A new species of the genus Noblella (Amphibia: Strabomantidae) from Ecuador, with new information for Noblella worleyae

We describe a new species of terrestrial-breeding frog of the genus Noblella from the northwestern slopes of the Andes of Ecuador, in the province of Pichincha, Ecuador, and report a new locality for the recently described N. worleyae. We include a detailed description of the osteology of both species and discuss their phylogenetic relationships. The new species is differentiated from other species of Noblella by having discs of fingers rounded, without papillae; distal phalanges only slightly T-shaped; toes slightly expanded and rounded distally, without papillae; dorsum uniform brown with irregular suprainguinal dark brown marks; venter yellowish cream, ventral surfaces of legs and thighs reddish to brownish cream; and dark brown throat. The new locality for N. worleyae is located in Los Cedros Reserve, an area highly threatened by mining. We highlight the importance of protecting endemic species of small vertebrates in northwestern Ecuador.

Terrestrial-breeding frogs of the genus Noblella Barbour 1930 are minute-size anurans (SVL < 22 mm), morphologically differentiated by having terminal discs on digits not or barely expanded, discs and circumferential grooves present distally (except in N. duellmani), terminal phalanges narrowly T-shaped, pointed tips of at least Toes III-IV, and an inner tarsal tubercle (De La Riva et al., 2008;Hedges et al., 2008;Duellman and Lehr, 2009). However, phylogenetic relationships of Noblella are not fully resolved and its monophyly is uncertain (De la Riva et al., 2017;Santa-Cruz et al., 2019). As currently defined, Noblella includes 16 species, fourteen distributed in the Andes of Ecuador, Peru, and Bolivia, and two (N. losamigos and N. myrmecoides) in the Amazonian lowlands from southeastern Colombia, Ecuador, Peru, Bolivia, and western Brazil (Frost, 2021). During the last 15 years, the number of species in the genus has doubled; and four new species have been described since 2019 (Catenazzi and Ttito, 2019;Reyes-Puig et al., 2019c, 2020bSanta-Cruz et al., 2019). Currently, the total number of species of the genus Noblella is 16, distributed in ten species in Peru, seven in Ecuador, three in Bolivia, and one in Colombia and Brazil (Frost, 2021).
Andean species of the genus Noblella show a high level of endemicity, with very restricted distributions. While some species of Noblella may apparently be able to survive in environments modified by humans (e.g., N. duellmani, N. losamigos, N. lochites, N. naturetrekii;Duellman and Lehr, 2009;Reyes-Puig et al., 2019c;Santa-Cruz et al., 2019); most species (e.g., N. coloma, N. heyeri, N. personina, N. pygmaea;Lynch, 1986;Guayasamin and Terán-Valdez, 2009;Harvey et al., 2013) seem to depend on undisturbed forest. Three species of Noblella have been described from western Ecuador, all from mature mountain forests: Noblella heyeri (Lynch, 1986) occurs in southwestern Ecuador and extreme northwestern Peru; Noblella coloma Guayasamin and Terán-Valdez, 2009 is known from its type locality and surroundings (Rio Guajalito and Chiriboga area; Ron et al., 2019); and Noblella worleyae, a recently described species is known just from seven specimens, all found in mature forest in the Río Manduriacu Reserve, province of Imbabura, Ecuador (Reyes-Puig et al., 2020b).
While the Ecuadorian Andes have suffered serious habitat destruction and fragmentation caused by expansion of deforestation, agriculture, mining, among others (Castellanos et al., 2011;Roy et al., 2018;Lessmann et al., 2019;Ortega et al., 2021), there are still some areas with mature forests that have not been exploited due to their complex topography, difficult access, private protection, or preservation for touristic activities. Unfortunately, all such sites are under strong anthropogenic pressure, including mining concessions and the expansion of agricultural boundaries, among others (Cuesta et al., 2017;Roy et al., 2018;Guayasamin et al., 2019;Ortega et al., 2021). These privileged areas have proven to keep an extremely high cryptic diversity of small vertebrates and contain the last remnant populations of numerous threatened species (Cisneros-Heredia and Yanez-Muñoz, 2010;Reyes-Puig et al., 2010, 2019a, 2019bYánez-Muñoz et al., 2010bGuayasamin et al., 2018Guayasamin et al., , 2019Guayasamin et al., , 2020Sánchez-Nivicela et al., 2018;Barrio-Amorós et al., 2020).
During the last five years, we have carried out surveys on the western slopes of the Andes in the provinces of Imbabura and Pichincha, Ecuador. As a result of this continuous effort, we found a new species of leaf-litter frog of the genus Noblella, which we describe herein based on a combination of morphological, molecular, and osteological features. We also document new information on distribution, external morphology and osteology for the recently described Noblella worleyae, information that was not described in detail in the original description. We also include intraspecific variation that will allow complete full with members of the same genus in the future.

Taxonomy
We followed the family taxonomy proposed by Heinicke et al. (2018) and, also we revised De la Riva et al. (2017) and Barrietos et al. (2021). For identifying species, we assumed the unified species concept (De Queiroz, 2005. Information for species comparisons was extracted from the original descriptions and cited once at the beginning of the comparison.

Study area and fieldwork
Over the last three years (i.e., 2018-2020), we have carried out field surveys at several localities in montane forests of northwestern Ecuador, mainly in the provinces of Imbabura and Pichincha. Specimens of two different species of Noblella were found in Mindo (province of Pichincha) and Los Cedros Biological Reserve (province of Imbabura). Mindo is a small town renowned for its adventure and nature-based touristic activities; thus the area has numerous reserves that protect cloud forests (Arteaga-Navarro et al., 2013). Los Cedros Biological Reserve is a protected area that contains 6,879 hectares of premontane humid tropical forest and cloud mountain forest. This reserve is located south of the Cotocachi-Cayapas Ecological Reserve (state protected area), and is also recognized by its endemic microfauna (Hutter and Guayasamin, 2015). Collected specimens were euthanized with benzocaine, fixed in 8% formalin, and preserved in 75% ethanol. Liver and leg muscle tissue samples were collected from all individuals prior to preservation. Tissues were preserved in 95% ethanol and stored at -20°C at the Laboratorio de Biología Evolutiva USFQ. Specimens were deposited in the Museo de Zoología, Universidad San Francisco de Quito, Ecuador (ZSFQ).

DNA extraction, amplification, and sequencing
We obtained new DNA sequences of Noblella sp. nov. (ZSFQ 050-051). DNA was extracted from muscle or liver tissue following the protocol by Peñafiel et al. (2019). Standard polymerase chain reaction (PCR) was performed to amplify a fragment of the mitochondrial gene 16S rRNA, using a combination of the following primers: 16L10, 16H36E, 16L34, 16H47 (Heinicke et al., 2007). Amplicons were sequenced in both directions by the Macrogen Sequencing Team (Macrogen Inc., Seoul, Korea).
The new sequences were assembled and edited with Geneious 7.1.7 (GeneMatters Corp). After assemblage, the sequences were combined with sequences from GenBank for all species of Noblella and representatives of the genera within the Terrarana clade (sensu Hedges et al., 2008) &Heinicke 2008, andMicrokayla De la Riva, Chaparro, Castroviejo-Fisher, Padial 2017. GenBank codes are shown in our inferred phylogenetic tree (Fig. 1).

Phylogenetic analyses
Phylogenetic relationships were inferred using maximum likelihood as the optimality criterion. The final matrix, with 52 terminals, was aligned with MAFFT v.7 (Multiple Alignment Program for Amino Acid or Nucleotide Sequences: http:// mafft.cbrc.jp/alignment/software/), with the Q-INS-i strategy. MacClade 4.07 (Maddison and Maddison, 2005) was used to visualize the alignment, which contained a total of 492 bp. Phylogenetic analyses were performed under the ML criteria in GARLI 2.01 (Genetic Algorithm for Rapid Likelihood Inference; Zwickl, 2006) for the mitochondrial gene 16S. GARLI uses a genetic algorithm that finds the tree topology, branch lengths, and model parameters that maximize lnL simultaneously (Zwickl, 2006). Individual solutions were selected after 10,000 generations with no significant improvement in likelihood, with the significant topological improvement level set at 0.01. Then, the final solution was selected when the total improvement in likelihood score was lower than 0.05, compared to the last solution obtained. Default values were used for other GARLI settings, as per recommendations of the developer (Zwickl, 2006). Bootstrap support was assessed via 1,000 pseudoreplicates under the same settings used in tree search. Pairwise genetic distances between species (uncorrected-p) for gene 16S were calculated with PAUP 4a (Swofford et al., 1996).

External morphology
Diagnosis and description of the new species follow formats proposed by Duellman and Lehr (2009) and Lynch and Duellman (1997). For comparisons, we examined specimens of other species of Noblella (see Appendix I). We followed the sequence of characters proposed by Guayasamin and Terán-Valdez (2009). We measured preserved specimens using digital calipers to the nearest 0.01 mm. These measurements are: snout to vent length (SVL), from the tip of the snout to the cloaca; head length (HL), measured from tip of snout to anterior edge of tympanum; head width (HW), measured at midorbital region; horizontal diameter of the eye (ED); eye-nostril distance (EN), from anterior ocular angle to posterior edge of nostril; horizontal diameter of tympanum (TD); minimum interorbital distance (MIOD); minimum eyelid width (MWE); hand length (LH), from posterior edge of palmar tubercle to tip of third digit; shank length (LS), from the tip of the ankle to the knee; and foot length (LF), from posterior edge of external metatarsal tubercle to tip of Toe IV. We determined sexual maturity by the presence of vocal slits or extended vocal sacs in males and by the presence of eggs or convoluted oviducts in females. Detailed illustrations of the head, hands and feet were done with Adobe InDesign ©.

Osteology
Osteological descriptions were based on one specimen of the new species (ZSFQ 050) and one of Noblella worleyae (MZUTI 1709). Both specimens were scanned using a highresolution micro-computed tomography (micro-CT) desktop device (Bruker SkyScan 1173, Kontich, Belgium) at the Zoologisches Forschungsmuseum Alexander Koenig (ZFMK, Bonn, Germany). To avoid movements during scanning, specimens were placed in a small plastic container and mounted with styrofoam. Acquisition parameters comprised: An X-ray beam (source voltage 43 kV and current 114 µA) without the use of a filter; 800 projections of 500 ms exposure time each with a frame averaging of 5 recorded over a 180° continuous rotation (rotation steps of 0.3 degrees), resulting in a scan duration of 49 min; a magnification setup generating data with an isotropic voxel size of 19.16 µm (MZUTI 1709) and 14.55 µm (ZSFQ 050), respectively. The CT-dataset was reconstructed with N-Recon software (Bruker MicroCT, Kontich, Belgium) and rendered in three dimensions using CTVox for Windows 64 bits version 2.6 (Bruker MicroCT, Kontich, Belgium). Osteological terminology follows Trueb (1973), Duellman and Trueb (1994), Fabrezi and Alberch (1996), Terán-Valdez (2009), Scherz et al. (2017), and Suwannapoom et al. (2018). Cartilage structures were omitted from the osteological descriptions because micro-CT does not render cartilage.

Etymology
The specific name "mindo" is a word of unknown meaning in Panzaleo, an extinct pre-Columbian language of northern Ecuador (Jijón y Caamaño 1940). It is used as a noun in apposition, and alludes to the valley of Mindo, where the type locality of the new species is located. The remnant forests of this emblematic valley protect several species of endemic amphibians and reptiles such as Pristimantis mindo, Noblella mindo, and Anolis proboscis.

Description of the holotype
Adult female (ZSFQ 050); head narrower than body, its length 40.8% of SVL; head longer than wide; head width 31.1% of SVL; snout round in dorsal and lateral  views; canthus rostralis straight, slightly concave in profile; loreal region slightly concave; upper eyelid 45.6% of interorbital distance; eye-nostril distance 54.8% of eye diameter; tympanum visible externally, tympanic membrane differentiated from surrounding skin; supratympanic fold indistinct. Dentigerous processes of vomers absent and vomerine teeth absent; choanae laterally oriented; tongue longer than wide, elongated, partially notched posteriorly.
Color of holotype in life (Fig. 4) Dorsum brown, grayish brown towards the flanks; well-defined cream middorsal stripe, extending from interparietal region to cloaca and continuing along posterior surfaces of hindlimb. Loreal region black, extending as homogeneous dark band to upper insertion of arm and into body flanks, narrowing towards groin and limited dorsally with a lighter brown line; flanks strongly light flecked; groin dark. Rictal gland white. Venter and ventral surfaces of hindlimbs yellowish cream; throat dark brown with large irregular yellowish cream marks and Color of holotype in ethanol (Fig. 5) Dorsum brown, darker towards middorsum, welldefined middorsal line cream, extending from interparietal region to cloaca where stripe continues along posterior surface of thighs and pes. Dorsal surfaces of forelimbs brown with black spots. Labial bars absent; rictal gland light brown. Loreal region black, extending as homogeneous dark band to upper insertion of arm and into body flanks, narrowing towards groin; flanks strongly light flecked; groin dark. Dorsal surfaces of hindlimbs lighter brown than dorsum to cream with dark fleck and spots. Throat dark brown with cream irregular marks and medium longitudinal stripe. Chest, venter and ventral surfaces of thigh and crus cream.  Adult females ZSFQ 050, 304-305 and the adult male ZSFQ 051 exhibit a cream middorsal stripe extending from interparietal region to cloaca; stripe of ZSFQ 305 is thinner and faintly defined. Dark suprainguinal marks are faint in ZSFQ 049. Throat, chest and ventral surfaces of forelimbs are dark brown with a white cross formed by a longitudinal, fine line running from chin to chest, crossing a similar line departing from midventral

Osteology
Osteological description of Noblella mindo sp. nov. is based on micro-CT images of the adult female holotype (ZSFQ 050). Details of skull morphology and osteological aspects of hand and foot are presented in Fig. 7 and main skeletal features are shown in Fig. 8. Skull (Fig. 7) Skull slightly longer than wide; widest part is at about where quadratojugal meets maxilla and is 89% of skull length. Rostrum short; distance from anterior edge of frontoparietals to anterior face of premaxilla is 16% of skull length. At level of midorbit, braincase is about 38% of maximum skull width. Braincase combines welland poorly ossified elements. Frontoparietals are welldeveloped bones, distinctly longer than broad, slightly narrower anteriorly than posteriorly; narrowly separated along most of their length and only fused in anterior region. Boarder between frontoparietals and prootics not well-resolved in micro-CT scan. Ventrally, prootics in contact with parasphenoid alae. Prootics well-separated from each other. Exoccipitals approximate one another ventromedially and dorsomedially but still clearly separated with about same distance ventrally and dorsally; separated from frontoparietals. Anterolaterally, frontoparietals in contact with sphenethmoid. Sphenethmoid ventrally at midline separated in anterior half and fused in posterior half; posterior margin does not reach midpoint of orbit and is broadly separated from prootic and in ventral contact with parasphenoid. Cultriform process of parasphenoid well-ossified posteriorly, thinning anteriorly, and about 28% width of braincase at mid-orbit. Lateral margins of process approximately parallel. Parasphenoid alae long but poorly ossified at their lateral ends. Neopalatines very thin and long, articulate with sphenethmoid and approximate but not contact maxilla. Columella (or stapes) large and well-ossified. Due to tiny size and fine structure, septomaxilla is not well-resolved in micro-CT scan. Dorsal investing bones moderately developed. Nasals thin and broadly separated from one another, posteriorly in contact with anterior end of frontoparietals and posterolaterally in thin contact with maxilla. They curve ventrally towards their lateral edges. Small prevomers broadly separated from one another medially, their anterior edge almost contacts a long and thin posterior projecting ramus of septomaxilla. Maxillary arcade bears many small, poorly resolved teeth on premaxillae and maxillae. Premaxillae separated medially, and their anterodorsal alary processes rise divergent from midline but still distinctly separated from nasals. Premaxilla and maxilla in lateral contact, with anterior edge of maxilla slightly overlapping lateral edge of premaxilla. Pars palatina of premaxilla broad, with two well-defined processes: medial process thin and acuminate, running about parallel to its counterpart, being distinctly separated from it; lateral process about the same length, but slightly broader, especially at its truncate posterior ending. Maxilla long, its posterior end acuminate and in contact with quadratojugal. Triradiate pterygoid bears a long, curved anterior ramus oriented anterolaterally toward maxilla, with which it articulates at ventral boarder slightly anterior to midline of orbit. Posterior ramus of pterygoid about same length as medial ramus and both about half length of anterior ramus; however, posterior ramus more robust than other two. Edge of medial ramus overlaps lateral edge of prootic. Quadratojugal slender, almost straight and articulating anteriorly with maxilla and posterodorsally with ventral ramus of squamosal. Squamosal T-shaped, with a long laminar otic ramus; zygomatic ramus much shorter and slender; ventral ramus about same length as otic ramus, laminar and broad, increasing in width ventrally. Mandible slim and edentate. Mentomeckelians small, medial- 3.5-3.9 (3.7 ± 0.2) 3.0-3.2 (3.1 ± 0.2) LS 9.0-9.3 (9.1 ± 0.2) 7.7-8.0 (7.8 ± 0.2) LF 8.4-9.0 (8.6 ± 0.4) 7.3-7.5 (7.4 ± 0.1) ly, and laterally slightly broadened, and separated medially by a narrow gap. Dentary long and thin, reaching to about anterior corner of orbit; it is posteriorly acuminate and overlapping angulospenial, seeming to be in contact with this bone for about the posterior half of its length; anteroventrally it contacts mentomeckelian bones. Angulosplenial long and arcuate. Coronoid process is a moderately long and slightly raised ridge. The only ossified portions of hyoid apparatus are two posteromedial processes, which are anteriorly slightly and posteriorly moderately expanded, approaching each other at anterior ends but being still moderately separated.
Postcranium (Fig. 8) Eight presacral vertebrae. All presacrals non-imbricate. Presacral I longer than posterior vertebrae. All except Presacral I bear well-developed diapophyses. Transverse processes of Presacrals V-VIII similar in size, being the shortest and thinnest, those of Presacrals II and IV also about similar in size and being slightly larger, and those of Presacral III being the longest and widest of all transverse processes. Transverse processes of Presacrals II and VIII have slightly anterolateral orientation, those of Presacral III are laterally oriented and the others are slightly posterolaterally oriented. Neural arch of Presacrals III-VIII bears a raised medial ridge. Sacrum bears slightly expanded diapophyses. Urostyle long, slender, slightly shorter than presacral portion of vertebral column and bearing a well-pronounced dorsal ridge along most of its length, beginning at its anterior end. The bone has a bicondylar articulation with the sacrum. Pectoral girdle with well-ossified coracoids, clavicles, scapulae and cleithra. Suprascapular and sternum unossified and not visible in micro-CT scan, and omosternum hardly visible. Clavicles long and slim, oriented anteromedially, slightly curved, with medial tips touching each other. Laterally, clavicles firmly articulating with scapulae. Coracoids stout and glenoidal and sternal ends about equally expanded. Anterior edges of coracoids slightly curved, posterior edges almost straight. Medial tips of coracoids broadly separated from another. Scapula long, with a prominent pars acromialis not separated from pars glenoidalis. Cleithrum long, broader, and thicker at scapular boarder, thinning posteriorly. In pelvic girdle, long, slender iliac shafts bearing conspicuous dorsolateral ridges along most of their length, except anteriormost region. Ilia are fused posteriorly with ischium and pubis. Ischium stout, whereas pubis is thin and blade-like. (Fig. 7) All phalanges are ossified with a phalangeal formula for fingers and toes: 2-2-3-3 and 2-2-3-4-3, respectively.

Manus and pes
Order of finger length: I < II < IV < III, and that of toes: I < II < V < III < IV. Distal knobs present on terminal phalanges of all fingers and toes. Terminal phalanges of all toes and fingers narrower than penultimate phalanges of all toes and fingers, respectively. Carpus and tarsus not well-resolved in micro-CT scan. However, carpus seems to be composed of a radiale, ulnare, Element Y, ossified prepollex element, Carpal 2 and a large post-axial element probably representing a fusion of Carpals 3-5. Tarsus seems to be composed of two tarsal elements: Tarsal 1 and Tarsal 2 + 3, with latter being distinctly larger than Tarsal 1. A moderately large centrale and small ossified prehallux are also present. In ventral view, three sesamoids of subequal sizes are overlaying proximal end of Metatarsals IV-V, a further smaller sesamoid is overlaying parts of Tarsal 1.

Distribution and Natural History
Noblella mindo sp. nov. is only known from El Cinto (0.09022°S, 78.81858°W; 1,673 m), Mindo, province of Pichincha, Ecuador (Fig. 2). Noblella mindo sp. nov. inhabits secondary cloud forests, with the presence of palmito (Bactris gasipaes) plantations and trees that have emerged after the massive logging of forests in the area. These forests have a high humidity index, dense leaf litter layer, and abundant epiphytes. It has a restricted distribution; sampling activities were carried out in a range up to 3km around the type locality, and no individuals nor calls of N. mindo sp. nov. were recorded. The gecko Lepidoblepharis conolepis was found in sympatry. The locality is surrounded by livestock areas and within the type locality forest, there are trails used by farmers to move their livestock. The population of Noblella mindo sp. nov. could be impacted if livestock activity or deforestion expands. Three individuals (ZSFQ 049-051) were found active during the day between 10:00 and 11:00 am; all frogs were on the ground in a 2-meter depth hole.

Osteology description
Osteological description of Noblella worleyae is based on micro-CT images of an adult female (MZU-TI 1709). Details of skull morphology and osteological aspects of hand and foot are presented in Fig. 11 and main skeletal features are shown in Fig. 12. Skull (Fig. 11) Skull almost as wide as long; widest part is at about where quadratojugal meets maxilla and is 97% of skull length. Rostrum short; distance from anterior edge of frontoparietals to anterior face of premaxilla is 18% of skull length. At level of midorbit, braincase is about 34% of maximum skull width. Braincase combines well-and poorly ossified elements. Prootic and exoccipital seem to be fused to form otoccipital. Frontoparietals are welldeveloped bones, distinctly longer than broad, slightly narrower anteriorly than posteriorly; narrowly separated along most of their length and only fused in anterior region. Posterior portion of braincase seems to be fully enclosed by partial fusion of frontoparietals with otoccipitals. However, there might still exist some traces of boarders between bones, but these parts are not wellresolved in micro-CT scans. Ventrally, otoccipitals are in contact with parasphenoid alae. Prootic part of otoccipitals are well-separated from each other. Exoccipital parts approximate one another ventromedially and dorsomedially but are still clearly separated with a broader ventral than dorsal gap between them. Anterolater-ally, frontoparietals are in contact with sphenethmoid. Sphenethmoid is well-ossified and ventrally fused at midline; posterior margin almost reaches midpoint of orbit but is still broadly separated from prootic part of otoccipitals and is in ventral contact with cultriform process of parasphenoid. Cultriform process of parasphenoid is well-ossified posteriorly, thinning anteriorly, and about 31% of width of braincase at mid-orbit. Lateral margins of process are approximately parallel. Parasphenoid alae are long and well-ossified. Neopalatines are very thin and long, articulating with sphenethmoid dorsomedially and maxilla laterally. Columella (or stapes) is large and well-ossified. Because of tiny size and fine structure, septomaxilla is not well-resolved in micro-CT scan. Dorsal investing bones are moderately developed. Nasals are thin and broadly separated from one another, posteriorly in contact with anterior end of frontoparietals and posterolaterally in very thin contact with maxilla. They curve ventrally towards their lateral edges. Small prevomers are broadly separated from one another medially, their anterior edge approximates a long and thin posterior projecting ramus of septomax-   , (B) ventral, (C) frontal, and (D) lateral views. alary p = alary process, angspl = angulosplenial, col = columella, dent = dentary, fpar = frontoparietal, max = maxilla, mmk = mentomeckelian bone, nas = nasal, npl = neopalatine, occ con = occipital condyle, otoc = otoccipital (fused prootic and exoccipital), pmax = premaxilla, prsph = parasphenoid, prvom = prevomer, pter =pterygoid, qj = quadratojugal, smax = septomaxilla, spheth = sphenethmoid, sq = squamosal. The right hand is shown in (E) dorsal, and (F) palmar aspects; and the left foot in (G) dorsal, and (H) plantar aspects. Digits numbered I-V. antbr = os antebrachii (radius + ulna), carp d = carpale distale, cent = centrale, fib = fibulare, mtc = metacarpalia, mtt = metatarsalia, ph d I-IV = finger phalanges F1-F4, ph d I-V = toe phalanges F1-F5, prhl =prehallux, prpl = prepollex, rad = radius, tar d = tarsale distale, tib = tibiale, uln = ulnare. Images prepared by Claudia Koch.
illa. Maxillary arcade bears many small, poorly resolved teeth on premaxillae and maxillae. Premaxillae are separated medially, and their anterodorsal alary processes rise divergent from midline but are still distinctly separated from nasals. Premaxilla and maxilla are in lateral contact, with anterior edge of maxilla slightly overlapping lateral edge of premaxilla. Pars palatina of premaxilla is broad, with two well-defined processes: medial process acuminate, and runs about parallel to its counterpart, being distinctly separated from it; lateral process is slightly shorter and broader. Maxilla is long, its posterior end acuminate and in contact with quadratojugals. Triradiate pterygoid bears a long, curved anterior ramus that is oriented anterolaterally towards maxilla, with which it articulates at ventral boarder anterior to midline of orbit. Posterior ramus of pterygoid is about same length as medial ramus and both are about half length of anterior ramus; however, posterior ramus is more robust than the other two. Edge of medial ramus overlaps lateral edge of prootic part of otoccipital. Quadratojugal is slender, slightly curved and articulates anteriorly with maxilla and posterodorsally with ventral ramus of squamosal. Squamosal is T-shaped with long laminar otic ramus; zygomatic ramus is much shorter and more slender; ventral ramus is about same length as otic ramus, laminar and broad, increasing in width ventrally. Mandible is slim and edentate. Mentomeckelians are small, medially and laterally slightly broadened, and medially contacting each other. Dentary is long and thin, reaching to about anterior corner of orbit; posteriorly acuminate and overlapping angulospenial, seeming to be in contact with this bone for most of its length, except the most anterior part; anteroventrally it contacts mentomeckelian bones. Angulosplenial is long and arcu- views. antbr = os antebrachii (radius + ulna), clav = clavicle, cle = cleithrum, cor = coracoid bone, crur = os cruris (tibia + fibula), fem = femoral bone, fib = fibulare, hm = humeral bone, il = ilium, isch = ischium, pr p-m = processus postero-medialis, prsac v = presacral vertebrae, pub = pubis, sac v = sacral vertebra, sc = scapula, ur = urostyle, tib = tibiale. Images prepared by Claudia Koch.
ate. Coronoid process is a moderately long and strongly raised ridge. The only ossified portions of hyoid apparatus are two posteromedial processes, which are anteriorly slightly more expanded than posteriorly, approaching each other at anterior ends but being still distinctly separated.
Postcranium (Fig. 12) Eight presacral vertebrae. All presacrals are nonimbricate. First presacral vertebra is longer than posterior vertebrae. All except Presacral I bear well-developed diapophyses. Transverse processes of Presacrals V-VIII similar in size and being the thinnest and second shortest, those of Presacral II being the shortest, and those of Presacral III being the longest and widest of all transverse processes. Transverse processes of Presacrals II and VIII have slightly anterolateral orientation, those of Presacrals III and VII are laterally oriented and others are slightly posterolaterally oriented. Neural arch of all presacrals bears a raised medial ridge. Sacrum bears slightly expanded diapophyses. Urostyle is long, slender, about similar in length as presacral portion of vertebral column and bearing a well-pronounced dorsal ridge along about two-thirds of its length, beginning at its anterior end, with a lateral foramen in anterior region. The bone has a bicondylar articulation with the sacrum. Pectoral girdle with well-ossified coracoids, clavicles, scapulae and cleithra. Suprascapular, omosternum, and sternum unossified and not visible in micro-CT scan. Clavicles are long and slim, oriented anteromedially, slightly curved, with medial tips approaching but not touching each other. Laterally, clavicles firmly articulating with scapulae. Coracoids are stout and glenoidal and sternal ends are about equally expanded. Anterior edges of coracoids are curved, the posterior edges are almost straight. Medial tips of coracoids are broadly separated from another. Scapula is long with a prominent pars acromialis that is not separated from pars glenoidalis. Cleithrum is long, broader and thicker at scapular boarder, thinning posteriorly. In pelvic girdle, long, slender iliac shafts bear conspicuous dorsolateral ridges along most of their length, except the anterior most region. Ilia is fused posterirly with ischium and pubis. Ischium is stout, whereas pubis is thinner and blade-like.
Manus and pes (Fig. 11) All phalanges are ossified, with phalangeal formula for fingers and toes: 2-2-3-3 and 2-2-3-4-3, respectively. Order of finger length: I < II < IV < III, and that of toes: I < II < V < III < IV. Distal knobs seem to be absent on Finger I but are present on terminal phalanges of all other fingers and toes. Nevertheless, they are not well-resolved in micro-CT scans and are sensitive to thresholds used during reconstruction. Terminal phalanges of all toes and fingers are narrower than penultimate phalanges of all toes and fingers, respectively. Carpus and tarsus are not well-resolved in micro-CT scan. However, carpus seems to be composed of a radiale, ulnare, Element Y, ossified prepollex element, Carpal 2 and a large post-axial element probably representing a fusion of Carpals 3-5. Tarsus seems to be composed of two tarsal elements: Tarsal 1 and Tarsal 2 + 3, with latter being larger than Tarsal 1. A moderately large centrale and small ossified prehallux are also present. In ventral view, three sesamoids of subequal sizes overlaying proximal end of Metatarsals II-IV, a further smaller sesamoid overlaying parts of Tarsal 1 and proximal end of Metatarsal I.

Natural History
We report a new locality for Noblella worleyae: Los Cedros Biological Reserve, province of Imbabura, Ecuador (Fig. 2), at approximately 8.4 km in a straight line between the type locality of N. worleyae (i.e., Manduriacu Reserve). The individuals were found at several point-localities between 1,417-1,790 m of elevation. The reserve presents an important track of mature Low Montane Evergreen Forest. All specimens were collected at night between 7:00 and 11:50 pm and were found on the ground, in areas covered with abundant leaf litter. Individuals appeared inactive, because they were located by movement only when litter was removed. Syntopic species were Pristimantis mutabilis (Guayasamin et al., 2017b), P. crenunguis, (Lynch, 1976a) and Alopoglossus viridiceps (Torres-Carvajal and Lobos, 2014). Noblella worleyae seems to be a rare species at Los Cedros Biological Reserve. Only two individuals were found in two different surveys with known sampling effort. The first sampling was carried out between 22-25 August 2019, with five people for approximately nine hours per day. The second sampling was carried out between 26-30 October 2019, with two people for about nine hours per day. Individuals were found in the lower forest stratum, camouflaged extremely well in leaflitter and having an evasive behavior, similar to other Noblella species (Reyes-Puig et al., 2019c). DISCUSSION Our phylogenetic analyses (Fig. 1) agree with previous studies that have shown that southern species of Noblella (N. losamigos, N. madreselva, N. pygmaea, N. thiuni) are more closely related to species of Psychrophrynella, rather than to northern species of Noblel-la (N. coloma, N. heyeri, N. lochites, N. mindo sp. nov., N. myrmecoides, N. personina, N. worleyae) (Reyes-Puig et al., 2019c, 2020Santa-Cruz et al., 2019). Noblella peruviana is the type species of the genus, which, based on geography, is most likely part of the Southern Clade. However, since there are no sequences of N. peruviana, we refrain from proposing a new generic arrangement.
Species richness of the genus Noblella has increased dramatically over the last decade (Frost, 2020). About a decade ago, only three species of Noblella were known in Ecuador and no species had been described from the northwestern slopes of the Andes of Ecuador (Cisneros-Heredia and Reynolds, 2007). Nowadays, there are eight species of Noblella reported from Ecuador, including three from the western Ecuadorian Andes (N. coloma, N. mindo sp. nov., and N. worleyae) that form a distinct clade among the northern Noblella (Fig 1). The diversity of the genus Noblella has also increased in the Peruvian Andes, where in recent years three species been described, forming a clade with the previously described Psychrophrynella and Noblella (Catenazzi et al., 2015;Santa Cruz et al., 2019;Catenazzi and Ttito, 2019).
Our new records of Noblella worleyae from Los Cedros reserve add important intraspecific variation to the original description, in terms of its body size, coloration and shape of tips of the digits. In particular, variation in the fingertips was found, with tips of Fingers I and IV varying from slightly acuminate (original description) to rounded (data presented herein), and tips of Fingers II and III from acuminate (original description) to slightly acuminate (data presented herein). We also strengthen the original publication (Reyes-Puig et al., 2020) with a detailed description of the osteology of the species.
Diversification in Noblella seems be related with the linearity of the Andes, with allopatric and parapatric populations being separated by ecological and geographic barriers. In Ecuador, all species of Noblella are allopatric and most of them are restricted to very specific geographic areas. Noblella mindo sp. nov. occurs in low montane forest in the valley of Mindo, in the Nambillo River watershed, western slopes of the Pichincha Massif, northwestern Andes, at 1,673 m; while N. worleyae inhabits low montane forest in the Manduriacu-Los Cedros watersheds, southern slopes of the Toisan massif; and N. coloma is restricted to the cloud forests of the Río Guajalito watershed, western slopes of the Atacazo volcano. Noblella worleyae is separated from N. coloma and N. mindo sp. nov. by the Guayllabamba River Valley (Fig. 2), an important biogeographic barrier, especially for frog species with low vagility (Hillman et al., 2014). Although the valley of Mindo (type locality of N. mindo sp. nov.) and the valley of Guajalito (type locality of N. coloma) are ca. 20 km apart in straight line, they are in different watersheds, separated by the Nambillo River and complex orogeny caused by the Pichincha massif and the Atacazo volcano.
All species currently recognized under the genus Noblella are miniaturized frogs, among the smallest Neotropical vertebrates (Duellman and Lehr, 2009). They are cryptic and adapted to live amidst or under leaf litter in forests, where they are often overlooked by amphibian visual surveys, being easier to locate by their calls or through pitfall traps (Reyes-Puig et al., 2019c). Although some species may be abundant locally, most species appear to have low densities. For example, yearly surveys between 2000-2012 at the type locality of N. coloma produced only three records, while up to 20 individuals of N. lochites were found in 2014 at a single locality in the province of Zamora-Chinchipe (D. F. Cisneros-Heredia pers. obs.).
Most remnants of mature forests in the Andes of Ecuador are nowadays either inside public or private protected areas or persist due to their inaccessibility. Private conservation initiatives have become extremely important in Ecuador (Betancourt et al., 2018;Guayasamin et al., 2018Reyes-Puig et al., 2019a, 2019b, 2019c, where public protected areas do not cover all critically important regions for biodiversity (Lessmann et al., 2014;Cuesta et al., 2017;Reyes-Puig et al., 2017).
Unfortunately, habitat loss due to unsustainable expansion of the agricultural frontier, mining and infrastructure projects have placed a heavy burden on several private reserves (Roy et al., 2018;. In the early 2000s, the region of Mindo was heavily threatened by the construction of an oil pipeline (Oleoducto de Crudos Pesados OCP). Fortunately, local, national and international protests managed to promote some actions to mitigate the largest impacts of the pipeline development. Eventually Mindo has transformed into one of the most popular ecotourism destinations in northwestern Ecuador, allowing several private protected areas to preserve large tracks of mature forest (Welford and Yarbrough, 2015). Unfortunately, twenty years later, history repeats itself, now at Los Cedros reserve, but this time with mining concessions, exploration and exploitation (Roy et al., 2018). Thus, biodiversity conservation is facing an uncertain future.
The increasing descriptions of new species within the Ecuadorian territory have a practical application in the conservation of biodiversity. By highlighting the presence of new vertebrates with restricted distributions in the Andes, the visualization of this unique biodiversity is indisputable.