DOI: 10.5061/dryad.c59zw3rjj
title: A comprehensive molecular phylogeny of the genus Sylvirana (Anura: Ranidae) highlights unrecognized diversity, revised classification and historical biogeography

description: The genus Sylvirana includes 12 species widely distributed in
 South China and Southeast Asia. The phylogenetic relationships
 and species diversity for Sylvirana and
 allied genera remain unresolved and controversial due to
 insufficient data and incomplete taxon sampling. Using a combined dataset
 of mitochondrial genes (16S and COI) and 101 nuclear genes obtained
 through the amplicon sequence capture approach, we generated the most
 comprehensive phylogenetic analysis for the genus Sylvirana to
 date, inferring diversity, phylogenetic relationships, and historical
 biogeography with unprecedented levels of taxon and geographic
 sampling. Our results conservatively reveal
 six undescribed species, mostly distributed in peninsular
 Indochina. Phylogenetic analyses strongly support the
 non-monophyly of Sylvirana with respect to Pterorana.
 Additionally, phylogenetic results place Sylvirana
 guentheri and Pelophylax lateralis into genus Humerana,
 supporting the inclusion of
 Hylarana latouchii, Papurana milleti, and
 Hylarana attigua within Pterorana + Sylvirana.
 The long-disputed species of Hylarana bannanica (previously
 Sylvirana) cluster with genus Papurana. Because the results of
 multiple non-monophyletic genera create taxonomic confusion, we
 suggest relegating all genera to subgenus rank
 of Hylarana. Sylvirana is a junior synonym of
 the Pterorana. Biogeographically, we trace the origin
 of Pterorana to Southeast Asia during the early Miocene,
 with subsequent dispersal thereafter. Our study shows that
 climatic changes may have profoundly inﬂuenced the diversification of
 Pterorana during the Miocene.
description: Library preparation, bait preparation, hybridization, and
 sequencing were conducted at Sun Yat-sen University. The protocol followed
 that of Zhang et al. (2019). In brief, we mixed 20 ng of genomic DNA of
 each of the 38 samples to make a DNA pool. The pooled DNA was used as
 template to amplify 101 nuclear protein-coding (NPC) markers using the
 primers and protocol developed by Shen et al. (2013). The 101 PCR products
 were then mixed together and purified. The purified PCR mixture was
 subjected to end-repair and A-tailing and then ligated with a biotinylated
 adapter. The biotinylated amplicons were subsequently immobilized on
 Dynabeads MyOne streptavidin magnetic beads (Life Technologies) to obtain
 bait-coated beads. For each hybridization reaction, 500
 ng of pooled libraries and 2.5 μl of bait-coated beads (containing 100 ng
 of biotinylated amplicons) were used. A touch-down hybridization program
 was adopted. After denaturation, the hybridization started from 65°C,
 decreased by 5°C every 6 hours and ended at 45°C, for a total duration of
 30 hours. The captured libraries were amplified, pooled in equal
 concentrations, and sequenced on an Illumina HiSeqX10 sequencer using the
 paired-end 150-bp mode. Data processing followed Zhang
 et al. (2019). In brief, the Illumina paired-end reads were
 quality-controlled using Trimmomatic (Bolger et al., 2014), FastQC
 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and FastUniq
 (Xu et al., 2012). Clean reads of each sample were assembled into contigs
 using the SPAdes v.3.8.1 genome assembler (Bankevich et al., 2012). The
 sequencing depths for the filtered contigs were calculated by SAMtools
 v.1.4.1 (Li et al., 2009). Only contigs with an average sequencing depth ≥
 5× were retained for further analysis. The 101 NPC marker sequences were
 used as the reference to call the orthologous sequences from the contigs
 set of each sample by tBLASTN. Then, a reversed BLASTN was performed with
 the orthologous contigs obtained from the preceding steps against the
 reference sequences to identify potential chimeras. Following this
 process, the orthologous contigs set for each sample contained no more
 than one sequence for each marker.
description: # A comprehensive molecular phylogeny of the genus Sylvirana (Anura:
 Ranidae) highlights unrecognized diversity, revised classification and
 historical biogeography
 [https://doi.org/10.5061/dryad.c59zw3rjj](https://doi.org/10.5061/dryad.c59zw3rjj) ## Description of the data and file structure To further resolve the phylogenetic relationship of genus Sylvirana, we obtained nuclear sequence capture data from 101 loci for a subset of 12 species, six allied species (H. latouchii, H. bannanica, H. attigua, Pterorana khare, Pelophylax lateralis, and Papurana milleti), and five candidate species. To better understand the temporal evolution of the genus Sylvirana, 15 species were selected as outgroup taxa. In total, 38 species and candidate species were sequenced for nuclear loci. ### Files and variables #### File: Nu\_Hylarana2.fas **Description:** 101 nuclear sequence data for a subset of 12 species, six allied species (*H.* *latouchii*, *H. bannanica*, *H. attigua*, *Pterorana khare*, *Pelophylax lateralis*, and *Papurana milleti*), and five candidate species