The complete mitochondrial genome of a ground beetle Synuchus nitidus (Carabidae: Harpalinae: Sphodrini) from South Korea

Abstract

The ground beetle Synuchus nitidus (Motschulsky, 1861) (Carabidae: Harpalinae: Sphodrini) is one of the most common species in the forests of South Korea, which has the potential to be utilized as an environmental indicator. Here, we characterized the complete mitochondrial genome (mitogenome) of S. nitidus, which is the first in the harpaline tribe Sphodrini. Its genome is 16,392 bp in length and composed of 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and an A + T rich region. In addition, we reconstructed a maximum likelihood tree to elucidate the phylogenetic position of Sphodrini among the seven harpaline tribes using nucleotide sequences of the 13 PCGs. The ML tree supported a monophyletic clade of the subfamily Harpalinae and showed a close relationship between Sphodrini and Lebinii with a low bootstrap value. The complete mitogenome of S. nitidus could be helpful for molecular species identification and exploring phylogenetic relationships among carabids.

Keywords:

• Synuchus nitidus
• Sphodrini
• ground beetle
• mitochondrial genome
• phylogeny

Introduction

The ground beetle Synuchus nitidus (Motschulsky, 1861) (Carabidae: Harpalinae: Sphodrini) distributed in East Asia (Löbl and Löbl 2017) is one of the most dominant species in the forests of South Korea (Yeon et al. 2005). It is an important species since it is regarded as a potential bioindicator of forest succession or chemical pollution (Yeon et al. 2005; Fujita et al. 2008; Okatsu and Tsutsumi 2019). Despite the ecological importance of S. nitidus, only a few of its molecular data have been published in the NCBI database by Ruiz et al. (2009) and Kudo et al. (2019). In addition, the tribe Sphodrini, which includes S. nitidus, has an uncertain phylogenetic relationship with other harpaline tribes, as the phylogeny of Harpalinae based on 18S rRNA, 28S rRNA, and wingless gene resulted in polytomy (Ober and Maddison 2008).

The mitochondrial genome (mitogenome) has been widely used for phylogenetic studies in not only insects (i.e. Cameron 2014; Yuan et al. 2016; Park and Hwang 2022; Raupach et al. 2022) but also other arthropod taxa (i.e. Park et al. 2007; Woo et al. 2007; Choi et al. 2007; Dermauw et al. 2009; Baek et al. 2014). Therefore, this study characterized the complete mitogenome of S. nitidus and identified the phylogenetic relationship of the tribe Sphodrini within the subfamily Harpalinae using nucleotide sequences of 13 protein-coding genes (PCGs). The complete mitogenome of S. nitidus can help molecular identification of the species and elucidate carabid phylogeny.

Materials and methods

The adult specimen of S. nitidus was collected from Geochang-gun, Gyeongsangnam-do, South Korea (35°48'23"N 127°46'07"E). The specimen was identified by comparing external and male genital characteristics with Habu (1978) and is preserved under the voucher number LEGOA050011 along with extracted genomic DNA at the Animal Molecular Phylogenetics Lab., Kyungpook National University (UWH, [email protected]). Total genomic DNA was extracted from the thoracic muscle of the specimen using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). The complete mitogenome was amplified by PCR using two universal primer pair sets: LCO1490: 5′-GGTCAACAAATCATAAAGATATTGG-3′ (Folmer et al. 1994) and 16Sa 5′-CGCCTGTTTATCAAAAACAT-3′ (Palumbi et al. 1991) to amplify the fragment of COX1 to 16S rRNA; HCO2198: 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′ (Folmer et al. 1994) and 16Sb 5′-CTCCGGTTTGAACTCAGATCA-3′ (Palumbi et al. 1991) to amplify the fragment of 16S rRNA to COX1 (Figure S1). The PCR products were then sequenced based on the primer walking method. The read coverage plot was not included since the data was obtained with Sanger sequencing. Sequences were assembled by BioEdit 7.2.5 (Hall 1999) using the mitogenome of Harpalus sinicus Hope, 1845 (Yu et al. 2019) as a reference. Twenty-two tRNA genes were predicted using the tRNAscan-SE (Chan and Lowe 2019) and 13 protein-coding genes (PCGs) and two rRNA genes were identified using orthologous sequences of Harpalus sinicus (Yu et al. 2019). After the annotation, the circular mitochondrial genome of S. nitidus was visualized using the Proksee (Grant et al. 2023).

To investigate the phylogenetic placement of S. nitidus, 13 PCG nucleotide sequences of 36 species were used to infer the phylogenetic tree, including 35 species of the family Carabidae and one outgroup species belonging to the family Dytiscidae. Each protein-coding gene was aligned with the ClustalW alignment method (Thompson et al. 1994). Poorly aligned sites were removed and the sequences of each PCG were concatenated using the Gblock 0.91b (Castresana 2000). The sequences of PCGs were edge-linked, the best substitution model was selected as GTR + F + I + G4 by ModelFinder (Kalyaanamoorthy et al. 2017), and the maximum likelihood tree was inferred using the IQ-TREE online webserver (Trifinopoulos et al. 2016).

Results

The complete mitochondrial genome of S. nitidus (GenBank with accession number OR755976) (Figure 1) was 16,392 bp in length. Its GC content was 19.7% and nucleotide composition was as follows: 41.0% for A, 11.1% for C, 8.6% for G, and 39.3% for T. According to the annotation, it encoded 13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes. All PCGs started with a typical ATN codon except for ND1, which started with a TTG codon. As for the stop codon, all PCGs stopped with a TAA codon except for ND3, CYTB, and ND1, which stopped with a TAG codon, and ND5, which stopped with an incomplete termination codon T. The 12S rRNA and 16S rRNA genes were 786 bp and 1,317 bp in length, respectively. An A + T rich region was 1,524 bp in length and located between 12S rRNA and tRNA-Ile.

Figure 1. Circular map of the complete mitochondrial genome of Synuchus nitidus. The total length of the complete mitochondrial genome is 16,392 bp. The two outer circular bands indicate the strand and order of the genes, of which the exterior ones are on the heavy strand and the interior ones are on the light strand. The inner circle indicates the GC-skew, which is the deviation from the average GC content of the entire sequence. The photograph of S. nitidus was taken by DK.

In the reconstructed maximum likelihood tree (Figure 2), the family Carabidae was not monophyletic but paraphyletic to Trachipachidae, which was nested within the clade of Carabidae. All examined subfamilies of Carabidae formed a monophyletic clade with high node support values (BP > 95) except for the subfamily Harpalinae (BP = 58). Within the clade Harpalinae, the tribe Sphodrini formed a monophyletic clade with Harpalini, Hexagonini, and Lebiini with a lower node support value (BP = 48), and Sphodrini was closest to Lebiini (BP = 38). Interestingly, a monophyly of Pterostichini was not supported.

Figure 2. Inferred maximum likelihood tree based on nucleotide sequences of 13 PCGs of 36 species. As for included taxa, the family Dytiscidae was used as an outgroup. The star mark indicates the species studied here. Numbers on the branch indicate maximum likelihood support values. The following sequences were used: Hydroporus planus MW465248 (Villastrigo et al. 2021), Trachypachus holmbergi EU877954 (Sheffield et al. 2008), Cicindela anchoralis MG253029 (Wang et al. 2018), Manticora tibialis MF497821 (López-López and Vogler 2017), Pogonostoma subtiligrossum MF497820 (López-López and Vogler 2017), Omus cazieri MF497813 (López-López and Vogler 2017), Carabus changeonleei MG253028 (Wang et al. 2019), Carabus lafossei KY992943 (Liu et al. 2018), Carabus mirabilissimus GQ344500 (Wan et al. 2012), Carabus smaragdinus MN480425 (Oh et al. 2019), Harpalus pennsylvaticus MN245975 (Kieran 2020), Harpalus sinicus MN310888 (Yu et al. 2019), Abax parallelepipedus KT876877 (Linard et al. 2016), Pterostichus madidus KT876910 (Linard et al. 2016), Nebria brevicollis KT876906 (Linard et al. 2016), Amara aulica MN335930 (Li et al. 2020), Notiophilus quadripunctatus MW800883 (Raupach et al. 2022), Omophron limbatum MW800882 (Raupach et al. 2022), Metrius contractus MF497817 (López-López and Vogler 2017), Scarites buparius MF497822 (López-López and Vogler 2017), Brachinus crepitans JX412826, Carabus hortensis MN122850, Carabus granulatus MN122870, Blethisa multipunctata KX087243, Elaphrus cupreus KX087286, Hexagonia terminalis JX412768, Craspedophorus nobilis JX412738, Pterostichus niger KX087231, Pterostichus oblongopunctatus MN122833, Stomis pumicatus KX087349, Amara communis KX035135, Lebia chlorocephala KX087304, Promecognathus crassus JX313665, Bembidion varium KX087242, Pogonus iridipennis KX087338, and Synuchus nitidus OR755976.

Discussion and conclusion

Here, we characterized the complete mitochondrial genome (mitogenome) of S. nitidus, which is the first in the tribe Sphodrini. The order and strand position of the genes were identical to Harpalus sinicus and Notiophilus quadripunctatus Dejean, 1826 (Yu et al. 2019; Raupach et al. 2022). According to the ML tree (Figure 2), the relationships among carabid subfamilies were almost identical to those reported by Raupach et al. (2022). The tribe Sphodrini was placed within the subfamily Harpalinae, which was consistent with the current classification system listed in Löbl and Löbl (2017) and previous phylogenetic studies based on 28S rRNA and wingless genes by Ober and Maddison (2008). Among the seven examined harpaline tribes, Sphodrini was a sister to Lebiini (BP = 38), which was grouped with the clade of Hexagonini and Harpalini (BP = 48). This result did not support the close relationship of Sphodrini with Pterostichini and Zabrini, as suggested by Ruiz et al. (2009) inferred from four nuclear genes and three mitochondrial genes. To clarify the phylogenetic position of Sphodrini within the subfamily Harpalinae, it seems necessary to obtain much more harpaline mitochondrial genome data through further studies.

The complete mitogenome of ecologically important S. nitidus will be helpful for molecular identification of the species and future carabid phylogenetic research using mitogenomes.

Author contributions

UWH and EHC designed this study and UWH and DK wrote the manuscript. DK, GK, CRS, and EHC conducted the experiments and analyzed the data. DK and BP performed the fieldwork and sampling. All authors revised the manuscript and agreed to be responsible for all aspects of the work.

Ethical approval

The material involved in this article does not involve any ethical conflicts. This species is not endangered according to the CITES catalogue or IUCN Red List, and the sample was not collected from a natural reserve, so the collection did not require any specific permissions or licenses.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Data availability statement

The data that support the findings of this study are openly available in GenBank at http://www.ncbi.nlm.nih.gov/, under the accession number OR755976. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA1051788, SRX22874416, and SAMN38797855, respectively.

Additional information

Funding

This work was supported by a grant for UWH from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE), South Korea [NIBR202333201].