Do Cryptic Species Exist in Hoplobatrachus rugulosus? An Examination Using Four Nuclear Genes, the Cyt b Gene and the Complete MT Genome

Abstract

he Chinese tiger frog Hoplobatrachus rugulosus is widely distributed in southern China, Malaysia, Myanmar, Thailand, and Vietnam. It is listed in Appendix II of CITES as the only Class II nationally-protected frog in China. The bred tiger frog known as the Thailand tiger frog, is also identified as H. rugulosus. Our analysis of the Cyt b gene showed high genetic divergence (13.8%) between wild and bred samples of tiger frog. Unexpected genetic divergence of the complete mt genome (14.0%) was also observed between wild and bred samples of tiger frog. Yet, the nuclear genes (NCX1, Rag1, Rhod, Tyr) showed little divergence between them. Despite this and their very similar morphology, the features of the mitochondrial genome including genetic divergence of other genes, different three-dimensional structures of ND5 proteins, and gene rearrangements indicate that H. rugulosus may be a cryptic species complex. Using Bayesian inference, maximum likelihood, and maximum parsimony analyses, Hoplobatrachus was resolved as a sister clade to Euphlyctis, and H. rugulosus (BT) as a sister clade to H. rugulosus (WT). We suggest that we should prevent Thailand tiger frogs (bred type) from escaping into wild environments lest they produce hybrids with Chinese tiger frogs (wild type).

Characterization of the mitochondrial genomes of two toads, Anaxyrus americanus (Anura: Bufonidae) and Bufotes pewzowi (Anura: Bufonidae), with phylogenetic and selection pressure analyses

“…Total DNA was extracted from the toe-clips samples of A. americanus or Bufotes pewzowi using an Ezup Column Animal Genomic DNA Purification Kit (Sangon Biotech Company, Shanghai, China). The 11 universal primers for standard polymerase chain reaction (PCR) amplification of mitogenomes were slightly modified according to Yu et al (2015) and Zhang et al (2013) and 15 specific primers were designed based on the sequenced fragments from universal primers using Primer Premier 5.0 (PREMIER Biosoft International, CA, USA) (Tables S1 and S2). All PCR amplifications were carried out in a 50 µL reaction mixture and the procedures were performed using an Eppendorf Thermal Cycler (Mastercycle R nexus GSX1, Hamburg, Germany).…”

“…We used both standard PCR and Long-PCR methods with TaKaRa Ex-Taq and TaKaRa LA-Taq Kits (Takara Biomedical, Dalian, China). These two methods were slightly modified from Yu et al (2015) and Zhou et al (2009). All PCR products were detected by electrophoresis on 1% agarose gels, and sequences were obtained in an automated DNA sequencer (ABI 3730) by Sangon Biotech Company (Shanghai, China).…”

Peer Review #1 of "The complete mitochondrial genome of Pyxicephalus adspersus: high gene rearrangement and phylogenetics of one of the world’s largest frogs (v0.2)"

“…Total DNA was extracted from the clipped toe of one specimen using a DNeasy Tissue Kit (Qiagen, Germany). We amplified thirteen overlapping gene fragments by normal polymerase chain reaction (PCR) and long-and-accurate (LA) PCR methods slightly modified from Yu et al (2015) and Zhang et al (2013). However, these methods failed to find some mitochondrial genes including ND3, ND5, and tRNA Ile , tRNA Cys , tRNA Lys , and tRNA Arg .…”

“…BI and ML analyses were performed with anuran mt genomes including P. adspersus. In total this included species as the ingroup from Ranidae, Dicroglossidae, Rhacophoridae, Mantellidae, Pyxicephalidae, Petropedetidae, Ptychadenidae, Ceratobatrachidae and Phrynobatrachidae (Alam et al, 2010;Chen et al, 2011;Hofman et al, 2012;Huang et al, 2016a;Huang et al, 2016b;Jiang et al, 2017;Kakehashi et al, 2013;Kurabayashi et al, 2006;Kurabayashi et al, 2010;Li et al, 2016b;Li et al, 2014b;Lin et al, 2014;Liu et al, 2017a;Liu et al, 2017b;Liu et al, 2005;Ni et al, 2015;Ren et al, 2009;Sano et al, 2004;Sano et al, 2005;Xia et al, 2014;Yan et al, 2016;Yang et al, 2018;Yu et al, 2015;Yu et al, 2012a;Yu et al, 2012b;Zhang et al, 2016;Zhang et al, 2018a;Zhang et al, 2009;Zhang et al, 2013;Zhang et al, 2005;Zhao et al, 2018;Zhou et al, 2009) and four species as outgroups from Microhylidae (Chen et al, 2016;Zhao et al, 2018). We used the nucleotide data to assess BI and ML topology to discuss the phylogenetic position of Pyxicephalus.…”

“…Manuscript to be reviewed species compared to other species of Scapharca, had largest mitochondrial genomes among the genus Scapharca (Liu et al, 2013). A species with a giant size within an animal group may be associated with a larger mitogenome (Alam et al, 2010;Yu et al, 2012b;Yu et al, 2015;Zhang et al, 2018a;).However, no studies in P. adspersus has examined the potential involvement of mt genome evolution. So we wondered whether this giant bullfrog had novel features of its mt genome.…”