2018). We also conducted a Bayesian Inference (BI) analysis with the software MrBayes 3.2.6 (Ronquist et al. 2012), using four chains on two runs for 10 million generations and a sampling frequency of 10,000 generations with a burn-in of 0.10. Stationarity was determined with the software Tracer 1.6 (Rambaut et al. 2018).
For species delimitation, we first calculated uncorrected pairwise genetic p-distances for the 16S and cyt b genes between the two distinct evolutionary lineages identified within B. adspersa, with 1,000 bootstrap replicates using MEGA X (Kumar et al. 2018). Then, we split genetic lineages into candidate species using the Automatic Barcode Gap Discovery (ABGD) method (Puillandre et al. 2012). This analysis was based on the 16S gene matrix, the best-represented gene in our dataset, using K2P distances, prior for maximum value of intraspecific divergence between 0.001 and 0.1, ten recursive steps, and a gap width of 1.5. We also used the tree-based method as implemented in the Species Delimitation plugin in Geneious (Masters et al. 2011), using the ML phylogeny as a guide tree to calculate the mean probability of the ratio of intra-to interspecific genetic distances for the initial two-species hypothesis within B. adspersa (Brame and Wake 1963).
The inferred phylogenetic relationships were largely consistent with those from recent studies (Meza-Joya et al. 2017;Cusi et al. 2020;Jaramillo et al. 2020), with incongruences likely resulting from differences in taxon sampling. The Bolitoglossa adspersa species group was rendered as monophyletic with significant support (UFB = 99, PP = 1.0). All species included in our analyses were monophyletic with strong support, yet their relationships remained largely unresolved. As expected, samples from the new species were within the adspersa group, forming a well-supported clade (UFB = 100, PP = 1.0) sister to samples of B. adspersa from its type-locality, Bogotá, Cundinamarca department, Colombia, and surroundings, with significant support (UFB = 98, PP = 0.99). This clade was recovered as a sister to a clade grouping species from northeastern Colombia and Venezuela, but this relationship was poorly supported. Gen-Bank sequences of a specimen from El Soche, Cundinamarca, Colombia, identified as Bolitoglossa sp. 1 (MVZ 167947, corrected here as MVZ 167997 based on the actual number on the MVZ catalog) by Parra-Olea et al. 2004, correspond with the new species described here. This specimen has long been recognized as different from adspersa (Hanken and Wake 1982;Parra-Olea et al. 2004).
With respect to species delimitation, all methods supported the two-species hypothesis within B. adspersa (Brame & Wake, 1963). Uncorrected pairwise p-distances between these two sister lineages were 2.1% (± 0.003%) for 16S and 7.0% for cyt b. The ABGD analysis resulted in five partitions separating the data into five (P ≤ 0.0028) or two putative species (P ≥ 0.0046), yet the new species described here was recovered as a candidate taxon in all partitions. Taxonomic distinctiveness for the new species was also supported under either relaxed or strict tree-based criteria (P ID Liberal = 1.00, CI = 0.86-1.00; P ID Strict = 0.79, CI = 0.62-0.97) with significant support: Rosenberg's P AB statistics = 0.01 and P RD (randomly distinct) > 0.05. Furthermore, as it is shown below, morphological comparisons consistently support the recognition of this lineage as a new species. Diagnosis. Bolitoglossa muisca is a member the subgenus Eladinea and of the adspersa species group. The new species is characterized by the following morphological characters: a large-size body; a broad head; a rounded snout in dorsal and ventral views; a very thick postocular fold; a moderate subgular fold; smooth skin texture; moderately long limbs; moderate webbing on third finger and toe; and a short, robust tail.

Bolitoglossa muisca
Even though Bolitoglossa adspersa and B. muisca share their moderate webbing on hands and feet, we regard the former as having less webbing than the latter. Moreover, the tips of the fingers and toes are separated from the distal margin of the webbing, exposing their subcircular-shaped digits; unlike those of B. muisca (Fig. 4). The new species is slightly larger on average than B. adspersa (mean SVL 52.8±3.4 mm; range 33.0-72.1 mm; n = 22 vs. 45.0±3.4 mm; range 30.7-66.3 mm; n = 26), additionally, the tail of B. adspersa is thin and long in relation to the trunk but thick and short in relation to the trunk in B. muisca (Table 1)  and toe of the latter are triangular and pointed in outline (vs. third digits and toes oval and webbed to a higher degree in B. muisca), by being a slightly larger species (mean SVL 52.8±3.4 mm; range 33.0-72.1 mm; n = 22 vs. 44.0±2.9 mm; range 35.9-52 mm; n = 12), and by having mostly smooth skin (vs. coarse skin in B. pandi); moreover, the tail of B. pandi tapers gradually and symmetrically from broad to slender anteroposteriorly, unlike that of the new species, which is slightly rectangular in outline, becoming abruptly wider than the base, and ending in a rounded tip (Table 1).
Description of the holotype. An adult female (SVL = 61.3 mm) with a broad head (HW/SVL = 0.16); head longer than wide (HW/HL = 0.90); neck with a small, faint gular fold; snout short and truncated in profile, and dorsal view, but less so in ventral view (SNL = 3.1 mm); large eyes that do not extend beyond the outline of the head in dorsal view and smaller than interorbital distance (EYD = 3.18 mm, IOD = 3.37 mm); with a thick post ocular fold that extends past the posterior commissure of the eye onto the anterior margin of the gular fold; canthus rostralis subtle, small, rounded in outline; 33 maxillary teeth, 16 to the right and 17 to the left; vomerine teeth 25, these are not arranged in a single row but grouped towards the margins of the parasphenoid bone; with three premaxillary teeth that pierce the upper lip in males; nasolabial grooves well developed; moderate interdigital webbing on hands but third finger extends slightly further than the other fingers; toes with less interdigital webbing than fingers, toes II-V with less membrane than toe I; with subterminal pads on digits, digits in order of increasing length I<II<IV>III; toes I<II<III<IV>V; longest digits of hand and feet are subcircular (L3T and L3F), limbs relatively long (FL/SVL = 0.23, HLL/SVL = 0.23); tail not exceeding standard length (TL/ SVL 0.93), narrower than the body at the base (posterior to the vent), slightly rectangular in outline, becoming abruptly wider than the base and ending in a rounded tip, but this condition is artefactual because the tip of the tail is missing; a long trunk (52.5 mm); with 13 costal grooves (Fig. 3). See Table 2 for meristic data of all type specimens. Coloration of the holotype in life. The color pattern of the holotype is described based on a photograph taken the day after capture. The dorsal surfaces of the head, the body and the tail are Raw Umber (280), strongly speckled with Dark Salmon (59); white stipples on the lateral surface of the head; the flanks, dorsum, legs, and tail have an irregular thin white stripe; the iris is Light Sky Blue (191)    Coloration of the holotype in preservative. The color pattern of the holotype was recorded after approximately five months stored in 70% ethanol. The dorsal surfaces of the head, the body and the tail are Raw Umber (280), strongly speckled with Dark Salmon (59); the flanks, dorsum, legs, and tail have an irregular Smoke Gray (266) stripe; the iris is Amber (51) with Orange Rufous (56) reticulations. The throat and ventral surface are Smoke Gray (266) with Raw Umber (280) speckles and reticulations; hands and feet soles are Grayish Horn (268) ventrally (Fig. 3).
Color variation. The specimens IAvH-Am-17414-16 have the dorsal surfaces of the head, flanks, dorsum, front legs and vertebral band Orange-Rufous (56), strongly speckled with Raw Umber (280); paravertebral area, tail, and hind legs back Light-Yellow Ocher (13) with Raw Umber (280) dashes, and bordered with a wide Raw Umber (280) band; white stipples on the lateral surface of the head and back of the legs. The specimen IAvH-Am-17425 has the dorsal surfaces of the head, body, legs, and tail Dark Salmon (59), strongly speckled with Raw Umber (280), with greater concentration at the nape of the neck. Ventral markings or blotches on the ventral surfaces of the body and tail vary in shape and size; often with irregular margins but are consistently white or cream-colored independent of sex and age (Figs 5-7).
Etymology. Named after the native human inhabitants of the Altiplano Cundiboyacense and Sabana de Bogotá. The Muiscas regarded amphibians as sacred creatures associated with sex, fertility, and the arrival of the rainy season. The specific epithet is used as a noun in apposition.
Distribution. At present, Bolitoglossa muisca is known only from small cloud forest remnants on the western slopes of the Cordillera Oriental of Colombia in Bojacá, Granada, San Antonio del Tequendama, Silvania, and Soacha municipalities, Cundinamarca department. All specimens collected between 2390-2700 m a.s.l. (Fig. 2).
Natural history. Individuals from Bojacá municipality were regularly found at night on the base (on the mantillo) and leaves of Cyatheaceae ferns, which are dominant in the cloud forests of the Tequendama region of Cundinamarca department (Fig. 8). Most individuals from San Antonio del Tequendama municipality were found active at night perching on small branches of shrubs (Araceae and Melastomataceae), usually far away from rivers or streams. During the day, a few individuals were found inactive inside bromeliads below two meters height. When handled, these salamanders produced a sticky whiteish mucoserous substance; we consider this to be a defense mechanism against potential predators (Arrivillaga and Brown 2018). Two frog species (Pristimantis sp. and P. uisae) and a lizard (Anolis heterodermus) were found in sympatry with Bolitoglossa muisca; no other salamander species were found within our fieldwork area.
Other material examined. Countries are indicated in bold capitals, departments in regular capitals, municipalities, and localities in plain text. * Denotes specimens examined via photographs.

Taxonomic background
Brame and Wake (1963: 44) examined a single specimen of Bolitoglossa from the Tequendama region of Cundinamarca (ICNB Tequendama), which was regarded as an  undescribed species morphologically similar to B. adspersa. Despite finding diagnostic morphological differences between this specimen and those from B. adspersa, the limited sample size precluded any attempt by these authors to describe it as a new species.
Our morphological and molecular results support Brame and Wake's (1963) hypothesis. Through our conversations with Giovanni Chaves-Portilla we came across an unpublished manuscript written by the late David B. Wake and Arden H. Brame, dated sometime between the late 1980s and early 1990s (John D. Lynch pers. comm.), in which they proposed descriptions of three new Bolitoglossa from Colombia. One of these new species corresponds to Bolitoglossa muisca and is known from a site called Hacienda 'El Soche', Granada municipality, Cundinamarca department, Colombia, 2600 m a.s.l., i.e., from the same locality of MVZ 167997 (Fig. 1). Wake and Brame had planned to designate four types for this new species housed at Colección de Anfibios, Instituto de Ciencias Naturales, Universidad Nacional de Colombia, Bogotá D.C., namely ICN 3544-46 and ICN 3550. We managed to examine these specimens and confirm them to be conspecific with Bolitoglossa muisca, but unfortunately these are not in the best condition and are therefore designated as referred material.

Phylogenetic status
Our taxonomic sampling indicates that Bolitoglossa muisca is sister to B. adspersa and both are reciprocally monophyletic (Fig. 1). These two species are close geographically and their genetic uncorrected p-distance for the 16S and cyt b (7.0%) fragments were relatively low (2.1 and 7%, respectively). Yet, lower distances between morphologically well-defined sister species of Bolitoglossa have been previously reported (Batista et al. 2014;Meza-Joya et al. 2017), with the smallest divergence reaching 0.5% for 16S (Parra-Olea et al. 2004). In addition, species delimitation analyses provided support to the distinctiveness of B. muisca as a new species.

Threats and conservation status
Deforestation, logging, and forest clearing are the main threats faced by the habitat of the new species in the remnant cloud forests of the Tequendama region in Cundinamarca department, Colombia. Nonetheless, the type locality of Bolitoglossa muisca is located within the regional protected area 'Distrito de Manejo Integrado Cerro Manjui -Salto del Tequendama', a conservation project led by Empresas Públicas de Medellín (EPM) and Fundación Natura that focuses on improving connectivity among cloud forests remnants of the Tequendama region. The calculated Extent of Occurrence (EOO) using the localities where the species is distributed is 102 km 2 (estimate made with GeoCAT; Bachman et al. 2011). Bolitoglossa muisca is only known from an area of 102 km 2 and only the type locality has a certain degree of protection. However, based on our results and field observation we consider that this species should be considered as Endangered (EN) using the IUCN criteria B1b(iii) of the IUCN, given its small known range (< 5000 km), and the current threats to its native habitat. The consequent loss of native vegetation may be causing the new species described here to be most likely threatened by habitat loss, and a monitoring program is warranted to better assess the current status of its few known populations (Liu et al. 2022).