Computational protocol: A new species of Rana from the Dabie Mountains in eastern China (Anura, Ranidae)

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Protocol publication

[…] Sampling: In total, 17 specimens of Rana sp. were collected from Yaoluoping National Nature Reserve in Dabie Mountains, Yuexi County, Anhui Province, China (30°58'16.92"N, 116°04'11.88"E, elevation 1150 m a.s.l.) (Fig. ), in August 2015 and August 2016. Those individuals were dipped in 10% formalin (10 seconds) for fixation and subsequently transferred into 75 % ethanol for storage. Before fixing in formalin, liver tissues from all individuals were sampled and preserved in 100% ethanol for molecular analyses. All specimens and tissue samples were deposited in the Anhui University Museum, Research Center for Biology. DNA extraction, PCR amplification and sequencing: Genomic DNA was extracted from liver tissues of seven Rana sp. specimens using the standard proteinase K/phenol-chloroform protocol (). Four mitochondrial genes (12S rRNA, 16S rRNA, ND2, and Cyt b) and three nuclear DNA markers (Tyr, BDNF and RAG1) were sequenced for one individual, while for six remaining specimens only Cyt b mtDNA gene was sequenced. The primers used for PCR and sequencing are summarized in Table . All PCRs were performed with the same conditions in 50 μL: 20 to 80ng of genomic DNA, 25 μL 2×EasyTaq PCR SuperMix polymerase (TransGen Biotech, containing 1.25U Ex Taq, 0.4mM dNTP, 4mM Mg2+) and 0.4 μM of primers. Reactions were performed with the following profile: PCR cycles were 5 min at 95 °C followed by 35 cycles of 30 s at 95 °C, 30 s at appropriate annealing temperature (Table ), and 1 min at 72 °C, with a final extension at 72 °C for 10 min. The PCR products were purified using an EasyPure PCR Purification Kit (TransGene), and sequenced directly using the primers used in PCRs and the BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems) following the manufacturer’s instructions on an ABI Prism 3730 automated sequencer. Phylogenetic analyses: 147 sequences were used for genetic analysis, which include 135 sequences within the Chinese Rana were downloaded from NCBI and 12 sequences in this study. The data are summarized in Table . All nucleotide sequences were aligned using MUSCLE () with default parameters and checked manually with MEGA 5.0 (), the length of the fragments was trimmed; newly obtained sequences were deposited in GenBank (Table ). Nucleotide sites with ambiguous alignments were deleted from the analyses. Bayesian inference (BI) and maximum likelihood (ML) analyses were conducted using the six concatenated gene fragments. BI analyses were performed in MRBAYES v3.1.2 () using the optimal partitioning strategy and best-fit nucleotide substitution model for each region (Table ) selected by PARTITIONFINDER v1.1.1 (). MRBAYES analyses simultaneously initiated two Markov Chain Monte Carlo (MCMC) model runs to provide additional confirmation of convergence of posterior probability distributions. Analyses were run for 10,000,000 generations. Chains were sampled every 1000 generations. The first 25% of the total trees were discarded as "burn-in" and the remaining trees were used to generate a majority-rule consensus tree and to calculate Bayesian posterior probabilities. Nodal support was further assessed with a maximum-likelihood (ML) analysis in RAXML V.7.0.3 with 1000 bootstraps. Pelophylax nigromaculatus sequences were downloaded from GenBank and used as outgroup.Apart from phylogenetic tree-based methods, we also calculated pairwise sequence divergence based on uncorrected p-distance using MEGA 5.0 () to determine the genetic distance between species. The analysis compared the 7 individuals of Rana sp. from the Dabie Mountains to other 22 species of the genus Rana inhabiting China. Morphological analyses: The morphometric data were examined for 10 individuals. Measurements were made by Yanan Zhang using a vernier caliper with a precision of 0.1 mm. 17 linear measurements () were taken as follows: SVL (snout-vent length, from tip of snout to vent); HL (head length, from posterior corner of mandible to tip of snout); HW (head width, the greatest cranial width); SL (snout length, from tip of snout to the anterior corner of the eye); IN (internarial distance); ED (horizontal eye diameter); IO (interorbital distance, the minimal distance between upper eyelids); UE (upper eyelid width, the maximal width of upper eyelid); TD (horizontal tympanic diameter); LAHL (length of lower arm and hand, from the tip of finger III to the elbow joint); HAL (hand length, from proximal end of outer palmar tubercle to tip of the third finger); LAD (diameter of lower arm); HLL (hind limb length, from the tip of the toe IV to groin); TL (tibia length); TW (tibia width, the greatest width of tibia); FL (foot length, from the proximal end of the inner metatarsal tubercle to the tip of the toe IV) and TFL (length of tarsus and foot, from the proximal end of tarsus to the tip of the fourth toe IV).The description of toe webbing followed Savage (1975) (Table ). The morphological characters of the individuals of Rana sp. from Dabie Mountains were compared with the members in the R. longicrus group, R. chaochiaoensis Liu, 1946, R. culaiensis Li, 2008, R. hanluica Shen, Jiang & Yang 2007, R. jiemuxiensis Yan, 2011, R. longicrus Stejneger, 1898, R. maoershanensis Lu, 2007, R. omeimontis and R. zhenhaiensis Ye, 1995. We also compared individuals collected in this area with other Rana species distributed in other parts of China R. arvalis Gislén, 1959 (previously listed as R. altaica, identification following ), R. amurensis Boulenger, 1886, R. luanchuanensis Zhao, 2017, R. asiatica Bedriaga, 1898, R. chensinensis David, 1875, R. dybowskii Guenther, 1876, R. huanrenensis Liu, 1993, R. kukunoris Nikolsky, 1918 and R. kunyuensis Lu, 2002. Considering the restrictions of samples, the morphological characteristics of the species in Rana were obtained from literature (, , , , , , ). […]

Pipeline specifications

Software tools MEGA, MrBayes, PartitionFinder, RAxML
Application Phylogenetics