Computational protocol: A new species of Phrynopus (Amphibia, Anura, Craugastoridae) from upper montane forests and high Andean grasslands of the Pui Pui Protected Forest in central Peru

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[…] Fieldwork. The puna of the PPPF was reached by walking 1.5 days along a trail from Toldopama (11°30'15.4"S, 74°55'32.7"W, 3670 m a.s.l., two hours by car from Satipo) to Tarhuish (11°23'23.2"S; 74°57'02.5"W, 3783 m a.s.l.; Fig. ) with the help of local guides. Fieldwork was conducted in puna and upper montane forests in 2012 between May 8 and 21 by EL and RvM, and in 2013 between June 21 and July 8 by EL, JM, and JCC. Collected specimens were preserved in 96% ethanol and stored in 70% ethanol. Morphological characters. The format for the description follows , except that the term dentigerous processes of vomers is used instead of vomerine odontophores (), and diagnostic characters are those of . Taxonomic classification follows , except that we followed for family placement. Sex and maturity of specimens were identified by observing gonads through dissections. The senior author measured the following variables to the nearest 0.1 mm with digital calipers under a stereomicroscope: snout-vent length (SVL), tibia length (TL, distance from the knee to the distal end of the tibia), foot length (FL, distance from proximal margin of inner metatarsal tubercle to tip of Toe IV), head length (HL, from angle of jaw to tip of snout), head width (HW, at level of angle of jaw), horizontal eye diameter (ED), interorbital distance (IOD), upper eyelid width (EW), internarial distance (IND), eye-nostril distance (E-N, straight line distance between anterior corner of orbit and posterior margin of external nares), and egg diameter. Fingers and toes are numbered preaxially to postaxially from I–IV and I–V, respectively. We compared the lengths of toes III and V by adpressing both toes against Toe IV; lengths of fingers I and II were compared by adpressing these fingers against each other. All drawings were made using a stereomicroscope and a camera lucida. Photographs of live specimens were used for descriptions of coloration in life and for evaluation of morphological characters that might have been impacted by the preservation process. Information on species for comparative diagnoses was obtained from and from original species descriptions. For specimens examined see Appendix I. Codes of collections are: MUSM – Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Lima, Peru; NMP6V – National Museum, Prague, Czech Republic; UMMZ – University of Michigan Museum of Zoology, Ann Arbor, USA. Field number code is: IWU – Illinois Wesleyan University, Bloomington, USA. Threat status was evaluated using the IUCN criteria (IUCN Standards an Petitions Subcommittee 2016). Maps. Maps were made with ArcGIS 10.0 (). The estimated area was calculated by a minimum convex polygon using known sites of occurrence of the species as defined by . Molecular phylogenetic analysis. Our analysis included DNA sequence data from Phrynopus species that were available in GenBank (as of 1 August 2017; Table ) as well as sequences from other closely related genera (Lynchius, Oreobates) and more distantly related ones (Ischnocnema guentheri, Hypodactylus brunneus, and H. dolops) as outgroups following the results of . Newly produced sequences include those obtained from seven specimens of the new species and one specimen of Phrynopus juninensis collected near Hacienda Cascas, Junín, the type locality of this species (Table ). Our analysis also included sequences from three mitochondrial and two nuclear genes for several species of Phrynopus included in a recent study (). The mitochondrial genes were a section of the 16S rRNA gene, a section of the 12S rRNA gene, and the protein-coding gene cytochrome c oxidase subunit I (COI). The nuclear genes were the recombination-activating protein 1 (RAG1) and Tyrosinase precursor (Tyr).Extraction, amplification, and sequencing of DNA followed protocols previously used for Neotropical terrestrial breeding frogs (, ). Primers used are listed in Appendix II. We employed the following thermocycling conditions to amplify DNA from each gene using the polymerase chain reaction (PCR). For 16S, we used: 1 cycle of 96 °C/3 min; 35 cycles of 95 °C/30 s, 55 °C/45 s, 72°C/1.5 min; 1 cycle 72°C/7 min. For 12S, we used: 1 cycle of 94°C/1.5 min; 35 cycles of 94°C/45 s, 50°C/1 min., 74°C/2 min; 1 cycle 72°C/10 min. For RAG1, we used: 1 cycle of 96°C/2 min; 40 cycles of 94°C/30 s, 52°C/30 s, 72°C/1.5 min; 1 cycle 72°C/7 min. For Tyr, we used: 1 cycle of 94°C/5 min; 40 cycles of 94°C/30 s, 54°C/30 s, 72°C/1 min; 1 cycle 72°C/7 min. We completed the cycle sequencing reactions by using the corresponding PCR primers and the BigDye Terminator 3.1 (Applied Biosystems), and obtained sequence data by running the purified reaction products in an ABI 3730 Sequence Analyzer (Applied Biosystems). We deposited the newly obtained sequences in GenBank (Table ).We used Geneious R6, version 6.1.8 (; http://www.geneious.com/) to align the sequences with the built-in multiple alignment program. Prior to conducting phylogenetic analysis, we used PartitionFinder, version 1.1.1 () to select the appropriate models of nucleotide evolution and used the Bayesian information criterion (BIC) to determine the best partitioning scheme and substitution model for each gene. The best partitioning scheme included five subsets (BIC value: 27719.16). The first partition subset included both the 12S and 16S sequences and the best fitting substitution model was GTR+I+G. The remaining four subsets were partitioned according to codon positions as follows (substitution model in parenthesis): one set including the 1st codon position of COI and the 3rd codon position of both RAG1 and Tyr (K80+G); one set with only the 2nd codon position of COI (HKY); one set with only the 3rd codon position of COI (HKY); one set including the 1st and 2nd codon position of RAG1 and the 1st and 2nd codon position of Tyr (HKY+I).We employed a Bayesian approach using MrBayes, version 3.2.0 () to infer a molecular phylogeny. Our analysis included 44 terminals and a 2684-bp concatenated partitioned dataset. We performed an MCMC Bayesian analysis that consisted of two simultaneous runs of 8 million generations, and we set the sampling rate to be once every 1000 generations. Each run had three heated chains and one “cold” chain, and the burn-in was set to discard the first 25% samples from the cold chain. At the end of the run, the average standard deviation of split frequencies was 0.002257. Following the completion of the analysis, we used Tracer 1.6 () to verify convergence. Subsequently, we used FigTree (http://tree.bio.ed.ac.uk/software/figtree/) to visualize the majority-rule consensus tree and the posterior probability values to assess node support. Additionally, we used the R package ‘APE’ () to estimate uncorrected p-distances (i.e., the proportion of nucleotide sites at which any two sequences are different). […]

Pipeline specifications

Software tools Geneious, PartitionFinder, MrBayes, FigTree, APE
Application Phylogenetics
Diseases Byssinosis