The complete mitochondrial genome information of Hynobius unisacculus (Amphibia, Caudata, Hynobiidae) and the phylogenetic implication.

Hynobius unisacculus is a hynobiid salamander species found only in a limited area at the southernmost part of the Korean Peninsula. Here, we characterized the complete mitochondrial genome of this species that was used to identify the phylogenetic relationship with other Hynobius species. The whole sequence was 16,411 bp and included 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. The gene arrangement was completely identical to those observed in other Hynobius species. Upon robust phylogenetic tree reconstructed based on 13 protein-coding genes, Hynobius species living on the Korean Peninsula showed close phylogenetic affinity; H. unisacculus and H. quelpaertensis formed a cluster that was the sister to the cluster of H. leechii and H. yangi.

Over two decades, several new Hynobius salamander species have been discovered on the Korean Peninsula, a very small area on the southern tip of East Asia (Kim et al. 2007; Baek, Lee, Lee et al. 2011a; Baek, Lee, Song et al. 2011b; Min et al. 2016). Hynobius unisacculus is the fourth Hynobius species officially recorded on this peninsula after H. leechii, H. quelpaertensis and H. yangi (Baek, Lee, Lee et al. 2011a; Baek, Lee, Song et al. 2011b; Min et al. 2016). Upon a phylogenetic tree reconstructed based on cyt b and 12S rRNA, this species formed a single cluster with H. quelpaertensis among these three previously known Hynobius species (Baek, Lee, Lee et al. 2011a; Baek, Lee, Song et al. 2011b; Min et al. 2016). Hynobius unisacculus is found only in limited areas (Naro Islands, Goheung-gun, Suncheon-si, and Boseong-gun, Jeollanam-do, South Korea) at the southernmost part of the Korean Peninsula (Min et al. 2016). This species has not yet been designated as a legal protection target in South Korea. The phylogenetic structure of this species can thus provide important data for estimating the biogeographical pathways on the Korean Peninsula and establishing the management strategies.

We characterized the complete mitochondrial genome of this species to identify the robust phylogenetic relationship with other Hynobius species. The DNA sample was isolated from an ethanol-immersed specimen stored in the Conservation Genome Resource Bank for Korean Wildlife (CGRB: http://www.cgrb.org/) that was collected (34.438139, 127.478556) in 2010. The specimen is accessible as cgrb15732 in CGRB. Mitogenomic sequences were extracted using the CLC genomics workbench 6.5 (http://www.qiagenbioinformatics.com/) from the reads (5,406,233,748 bp) generated from the MiSeq platform. Each mitochondrial region was annotated using the MITOS web server (Bernt et al. 2013) and manually checked using the mitochondrial information of H. leechii (Zhang et al. 2006). The sequence information was deposited at NCBI GenBank under the accession number of MN242821.

The whole sequences were 16,411 bp and included 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. L-strand was observed in eight tRNA genes and ND6. Every protein-coding gene contained ATG start codon with a single exception in COX1 starting with GTG. ND1, COX1, APT8, APT6, ND3, and ND4L were terminated by TAA, ND1, and COX2 by TAG, and ND6 by AGA. Incomplete stop codon was detected at COX3 (ATA), ND4 (CAT), ND5 (TTA) and Cyt b (ATA). The gene arrangement was completely identical to those observed in other Hynobius species (Zhang et al. 2006). Based on 13 protein-coding genes, we examined the phylogenetic placement of this species in the genus Hynobius (Figure 1). Korean species showed close phylogenetic affinity; H. unisacculus and H. quelpaertensis formed a cluster that was the sister to the cluster of H. leechii and H. yangi (Figure 1). Our study is predicted to be useful to reconstruct the consensus phylogenetic tree that is essential for the future study of biogeographic dispersal of terrestrial animals on the Korean Peninsula, which also can provide an important guideline to establish the management strategies for Hynobius species conservation.

Figure 1.

Bayesian inference tree of the genus Hynobius reconstructed by MrBayes 3.2 (Ronquist et al. 2012) using 13 protein-coding genes of H. unisacculus (bold) and 11 Hynobius species extracted from our analysis and NCBI GenBank. Onychodactylus fischeri in the same family was used as outgroup. GTR + I+G was selected as the best-fit substitution model by jModeltest 2.1.4 (Darriba et al. 2012) under Akaike information criterion (Akaike 1974), and two parallel runs were performed for one million Markov Chain Monte Carlo (MCMC) generations with sampling every 1000 steps. Posterior probabilities were indicated on the nodes.