The complete mitochondrial genome Hainan Gymnure Neohylomys hainanensis

Introduction

The Hainan Gymnure Neohylomys hainanensis Shaw and Wang, 1959 is mainly distributed in Hainan, China and north Vietnam (Liu and Wu 2019; Abramov et al. 2018). It mainly inhabits the tropical forests and subtropical forests (Smith et al. 2009). To date, the phylogenetic relationships of three species (N. hainanensis, Hylomys suillus and Neotetracus sinensis) within Erinaceidae have been debated (Cobert 1988; Gould 1995; Grenyer and Purvis 2003; He et al. 2012). Previous studies (Cobert 1988; Gould 1995; Grenyer and Purvis 2003) indicated sister relationships of N. hainanensis and N. sinensis based on morphological data. However, He et al. (2012) showed that N. sinensis clustered with H. suillus rather than N. hainanensis based on combined data of mitochondrial genes (CYTB, ND2 and 12S). Complete mitochondrial genome sequences are highly informative markers for resolving high level phylogenetic relationships among taxa (Kumazawa 2007; Shen et al. 2010; Yue et al. 2015). Though the complete mitochondrial genome of N. hainanensis is available in GenBank (MW429379), we sequenced the mitochondrial genome using our sample based on primer walking method to confirm the accuracy of the data. Additionally, we investigated phylogenetic relationships of N. hainanensis among family Erinaceidae to solve the disputes.

Methods

The animal was captured using a mouse cage trap at Yinggeling Mountains (109.289°E, 18.878°N), Hainan province, China in May, 2022. The animal was euthanized in the euthanasia cage (10L), which was gradually filled with 99% purity of CO₂ within 2-3 minutes.

A total of 2 g of femoral muscle sample was clipped from the specimen using surgical scissors, and the total genomic DNA was extracted from the muscle tissue using the phenol–chloroform extraction (Sambrook et al. 1989), which contained three main procedures: 1) Sodium dodecylsulfate (SDS) and proteinase K were used for the enzymatic digestion of proteins; 2) A mixture of phenol: chloroform:isoamyl alcohol (25:24:1) was then added to promote the partitioning of lipids into the organic phase; 3) The DNA was rinsed using analytical alcohol of different solubility and then otained the purified DNA for PCR. Seven primer combinations were used for the generation of PCR products (see Extended data (Tu 2023)). PCR amplifications of mitochondrial genome of N. hainanensis were listed as follows: PCR amplifications were carried out in 25 uL reaction volumes containing 1×^EX Taq buffer (Mg²⁺ Free; TaKaRaBiotech, Dalian, China), 2.5 mM MgCl₂, 0.1-0.4 mM dNTP, 0.4 uM each primer, 0.04 u/uL ^EX Taq polymerase, and ~5 ng/uL total genomic DNA as template. PCR protocol was set as follows: an initial denaturation at 94°C for 4 min; 35 cycles of amplification as the main procedure: a denaturation at 94°C for 45 s, an annealing at 50-60°C for 50 s, an elongation at 72°C for 2-3 min, and a final extension at 72°C for 4 min. All PCR products were examined through electrophoresing on a 1.0% agarose gel and then directly sequenced using an ABI 3730xl sequencer. All sequences were assembled and edited using SeqMan (DNASTAR 7.1.0) (Swindell and Plasterer 1997). The boundaries of protein-coding genes were predicted via comparison with homologs using MEGA 6.0 (Tamura et al. 2013). The transfer RNA (tRNA) genes were identified using tRNAscan-SE 2.0 (Lowe and Chan 2016).

To better understand the phylogentic position of N. hainanensis within Eulipotyphla, we constructed a phylogenetic tree by using neighbor-joining (NJ) method implemented in MEGA6.0 (Tamura et al. 2013) based on 13 complete mitochondrial genome sequences of Eulipotyphla using Kimura 2-parameters (K2P) model.

Results

The whole mitochondrial genome of N. hainanensis is 17,337 bp in length (GenBank Accession number ON054206.2 (Tu and Hou 2022)) and contains 13 protein-coding genes (PCGs), 22 tRNAs genes, two rRNAs genes, and one control region. The base composition of N. hainanensis mitogenome is as follows: A 33.4%, G 12.2%, T 33.1%, and C 21.3%. Typically, an A+T rich pattern (66.5%) was observed. Comparison with a previously published mitochondrial genome sequence (MW429379) for the same species, these two sequences differ in length (17,337 bp vs 16,795 bp), which may be due to mitochondrial control region sequence variations (1,868 bp vs 1334bp). Within 37 mitochodrial genes, the ND6 gene and eight tRNAs (tRNA ^Gln, tRNA ^Ala, tRNA ^Asn, tRNA ^Cys, tRNA ^Tyr, tRNA ^Ser, tRNA ^Glu and tRNA ^Pro) were encoded on the L-strand, whereas all other genes were encoded on the H-strand. Most mitochondrial PCGs, use ATG as its start codon, ND2 and ND3 begin with ATT, and ND5 begin with ATA. Five genes (COX2, ATP6, ND3, ND4L and ND5) use TAA as stop codons, whereas four genes (ND1, ND2, COX1 and ATP8) end with TAG, and ND6 ends with AGA. COX3, ND4 and CYTB use T as an incomplete stop codon.

A phylogenetic tree showed that two sequences of N. hainanensis shared a common evolutionary ancestry. Within subfamily Galericinae, H. suillus and the common ancestors of N. hainanensis and N. sinensis shaped sister groups (Figure 1). The results were consistent with morphological studies (Cobert 1988; Gould 1995; Grenyer and Purvis 2003). Our results support current taxonomy that places the hainanensis, sinensis and suillus in different genera (Smith et al. 2009; Wei et al. 2021). This mitochondrial genome sequence determined in this study will benefit conservation genetics and phylogenetics of the species in future.

Figure 1. A phylogenetic tree of ten species based on the complete mitochondrial genomes.

Nodal support indicated by bootstrap.