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. 2025 Sep 2;10(10):909–913. doi: 10.1080/23802359.2025.2554217

Complete mitochondrial genome of the firefly Luciola parvula Kiesenwetter (Coleoptera, Lampyridae) from Japan and its phylogenetic analyses

Yuki Selene Saito a,b,f,g,*, Yuki Kaneko-Ochi a,*, Masayuki Iigo a,b,c,d,e,f,✉
PMCID: PMC12406331  PMID: 40909384

Abstract

We have sequenced the complete mitochondrial genome of the firefly Luciola parvula Kiesenwetter, 1874 (Coleoptera, Lampyridae, Luciolinae) from Niigata Prefecture, Honshu, Japan, and inferred the Luciolinae phylogeny. The circular genome of 16,685 bp (GenBank accession number, LC677171) has a base composition of A (43.52%), C (13.21%), G (8.52%) and T (34.74%). Our sequence is similar to other Metazoa, which contains 13 protein coding genes (PCGs), 2 rRNA genes, 22 tRNA genes and D-loop. Molecular phylogenetic analyses based on mitochondrial whole genome sequences revealed L. parvula as a sister group of (L. unmunsana [MT134039] + L. parvula [OL944082]). Identity of the two L. parvula sequences (LC677171 and OL944082) is only 90.5% (14,064/15,537 bp), suggesting the two L. parvula might be different species. Further taxonomical examination including morphological analyses should be required to test if the two populations are different species.

Keywords: Firefly, Luciola parvula, Luciolinae, mitogenome, molecular phylogenetic analysis

Introduction

About 50 species of fireflies are known in Japan (Fukuta et al. 2024). Luciola parvula Kiesenwetter (Japanese name, Hime-botaru; Coleoptera, Lampyridae, Luciolinae) (Figure 1) is a terrestrial firefly species widely distributed in Japan (Kawashima et al. 2003). The adult male L. parvula emits from their lanterns a yellow light, one of the most red-shifted colors found among fireflies that belongs to a specific subfamily Luciolinae (Bessho-Uehara and Oba 2017). They emit the flash at interval of 0.5 to 0.8 s (Ohba 2016), indicating that this species is an interesting model to investigate light-communication systems and ultradian rhythms in light emission.

Figure 1.

Figure 1.

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Photographs of a male Luciola parvula. Left, dorsal view; right, ventral view. These photographs were taken by the corresponding author (Masayuki Iigo).

Several studies have demonstrated genetic diversity of local populations of L. parvula in Japan using mitochondrial 16S (Suzuki 1997; Choi et al. 2003), ND5 (Hiyori et al. 2016) and COI (Oba et al. 2011) genes. However, since the complete genome remained to be sequenced, we undertook to sequence and characterize the complete mitochondrial genome of L. parvula for subsequent comparison with other mitochondrial genomes and construction of a phylogeny based only on mitochondrial genomes.

Materials and methods

The firefly L. parvula (#1194 M, a single male) was caught in Uonuma (Okutadami), Niigata Prefecture, Honshu, Japan (37.06 N, 139.25 E; altitude of 780 m) on 17 July 2014 and stored in ethanol. A specimen was deposited at Utsunomiya University (Masayuki Iigo, iigo@cc.utsunomiya-u.ac.jp) under the voucher number #1194 M]. DNA was extracted from the muscle using DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany). The extracted DNA was sheared using the M220 Focused-ultrasonicator (Covaris, Woburn, MA, USA). A library for next-generation sequencing was prepared using TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA). The nucleotide sequence was determined by 301 bp × 2 pair-end sequence using MiSeq (Illumina). Trimming, de novo sequence assembly, local BLAST search and mapping of trimmed reads to the identified mitochondrial complete genome were performed using CLC Genomics Workbench ver 11.0.1 (QIAGEN). The mitochondrial genome was annotated using MITOS (http://mitos.bioinf.uni-leipzig.de/index.py; Bernt et al. 2013) and manually curated. The genome map of L. parvula was visualized using the Proskee (Grant et al. 2023). The completeness of the mitochondrial genome was verified using sequencing depth coverage data calculated from raw read mapping to the complete genome map.

Molecular phylogenetic analyses based on mitochondrial whole genome sequences were performed with published mitogenomes of 19 Luciolinae species with 2 Lampyrinae species as an outgroup, to assess the phylogenetic position of L. parvula. MEGA12 program (Kumar et al. 2024) was used. Multiple sequence alignment was performed by MUSCLE (Edgar 2004). Molecular phylogenetic analysis was performed using maximum-likelihood (ML) method, and the best model GTR + G + I was finally used to construct phylogenetic tree with 1,000 bootstrap replications. The partial deletion option was applied to eliminate all positions with less than 95% site coverage resulting in a final data set comprising 15,085 positions.

Results

Total of 30,764,808 raw reads were yielded by MiSeq. Raw reads were filtered using Genomics Workbench to remove adapter sequences and low-quality reads, which resulted in a total of 29,138,179 clean reads with the mean length of 195.7 bp. Local BLAST search of de novo assembled contigs using complete mitochondrial genome of Nipponoluciola cruciata (AB849456; Maeda et al. 2017a) identified the complete mitochondrial genome of L. parvula. The complete mitochondrial genome (DDBJ/EMBL/GenBank accession number: LC677171) is a double-stranded and circular molecule with 16,685 bp in length, containing 13 PCGs, 2 rRNA genes, 22 tRNA genes and D-loop (Figure 2). 122,238 reads (0.44%, 196.6 bp in average length) were mapped to the complete mitochondrial genome (Figure S1). The mean coverage was x1,440. The nucleotide composition is A (43.52%), T (34.74%), C (13.21%) and G (8.52%) with A + T bias (78.26%). The light strand encodes 4 PCGs (ND5, ND4, ND4L and ND1) and eight tRNA genes (tRNA-Gln, tRNA-Cys, tRNA-Tyr, tRNA-Phe, tRNA-His, tRNA-Pro, tRNA-Leu and tRNA-Val), and heavy strand encodes the other tRNA genes. Among PCGs, ATG is used as the start codon for COII, ATP6, COIII, ND4, ND4L and Cytb, ATA for ND2, ND3 and ND6, ATT for COI, ATP8 and ND5, and TTG for ND1. TAA is used as the stop codon for ND2, COI, ATP8, ATP6, ND4, ND4L and ND6, TAG for Cytb and ND1. An incomplete stop codon (a single T codon) was found in 4 PCGs (COII, COIII, ND3 and ND4).

Figure 2.

Figure 2.

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Gene map of the mitochondrial genome for Luciola parvula (DDBJ/EMBL/GenBank accession number: LC677171). Arrows indicate the orientation of gene transcription. Protein coding sequences (CDS), tRNA, rRNA and D-loop are marked with different colors. The tRNAs are labeled according to the three-letter amino acid codes.

The ML trees (Figure 3) revealed the phylogeny of Luciolinae based only on complete mitochondrial genomes. L. parvula (LC677171) and (L. unmunsana [MT134039] + L. parvula [OL944082]) were most closely related, followed by (Luciola kagiana [OQ184181] + L. curtithorax [MG770613]. These Luciola species formed a monophyletic group with the highest nodal support. Sclerotia substriata (formerly, Luciola substriata) [KP313820], Nipponoluciola cruciata (formerly, Luciola cruciata) [AB849456], Aquatica lateralis (formerly, Luciola lateralis) [LC306678], Aquatica wuhana [KX758086], Aquatica ficta [KX758085] and Aquatica leii [KF667531] were more distantly related.

Figure 3.

Figure 3.

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Molecular phylogenetic ML tree for the subfamily Luciolinae. The numbers above the branch meant bootstrap value (1000 replicates). GenBank accession numbers of complete mitochondrial genomes of luciolinae used are as follows: Emeia pseudosauteri, MK292112 (Chen et al. 2019); Asymmetricata circumdata, KX229747 (Luan and Fu 2016); Curtos bilineatus, MK292114 (Chen et al. 2019); Pteroptyx maipo, MF686051 (Fan and Fu 2017); inflata indica, MH427718 (Sriboonlert and Wonnapinij 2019); Pygoluciola qingyu, MK292093 (Chen et al. 2019); Abscondita cerata, MW751423 (Wang et al. 2021); Abscondita anceyi, MH020192 (Hu and Fu 2018b); Abscondita terminalis, MK292092 (Chen et al. 2019); Sclerotia substriata (formerly, Luciola substriata), KP313820 (Mu et al. 2016); Nipponoluciola cruciata (formerly, Luciola cruciata), AB849456 (Maeda et al. 2017a); Aquatica lateralis (formerly, Luciola lateralis), LC306678 (Maeda et al. 2017b); Aquatica wuhana, KX758086 (Wang et al. 2017); Aquatica ficta, KX758085 (Wang et al. 2017); aquatica leii, KF667531 (Jiao et al. 2015); Luciola kagiana, OQ184181; Luciola curtithorax, MG770613 (Hu and Fu 2018a); Luciola unmunsana, MT134039 (Kim et al. 2020); Luciola parvula, OL944082. Lampyrinae (Pyrocoelia rufa, AF452048, Bae et al. 2004; Pyrocoelia thibetana, MK292117) is used as an outgroup.

Discussion and conclusion

In this study, complete mitochondrial genome of L. parvula was successfully sequenced and annotated, revealing a comprehensive mitogenome containing 13 PCGs, 2 rRNA genes and 22 tRNA genes as in other Metazoa. The mean coverage depth was x1,440 and relatively uniform across the entire mitochondrial genome, indicating the sequence is reliable. The nucleotide composition is A + T biased (78.26%) as in the mitochondrial genome of other Luciola species studied to date (79.78% in L. kagiana, OQ184181; 80.01% in L. curtithorax, MG770613, Hu and Fu 2018a); 77.94% in L. unmunsana, MT134039, Kim et al. 2020; 78.34% in Chinese L. parvula, OL944082).

During the course of this research, complete mitochondrial genome for L. parvula (probably Chinese) was deposited to NCBI (OL944082) by Chinese researchers. In the ML molecular phylogenetic tree, the Japanese L. parvula (LC677171) is the sister to the clade of L. unmunsana (MT134039) and L. parvula (OL944082). The identity of nucleotide sequences of Chinese L. parvula (OL944082) and Korean L. unmunsana (MT134039, Kim et al. 2020) is 98.1% (15,265/15,561 bp) while the identity of the two L. parvula sequences (LC677171 and OL944082) is only 90.5% (14,064/15,537 bp), indicating the two L. parvula are different species. Type locality of L. parvula is Japan and they distribute in Honshu, Shikoku, Kyushu and Goto Islands in Japan (Kawashima et al. 2003; Ballantyne et al. 2022). Thus, it is highly probable that Chinese L. parvula (OL944082) is misidentified species. Further taxonomic investigation including characterization of morphology should be required to discriminate Japanese and Chinese L. parvula in future.

In conclusion, we have sequenced and characterized the complete mitochondrial genome sequence of L. parvula collected in Niigata Prefecture, Honshu, Japan. This would provide valuable information for investigating the genetic diversity of local populations of L. parvula and molecular framework for further phylogenetic analyses of Lampyridae.

Supplementary Material

Supplemental Material
TMDN_A_2554217_SM8815.docx (232.3KB, docx)

Acknowledgments

We thank Takuya Sakurai, Koshirazawa Hutte, for advices to collect L. parvula. Y.

Funding Statement

The study was supported in part by University/Regional Cooperative Action Support Project (2015–2024) from Tochigi Prefecture.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethical approval

The species used in this study is a common and unprotected species in Niigata Prefecture, Japan. We confirm that all researches are conducted in accordance with ethical guidelines and the legal requirements of the country.

Data availability statement

The genome sequence data that support the findings of this study are openly available in DDBJ/EMBL/GenBank under the accession no. LC677171. The associated BioProject, SRA, and BioSample numbers are PRJDB20559, DRX638461, and SAMD00897592, respectively.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Material
TMDN_A_2554217_SM8815.docx (232.3KB, docx)

Data Availability Statement

The genome sequence data that support the findings of this study are openly available in DDBJ/EMBL/GenBank under the accession no. LC677171. The associated BioProject, SRA, and BioSample numbers are PRJDB20559, DRX638461, and SAMD00897592, respectively.

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