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Molecular phylogeny of stream treefrogs (Hylidae: Hyloscirtus bogotensis Group), with a new species from the Andes of Ecuador

We present a new molecular phylogeny of the stream treefrog genus Hyloscirtus, with an improved taxon sampling in the Hyloscirtus bogotensis group. The tree supports the existence of three clades within the genus (Hyloscirtus armatus group, H. bogotensis group and Hyloscirtus larinopygion group) in congruence with previous studies, and suggests the presence of at least three new species in the H. bogotensis group. Herein, we describe one of these species, Hyloscirtus mashpi n. sp. from the Pacific slope of the Ecuadorian Andes. The validity of the latter is supported by molecular, morphological and acoustic data. We also tested individuals of the new species for the chytrid fungus Batrachochytrium dendrobatidis, finding a prevalence of 17.6% (6 positives and 28 negatives). However, at sampled streams, frog densities were high, suggesting that H. mashpi n. sp. may be tolerant to the infection.


Introduction
The genus Hyloscirtus [1] is part of the diverse tree frog family Hylidae, and represents a conspicuous component of the anuran fauna in the Andean foothills and cloud forests. This genus currently contains 34 recognized, extant species, [2] all of which reproduce in streams.
In this study, we present a new molecular phylogeny for the H. bogotensis group, as well as morphological and acoustic data that support the validity of at least one new species, which has been previously confused with H. alytolylax. Two other potentially new species are also revealed by genetic data, but we refrain from describing them until complementary evidence becomes available. Finally, we present information on the prevalence of Batrachochytrium dendrobatidis in the new taxon.

Methods Nomenclature
Generic names follow the taxonomy proposed by Faivovich et al. [3]. For an updated list of the species in the genus, see Frost [2] and AmphibiaWeb. [21] Morphology We examined comparative alcohol-preserved specimens from the herpetology collection at the Museo de Zoología of the Universidad Tecnológica Indoamérica (MZUTI), Instituto de Ciencias Naturales of the Universidad Nacional de Colombia (ICN) and the University of Kansas Biodiversity Institute (KU). See Appendix 1. Morphological characters studied follow the definitions provided by Duellman and Hillis [22] and Kizirian et al. [23]. Fingers are numbered from I to IV. Webbing formulae are described following Savage and Heyer, [24] with modifications by Myers and Duellman.[25] Morphological measurements were taken with Mitutoyo® digital caliper to the nearest 0.1 mm, as described by Guayasamin and Bonaccorso [26], except when noted, and are as follows: (1) snout-vent length (SVL); (2) tibia length; (3) foot length; (4) head length; (5) head width; (6) interorbital distance; (7) upper eyelid width; (8) internarial distance; (10) eye diameter; (11) tympanum diameter; (12) radioulna length; (13) hand length; (14) finger I length; (15) finger II length = distance from outer margin of palmar tubercle to tip of finger II; and (16) width of disc of finger III. Sexual maturity was determined by the presence of vocal slits in males and by the presence of eggs or convoluted oviducts in females.

DNA extraction, amplification and sequencing
Genomic DNA was extracted from frozen tissue with a salt precipitation method (M. Fujita, unpubl.) based on the Puregene DNA purification kit (Gentra Systems). We amplified and sequenced the mitochondrial 12S and 16S gene regions. The 12S marker was amplified using the primers 12L29E-F (AAAGCRTAGCACTGAAAATGC-TAAGA) and 12H46E-R (GCTGCACYTTGACCTG-ACGT) developed by Heinicke et al. [30], whereas the 16S gene was obtained with the primers 16SC (GTRG-GCCTAAAAGCAGCCAC) and 16Sbr-H (CCGGTCTG-AACTCAGATCACGT) developed by Darst and Cannatella [31] and Palumbi et al. [32], respectively. Each polymerase chain reaction (PCR) reaction contained a final concentration of 3-mM MgCl 2 , 0.2-mM dNTPs, 0.05 U/µL Taq DNA polymerase (Invitrogen) and 0.2-µM each primer, in a total volume of 25 µL. DNA amplification was achieved using the following touchdown protocol: 3-min denaturation at 94°C; 10 cycles of 30 s at 93°C, 30 s at 67°C decreasing 1°C/cycle and 1 min at 72°C; 18-28 cycles (depending on initial DNA template amount) of 30 s at 93°C, 30 s at 58°C and 1 min at 72°C; and final extensión of 7 min at 72°C (this work). Single PCR products were visualized in 1.5% agarose gel, and unincorporated primers and DNTPs were removed from PCR products with ExoSap (ExoSap-it, Affimetrix). Cycle sequencing reactions were conducted by a commercial company Macrogen Inc. Data from heavy and light stands were compared to generate a consensus sequence for each DNA fragment using Geneious 6.05. [33] Phylogenetic analyses Sequences were aligned using MAFFT v. 7, [34] with the Q-INS-i strategy. Maximum likelihood (ML) trees were estimated using GARLI 2.01 (genetic algorithm for rapid likelihood inference [35]). GARLI uses a genetic algorithm that finds the tree topology, branch lengths and model parameters that maximize lnL simultaneously. [35] Individual solutions were selected after 10,000 generations with no significant improvement in likelihood, with the significant topological improvement level set at 0.01; 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. [35] Bootstrap support was assessed via 1000 pseudoreplicates under the same settings used in tree search. GenBank accession numbers are listed in Appendix 2.

Vocalizations
Sound recordings were made with an Olympus LS-10 Linear PCM Field Recorder and a Sennheiser K6-ME 66 unidirectional microphone. The calls were recorded in WAV format with a sampling rate of 44.1 kHz/second with 16 bits/sample. Audio of KU specimens were recorded by W.E. Duellman with an Uhler 4000 S and microphone onto analogue tapes. The tapes were digitized at the Macaulay Library using their standard protocols (http://macaulaylibrary.org). A call is defined as the collection of acoustic signals emitted in sequence and produced in a single exhalation of air. A note is a temporally distinct segment within a call; notes are separated between them by a return to the background noise. Pulsed notes are considered those having one or more clear amplitude peaks, while tonal notes have relatively constant amplitude throughout the call. A call series is defined as a sequence of calls that are separated by a consistent time interval of background noise between calls.
The parameters above were measured using the R package SEEWAVE, [37] with a custom script to quickly batch analysed calls (available upon request). The analysis of calls was automated with the following routine: (1) audio file is normalized and filtered of background noise and other non-target frog sounds (insects, stream noise, rain, etc.), using a band-pass filter set to 2000-5000 Hz (removes sound that generates frequencies outside this range); (2) target calls are located in the filtered audio file by recording the start time of each amplitude increase above a 10% threshold; (3) each identified call was vetted to ensure that it was a call of the target species and not other sounds; (4) each individual call is normalized to a relative scale, which removes the effects of amplitude variation between calls; (5) using the call start times and the original recording, each call was saved as an unfiltered file; (6) recordings were analysed separately using various functions (for the call parameters above) available in SEEWAVE; and (7) significant outliers falling outside the 95% confidence intervals of the measurement data were manually inspected to ensure accuracy. Additionally, call durations and inter-note/call intervals were measured manually as it was difficult to detect the start and end of calls due to background noise. Recordings with excessive background noise (~25% amplitude) were carefully examined using frequency spectra and pulses above the background noise.  Finally, we evaluated the amount of bioacoustic differences between species using several criteria. We considered call variables influenced by body size (e.g. frequencies), motivation (e.g. call rate) and/or temperature (e.g. call rate) to be inadequate when left uncorrected for these factors. We considered differences to be important when the general call structure varied (pulsed vs. tonal notes, note arrangement and call arrangement) or when temporal variables varied independent of body size, temperature and motivation.

Population size
During four consecutive nights (24-27 February 2015; rainy season), we surveyed a 200 × 4 m transect along the stream La Laguna (0.1665°N, 78.8713°W; 1015 m). Each night, the stream was sampled by two people (LB and Frank Pichardo) for four hours starting at 20:00 h. All adult frogs were marked with standard procedures (toe-clipping); sex of marked individuals was assessed by external features (e.g. coloration, body size and presence/absence of vocal sac). Located frogs were swabbed for B. dendrobatidis (see below) and photographed. The population size of Hyloscirtus mashpi n. sp. was calculated using the program Mark v. 8.0. [38] We assumed that the population was closed, meaning that the model assumes that no births, deaths, immigration or emigration occur; given that the sampling period was only four days, this is a reasonable assumption. We used the Full Likelihood p and c model described in Otis et al. [39].

Diagnosis of B. dendrobatidis (Bd)
In the field, all captured individuals were swabbed, following the procedures described in Hyatt et al. [40]. Dry swabs were stored in −4°C until analyses. Testing for Bd was carried out using end-point PCR. DNA extractions were obtained with a salt precipitation method (M. Fujita, unpubl.) based on the Puregene DNA purification kit (Gentra Systems). Bd presence was tested using the primers Bd1a (5′-CAGTGTGCCATATGTCACG-3′) and Bd2a (5′-CATGGTTCATATCTGTCCAG-3′) and PCR protocol described by Annis et al. [41]. PCR reaction was set up to a final concentration of 3-mM MgCl 2 , 0.2-mM dNTPs, 0.05 U/µL Taq DNA polymerase (Invitrogen) and 0.5-µM of each primer in a 25 µL total volume. PCR protocol followed Annis et al. [41], except that 35 cycles were performed. When the PCR product retrieved was insufficient or dubious, an additional PCR was carried out, using a 1:50 dilution of the cleaned-up product from the first PCR as the template. The conditions of this second PCR were the same as described above, but fewer cycles were performed. A negative control, containing distilled water instead of DNA, was used each time. The presence/absence of Bd was determined by visualization of the amplified band in agarose gel electrophoresis.

Ethics statement
Animal research was performed under the approval and supervision of the Centro de Investigación de la

Phylogenetic analyses
The inferred topology ( Figure 1) generally agrees with those presented in studies with similar taxon and gene sampling, [3,6,27,28] and differences are mostly explained by more complete taxon sampling in this study (i.e. sister relationship between H. alytolylax and new species).
H. mashpi n. sp. can be distinguished from H. palmeri (characters of the latter in parenthesis) mainly by having a noticeably smaller, non-overlapping body size (male SVL 34.9-41.4 mm, female SVL 35.7-50.0), mid-dorsal stripe usually present (absent), foot webbing pale yellowish-green (orange) and by lacking a heel calcar (present; Figure 5). In Ecuador, H. mashpi n. sp. and H. palmeri have overlapping elevational ranges, and have been found in sympatry in one locality (Milpe).
Finally, when comparing H. mashpi n. sp. to its sister species (H. simmonsi; Figure 6C), H. simmonsi differs by having four notes in all recorded calls and longer, non-overlapping call duration than H. mashpi n. sp. (Table 2). Also, H. simmonsi is a larger species, with a male SVL of 35.0-37.0 mm, and is only known from the Pacific flank of Andes in Colombia at elevations of 1100-2000 m. [9] Description of holotype. An adult male of 32.7 mm SVL. Body relatively slender. Head slightly longer than wide (Head length = 35% of SVL; Head width = 34% of SVL). Snout rounded in dorsal view and profile; canthus rostralis distinct, slightly concave; lips rounded, not flared. Pale canthal stripe present. Nostrils not protuberant, directed anterolaterally at the level of the anterior margin of lower jaw. Internarial region and top of head flat. Interorbital distance longer than upper eyelid. Eye prominent, its diameter about 11% of SVL. Tympanum visible, tympanic annulus inconspicuous, rounded and subtly discernible; its diameter is about 4% of SVL. Supratympanic fold developed, starting at posterior end of upper eyelid and reaching posterior margin of insertion of arm; supratympanic stripe present, lighter than the rest of the body. Mental gland present, diamond shaped, partially covering gular area and extending about half the length of throat.
Dentigerous processes of vomers conspicuous, straight, narrowly separated from each other; each process bears 11 (right) and 12 (left) teeth. Choanae large, elliptical, not concealed by palatal shelf of maxillary arch. Tongue cordiform, attached overall (narrowly free around lateral and posterior margin); vocal slits present, longitudinal, originating on sides of tongue and extending to posterolateral corner of mouth. Vocal sac moderately distensible, evident externally, single, median and subgular. Forearm moderately robust; axillary membrane absent. Outer ulnar fold present. Fingers relatively short, thick, bearing small, ovoid discs; each disc only slightly expands laterally, and with clearly defined circumferential groove; disc on finger III about same width as tympanum diameter. Relative lengths of fingers I < II < IV < III. Fingers with fleshy dermal fringes; webbing present only between outer fingers; webbing formula II 2 --3 III 2 1/2 -2 -IV. Subarticular distal tubercles large and elliptical. Supernumerary tubercles present, fleshy and small. Palmar tubercle poorly differentiated. Inner metacarpal tubercle large, thick, elliptical; broad elliptical prepollex, not modified as a spine. Nuptial excrescencies absent.
Colour in life of the holotype. Dorsum pale yellowishgreen, with dark brown interorbital and mid-dorsal stripes. Yellowish-white line on canthus, external border of upper eyelid and supratympanic fold. Venter whitishcream. Iris brown with thin black reticulation (Figure 2).
Colour in preservative of the holotype. Head cream with small brown blotch at tip of the snout and irregular dark brown interorbital stripe; whitish supratympanic fold that matches the coloration of the external border of upper  Figure 3). Colour variation. The two observed females have a brown dorsal coloration, with a darker mid-dorsal stripe (Figure 7). In males, dorsal coloration varies from pale green with a faint brown mid-dorsal stripe to brown with a darker mid-dorsal stripe (Figures 5 and 7). In few individuals, the mid-dorsal stripe is nearly indistinguishable in life. All observed metamorphs and juveniles have a dark brown mid-dorsal stripe (Figure 7). Under stress conditions (e.g. handling), dorsal surfaces of individuals turn darker ( Figure 5(C)).

Measurements of the holotype (in mm
Variation and sexual dimorphism. Females are larger than males (male SVL 28.7-33.8; female SVL 37.0-38.5).
Vocalizations. We recorded 83 calls from four individuals of H. mashpi n. sp. ( Table 2). The call of this species sounds like a rapid set of rings to the ear. Hyloscirtus mashpi n. sp. emits calls with two or three notes per call ( Figure 7A). The call duration is 330.9-380.2 ms (353 ± 11.2), with a note interval of 115-162 (139 ± 13) ms. The fundamental frequency corresponds with the dominant frequency, which has a lower bound at 2721-2773 (2746 ± 13) Hz and an upper bound at 3024-3065 (3043 ± 10) Hz. The dominant frequency at peak amplitude is 2842-2929 (2907 ± 37) Hz, with no frequency modulation throughout the call. The first harmonic is 6091-6147 (6115 ± 15) Hz. See Table 2.
Conservation. The main threats to amphibian diversity are habitat destruction, climate change, infectious diseases and introduced species.[54] The known distribution of H. mashpi n. sp. lies within three protected areas, Reserva de Biodiversidad Mashpi, Área de Conservación y Uso Sustentable Mashpi-Guaycuyacu-Saguangal (ACUS) and Reserva Milpe. Thus, the habitat of the species at type localities is mostly protected. Also, as mentioned above, H. mashpi n. sp. is an abundant species with seemingly healthy populations, including reproductive adults, juveniles and tadpoles. We assessed the presence of the chytrid fungus B. dendrobatidis (Bd) in 34 adults of H. mashpi n. sp., 6 of which tested positive for the fungus. This means that the prevalence of Bd in this sample is 17.6%. None of the frogs that tested positive for the chytrid showed any obvious sign of sickness; on the contrary, all individuals were active and calling when found. Given the characteristics of the populations of H. mashpi n. sp., we speculate that this species is tolerant to Bd, as observed in other species. [42][43][44] We suggest that the most appropriate IUCN conservation category for H. mashpi n. sp. is Data Deficient, mainly because assessment of the distribution of H. mashpi n. sp. still requires an extensive re-examination of collections of H. alytolylax.
Etymology. The specific epithet mashpi is used as a noun in apposition and refers to one of the localities where the species is found, Reserva de Biodiversidad Mashpi, a protected area where research and conservation efforts are carried out by Reserva de Biodiversidad Mashpi S.A., Universidad Tecnológica Indoamérica, Tropical Herping and other institutions. The word mashpi is a Yumbo word that means 'friend of water', which is a precise description of this treefrog, which is always found along pristine streams.

Discussion
Given that our study has a similar taxon and gene sampling than previous ones, it is not surprising that the recovered phylogenies are mostly congruent. As shown before, [3,4,6,7,27,28] the genus Hyloscirtus is divided into three clades (H. armatus group, H. bogotensis group and H. larinopygion group). The two species of the H. armatus group (H. armatus and Hyloscirtus charazani) form a well-supported clade that is sister to the species in the H. larinopygion group, although this relationship has weak support (bootstrap = 61). The close relationship between the H. armatus and H. larinopygion groups has been recovered in recent studies by Faivovich et al. [3] using parsimony, and Coloma et al. [28] using both parsimony and Bayesian criteria, although Bayesian support values in the later study were not significant. ML analysis by Coloma et al. [28] suggests, with low support, that the H. armatus group is sister to the H. bogotensis group. Thus, at the moment, it is premature to conclude about the relationships among the three groups in Hyloscirtus, mainly because most of the analyses show non-significant support values at this level and, as described above, even topological contradictions (e.g. Coloma et al. [28]: Figure 2). In contrast, the monophyly of each of these groups (i.e. armatus, bogotensis and larinopygion) has been recovered in all recent analyses. [3, 5, 28, this study] Within the Hyloscirtus bogotensis group, genetic data support the existence of at least two cryptic species in what is currently recognized as H. alytolylax, as suggested by Arteaga et al. [20]. The undescribed species are: Hyloscirtus sp., from South-Western Ecuador (El Oro province), and H. mashpi n. sp., described above. We have decided not to describe the species from El Oro province until more data (such as specimens and calls) are obtained. Other noteworthy result includes the genetic separation between the populations of H. phyllognathus from Northern and Southern Ecuador and the existence of a potential new species (MZUTI 192, Napo province, Ecuador) that morphologically resembles H. phyllognathus. Within H. palmeri, the reciprocal monophyly and genetic distance (uncorrected p distance = 5.7-6.3%) between Panamanian and Ecuadorian populations is not surprising since we lack sampling from Colombia, and thus the effect of isolation by distance is expected.
The diversity of neotropical frogs, particularly Andean groups such as Hyloscirtus, has been drastically underestimated, as revealed by the number of recent descriptions of new species. [28, 29, this work] This underestimation of diversity highlights the continued need for field expeditions to under-studied areas and habitats such as Andean torrents. [9] Additionally, the cryptic nature of the Hyloscirtus group emphasizes the necessity of detailed comparisons in morphologically and ecologically conserved groups that may harbour significant cryptic diversity. For organisms such as frogs, this will often require data from molecular markers, external morphology and advertisement calls to establish the presence of distinct species boundaries. [23,28] These data may also be useful in future comparative analyses to determine the influence of traits like advertisement call to speciation via sexual selection.
Speciation process in Hyloscirtus seems to be influenced mainly by a combination of the following factors: (1) Linearity of the Andes [45] Finally, our data indicate that Hyloscirtus mashpi n. sp. is Bd positive, but maintains apparently healthy and robust populations at numerous sites. This suggests that H. mashpi n. sp. may exhibit total or partial tolerance to Bd, as do other amphibian species. [42,43,50,51] Within Hyloscirtus, Bd has been diagnosed in few additional species [44,52,53]; however, further studies are needed to determine the impact, if any, of the infection on population dynamics. In any case, monitoring of H. mashpi n. sp. is in place, and our preliminary results so far indicate that the populations are stable. This is heartening, as Hyloscirtus are generally considered rare and elusive species, [28] and would otherwise have been thought strongly susceptible to the general decline and extinction threatening other amphibians worldwide. [54][55][56] Author contributions JMG conceived and designed the study. JMG wrote the first draft of the manuscript. MR, AA, JC, LB, RAP, NP, CM and CRH reviewed and improved the manuscript. JMG, NP and CRH analysed the data. JMG, MR, AA, JC, LB, RAP, NP and CM collected specimens, calls and swabs.